Indraneel Ghosh

Interests

Teaching

I aspire to teach all my students to be intellectually engaged and independent, ask questions that we cannot presently answer, formulate and test their hypothesis, work hard and with enthusiasm, and derive pleasure in teaching others. Building and nurturing these abilities is a central objective both in a classroom setting as well as one-on-one teaching/mentoring of students engaged in research in our laboratory. Central to this quest for motivating students to ask difficult questions requires their thorough engagement in the material that they need to master. Thus, I make every effort to make the instructional material relevant to the students while presenting them with opportunities for independent discovery that allows them to clearly visualize the threads that connect abstract concepts, historical experiments, and modern science. I serve as an Honors Professor and have had the pleasure of teaching freshman colloquiums, where we discuss and debate both socially and scientifically relevant topics. I have also had the pleasure of teaching both the regular and Honors sections of sophomore Organic Chemistry, a fundamental class for all areas of science. Almost all undergraduate students that have worked with us in our laboratories have published their research in one or more peer reviewed publications and have gone on to graduate and/or medical schools. For example, Thom (PhD w/Carlos Barbas, Scripps), Eri (PhD w/Carlos, Catalano, Univ. Washington) Luisa (PhD w/Dan Kahne, NSF Fellow, Harvard), Ahmed (NSF Fellow w/David Liu, Harvard), Kurt (NSF Fellow, w/Alice Ting, MIT), Blake (NSF Fellow, Harvard), Mark (Johns Hopkins, MD), Kevin (Univ of Virginia, MD) etc. All graduate students that have started with me have completed their PhDs and gone on to postdoctoral positions with Min (David Craik. UCSF), Ben (Stanley Fields, Univ. of Wash), Vandana (Dan Herschlag, Stanford), Cliff (Barbara Imperiali, MIT), Jennifer (Peter Schultz, Scripps) and Jason (Jim Wells, UCSF) among others and have subsequently secured positions in both academia and industry

Research

The central motivation of our research program is to develop new methodologies that have the long term potential for detecting as well as controlling the biological activity of a variety of proteins and DNA. These designed proteins have the potential to provide new approaches to both understand fundamental biology and help provide diagnostics and therapeutics.Towards this goal, we explore new paradigms for designing protein and peptide architectures that can serve as:I) Turn-on sensors that respond to a variety of macromolecules. Our targets include DNA, RNA, proteins, enzymes and their inhibitors.II) Peptide and protein reagents for selectively targeting protein surfaces.III) Designed Kinases and Phosphatases that respond to small molecules allowing for controlling their activity in cells.Our research efforts have been disseminated in over 50 publications and reviews, with 13 appearing in J. Am. Chem. Soc., the flagship journal of the American Chemical Society. Our research has also been highlighted on journal covers and has been the subject of editorials and highlighted in Nature Methods, Molecular BioSystems, ACS Chemical Biology, Biotechniques, and the Faculty of 1000 among others.

Courses

Scholarly Contributions

Journals/Publications

• Campbell, S. T., Carlson, K. J., Buchholz, C. J., Helmers, M. R., & Ghosh, I. (2015). Mapping the BH3 Binding Interface of Bcl-X-L, Bcl-2, and Mcl-1 Using Split-Luciferase Reassembly. BIOCHEMISTRY, 54(16), 2632-2643.
• Meyer, S. C., Shomin, C. D., Gaj, T., & Ghosh, I. (2007). Tethering small molecules to a phage display library: Discovery of a selective bivalent inhibitor of protein kinase A. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 129(45), 13812-13813.
• Restituyo, E., Camacho-Soto, K., & Ghosh, I. (2015). A fragment-based selection approach for the discovery of Peptide macrocycles targeting protein kinases. Methods in molecular biology (Clifton, N.J.), 1248, 95-104.
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  Protein kinases are implicated in diverse signaling cascades and have been targeted with small molecules that typically bind the conserved ATP-binding active site. These inhibitors are often promiscuous and target multiple protein kinases, which has led to the development of alternate strategies to discover selective ligands. We have recently described a fragment-based selection approach, where a small-molecule warhead can be non-covalently tethered to a phage-displayed library of cyclic peptides. This approach led to the conversion of the promiscuous kinase inhibitor, staurosporine, into a selective bivalent inhibitor.
• Camacho-Soto, K., Castillo-Montoya, J., Tye, B., & Ghosh, I. (2014). Ligand-Gated Split-Kinases. Journal of the American Chemical Society, 136(10), 3995-4002.
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  Abstract: The activity of protein kinases are naturally gated by a variety of physiochemical inputs, such as phosphorylation, metal ions, and small molecules. In order to design protein kinases that can be gated by user-defined inputs, we describe a sequence dissimilarity based approach for identifying sites in protein kinases that accommodate 25-residue loop insertion while retaining catalytic activity. We further demonstrate that the successful loop insertion mutants provide guidance for the dissection of protein kinases into two fragments that cannot spontaneously assemble and are thus inactive but can be converted into ligand-gated catalytically active split-protein kinases. We successfully demonstrate the feasibility of this approach with Lyn, Fak, Src, and PKA, which suggests potential generality. © 2014 American Chemical Society.
• Camacho-Soto, K., Castillo-Montoya, J., Tye, B., Ogunleye, L. O., & Ghosh, I. (2014). Small molecule gated split-tyrosine phosphatases and orthogonal split-tyrosine kinases. Journal of the American Chemical Society, 136(49), 17078-86.
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  Protein kinases phosphorylate client proteins, while protein phosphatases catalyze their dephosphorylation and thereby in concert exert reversible control over numerous signal transduction pathways. We have recently reported the design and validation of split-protein kinases that can be conditionally activated by an added small molecule chemical inducer of dimerization (CID), rapamycin. Herein, we provide the rational design and validation of three split-tyrosine phosphatases (PTPs) attached to FKBP and FRB, where catalytic activity can be modulated with rapamycin. We further demonstrate that the orthogonal CIDs, abscisic acid and gibberellic acid, can be used to impart control over the activity of split-tyrosine kinases (PTKs). Finally, we demonstrate that designed split-phosphatases and split-kinases can be activated by orthogonal CIDs in mammalian cells. In sum, we provide a methodology that allows for post-translational orthogonal small molecule control over the activity of user defined split-PTKs and split-PTPs. This methodology has the long-term potential for both interrogating and redesigning phosphorylation dependent signaling pathways.
• Ogunleye, L. O., Jester, B. W., Riemen, A. J., Badran, A. H., Wang, P., & Ghosh, I. (2014). When tight is too tight: Dasatinib and its lower affinity analogue for profiling kinase inhibitors in a three-hybrid split-luciferase system. MedChemComm, 5(3), 328-332.
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  Abstract: The development of methods for profiling inhibitors of protein kinases has seen tremendous progress over the last decade. We have previously reported a split-luciferase based three-hybrid approach for determining kinase inhibitor selectivity that utilized the promiscuous staurosporine warhead for designing chemical inducers of dimerization (CID). Herein we describe the extension of this methodology to target the tyrosine kinase (TK) group using a Dasatinib warhead based CID. We found that though the Dasatinib enabled CID provided a means for assembling the split-protein fragments, it had too tight an affinity in the context of the three-hybrid system for several TKs and could not be displaced with inhibitors. By tuning the affinity of Dasatinib, we were able to successfully target multiple TKs that could subsequently be assayed for inhibition by small molecules. We further demonstrated that the new CID allowed for the screening and identification of inhibitors against ABL. © 2014 The Royal Society of Chemistry.
• Stains, C. I., Furman, J. L., Segal, D. J., & Ghosh, I. (2006). Site-specific detection of DNA methylation utilizing mCpG-SEER. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 128(30), 9761-9765.
• Yu, Y., Blair, S., Gillespie, D., Jensen, R., Myszka, D., Badran, A. H., Ghosh, I., & Chagovetz, A. (2010). Direct DNA Methylation Profiling Using Methyl Binding Domain Proteins. ANALYTICAL CHEMISTRY, 82(12), 5012-5019.
• Gaj, T., Meyer, S. C., & Ghosh, I. (2007). The AviD-tag, a NeutrAvidin/avidin specific peptide affinity tag for the immobilization and purification of recombinant proteins. PROTEIN EXPRESSION AND PURIFICATION, 56(1), 54-61.
• Ghosh, I., Jester, B. W., Gaj, A., Shomin, C. D., Cox, K. J., & Ghosh, I. -. (2012). Testing the promiscuity of commercial kinase inhibitors against the AGC kinase group using a split-luciferase screen. Journal of medicinal chemistry, 55(4).
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  Using a newly developed competitive binding assay dependent upon the reassembly of a split reporter protein, we have tested the promiscuity of a panel of reported kinase inhibitors against the AGC group. Many non-AGC targeted kinase inhibitors target multiple members of the AGC group. In general, structurally similar inhibitors consistently exhibited activity toward the same target as well as toward closely related kinases. The inhibition data was analyzed to test the predictive value of either using identity scores derived from residues within 6 Å of the active site or identity scores derived from the entire kinase domain. The results suggest that the active site identity in certain cases may be a stronger predictor of inhibitor promiscuity. The overall results provide general guidelines for establishing inhibitor selectivity as well as for the future design of inhibitors that either target or avoid AGC kinases.
• Ghosh, I., Lamba, V., & Ghosh, I. -. (2012). New directions in targeting protein kinases: focusing upon true allosteric and bivalent inhibitors. Current pharmaceutical design, 18(20).
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  Over the past decade, therapeutics that target subsets of the 518 human protein kinases have played a vital role in the fight against cancer. Protein kinases are typically targeted at the adenosine triphosphate (ATP) binding cleft by type I and II inhibitors, however, the high sequence and structural homology shared by protein kinases, especially at the ATP binding site, inherently leads to polypharmacology. In order to discover or design truly selective protein kinase inhibitors as both pharmacological reagents and safer therapeutic leads, new efforts are needed to target kinases outside the ATP cleft. Recent advances include the serendipitous discovery of type III inhibitors that bind a site proximal to the ATP pocket as well as the truly allosteric type IV inhibitors that target protein kinases distal to the substrate binding pocket. These new classes of inhibitors are often selective but usually display moderate affinities. In this review we will discuss the different classes of inhibitors with an emphasis on bisubstrate and bivalent inhibitors (type V) that combine different inhibitor classes. These inhibitors have the potential to couple the high affinity and potency of traditional active site targeted small molecule inhibitors with the selectivity of inhibitors that target the protein kinase surface outside ATP cleft.
• Porter, J. R., Stains, C. I., Jester, B. W., & Ghosh, I. (2008). A general and rapid cell-free approach for the interrogation of protein-protein, protein-DNA, and protein-RNA interactions and their antagonists utilizing split-protein reporters. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 130(20), 6488-6497.
• Badran, A. H., Furman, J. L., Ma, A. S., Comi, T. J., Porter, J. R., & Ghosh, I. (2011). Evaluating the global CpG methylation status of native DNA utilizing a bipartite split-luciferase sensor. Analytical Chemistry, 83(18), 7151-7157.
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  PMID: 21797230;PMCID: PMC3206592;Abstract: Epigenetic modifications play an essential role in the regulation of gene expression and ultimately cell fate. Methylation of cytosine at CpG dinucleotides (mCpG) is an important epigenetic mark that has been correlated with cancer when present at promoter sites of tumor suppressor genes. To develop a rapid methodology for the direct assessment of global levels of DNA methylation, we first interrogated the methyl-CpG binding domains (MBDs), the Kaiso family of Cys 2-His 2 zinc fingers, and an SET- and RING-associated domain using a split-luciferase reassembly methodology. We identified MBD1 as the most selective domain for the discrimination between mCpG and CpG sites with over 90-fold selectivity. Utilizing a bipartite strategy, we constructed a purely methylation-dependent bipartite sensor for the direct detection of global levels of DNA methylation by attaching MBD1 domains to each of the split-luciferase halves. This new sensor was validated for the direct determination of genomic DNA methylation levels in in vitro studies without any intervening chemical or enzymatic processing of DNA. Finally, we demonstrated that this bipartite sensor can be utilized for monitoring dose-dependent changes in global levels of methylation in DNA from HeLa cells challenged with 5-aza-2′-deoxycytidine, a DNA methyltransferase inhibitor. © 2011 American Chemical Society.
• Camacho-Soto, K., Castillo-Montoya, J., Tye, B., Ogunleye, L. O., & Ghosh, I. (2011). Small Molecule Gated Split-Tyrosine Phosphatases and Orthogonal Split-Tyrosine Kinases. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 136(49), 17078-17086.
• Cox, K. J., Shomin, C. D., & Ghosh, I. (2011). Tinkering outside the kinase ATP box: Allosteric (type IV) and bivalent (type V) inhibitors of protein kinases. Future Medicinal Chemistry, 3(1), 29-43.
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  PMID: 21428824;Abstract: Many members of the protein kinase family have emerged as key targets for pharmacological intervention, most notably in cancer. However, the high sequence and structural homology shared by the more than 500 human protein kinases renders it exceedingly difficult to develop selective inhibitors. Most, if not all, existing inhibitors target multiple protein kinases. Current paradigm suggests that an inhibitor that targets multiple kinases and displays polypharmacology is not only acceptable but also often desirable as a therapeutic agent. However, as we move toward personalized medicine the currently acceptable promiscuity is likely to pose significant hurdles in terms of their therapeutic index, especially for diseases that necessitate long-term drug administration. Moreover, selective inhibitors are the only pharmacologically relevant route toward reagents for the dissection of complex signal transduction pathways. This article provides an overview of recent developments in the design of kinase inhibitors that display increasing selectivity by targeting regions outside the highly conserved ATP-binding pocket. These new approaches may pave the way to potentially new avenues for drug discovery while providing valuable tools for studying signal transduction.
• Furman, J. L., Mok, P., Badran, A. H., & Ghosh, I. (2011). Turn-on DNA damage sensors for the direct detection of 8-oxoguanine and photoproducts in native DNA. Journal of the American Chemical Society, 133(32), 12518-12527.
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  PMID: 21520929;PMCID: PMC3206586;Abstract: The integrity of the genetic information in all living organisms is constantly threatened by a variety of endogenous and environmental insults. To counter this risk, the DNA-damage response is employed for repairing lesions and maintaining genomic integrity. However, an aberrant DNA-damage response can potentially lead to genetic instability and mutagenesis, carcinogenesis, or cell death. To directly monitor DNA damage events in the context of native DNA, we have designed two new sensors utilizing genetically fragmented firefly luciferase (split luciferase). The sensors are comprised of a methyl-CpG binding domain (MBD) attached to one fragment of split luciferase for localizing the sensor to DNA (50-80% of the CpG dinucleotide sites in the genome are symmetrically methylated at cytosines), while a damage-recognition domain is attached to the complementary fragment of luciferase to probe adjacent nucleotides for lesions. Specifically, we utilized oxoguanine glycosylase 1 (OGG1) to detect 8-oxoguanine caused by exposure to reactive oxygen species and employed the damaged-DNA binding protein 2 (DDB2) for detection of pyrimidine dimer photoproducts induced by UVC light. These two sensors were optimized and validated using oligonucleotides, plasmids, and mammalian genomic DNA, as well as HeLa cells that were systematically exposed to a variety of environmental insults, demonstrating that this methodology utilizing MBD-directed DNA localization provides a simple, sensitive, and potentially general approach for the rapid profiling of specific chemical modifications associated with DNA damage and repair. © 2011 American Chemical Society.
• Furman, J. L., Mok, P., Shen, S., Stains, C. I., & Ghosh, I. (2011). A turn-on split-luciferase sensor for the direct detection of poly(ADP-ribose) as a marker for DNA repair and cell death. Chemical Communications, 47(1), 397-399.
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  PMID: 20830433;Abstract: Designed sensors comprising split-firefly luciferase conjugated to tandem poly(ADP-ribose) binding domains allow for the direct solution phase detection of picogram quantities of PAR and for monitoring temporal changes in poly(ADP-ribosyl)ation events in mammalian cells. © 2011 The Royal Society of Chemistry.
• Ghosh, I., Badran, A. H., Furman, J. L., Ma, A. S., Comi, T. J., Porter, J. R., & Ghosh, I. -. (2011). Evaluating the global CpG methylation status of native DNA utilizing a bipartite split-luciferase sensor. Analytical chemistry, 83(18).
  More info

  Epigenetic modifications play an essential role in the regulation of gene expression and ultimately cell fate. Methylation of cytosine at CpG dinucleotides (mCpG) is an important epigenetic mark that has been correlated with cancer when present at promoter sites of tumor suppressor genes. To develop a rapid methodology for the direct assessment of global levels of DNA methylation, we first interrogated the methyl-CpG binding domains (MBDs), the Kaiso family of Cys(2)-His(2) zinc fingers, and an SET- and RING-associated domain using a split-luciferase reassembly methodology. We identified MBD1 as the most selective domain for the discrimination between mCpG and CpG sites with over 90-fold selectivity. Utilizing a bipartite strategy, we constructed a purely methylation-dependent bipartite sensor for the direct detection of global levels of DNA methylation by attaching MBD1 domains to each of the split-luciferase halves. This new sensor was validated for the direct determination of genomic DNA methylation levels in in vitro studies without any intervening chemical or enzymatic processing of DNA. Finally, we demonstrated that this bipartite sensor can be utilized for monitoring dose-dependent changes in global levels of methylation in DNA from HeLa cells challenged with 5-aza-2'-deoxycytidine, a DNA methyltransferase inhibitor.
• Ghosh, I., Cox, K. J., Shomin, C. D., & Ghosh, I. -. (2011). Tinkering outside the kinase ATP box: allosteric (type IV) and bivalent (type V) inhibitors of protein kinases. Future medicinal chemistry, 3(1).
  More info

  Many members of the protein kinase family have emerged as key targets for pharmacological intervention, most notably in cancer. However, the high sequence and structural homology shared by the more than 500 human protein kinases renders it exceedingly difficult to develop selective inhibitors. Most, if not all, existing inhibitors target multiple protein kinases. Current paradigm suggests that an inhibitor that targets multiple kinases and displays polypharmacology is not only acceptable but also often desirable as a therapeutic agent. However, as we move toward personalized medicine the currently acceptable promiscuity is likely to pose significant hurdles in terms of their therapeutic index, especially for diseases that necessitate long-term drug administration. Moreover, selective inhibitors are the only pharmacologically relevant route toward reagents for the dissection of complex signal transduction pathways. This article provides an overview of recent developments in the design of kinase inhibitors that display increasing selectivity by targeting regions outside the highly conserved ATP-binding pocket. These new approaches may pave the way to potentially new avenues for drug discovery while providing valuable tools for studying signal transduction.
• Ghosh, I., Furman, J. L., Mok, P., Badran, A. H., & Ghosh, I. -. (2011). Turn-on DNA damage sensors for the direct detection of 8-oxoguanine and photoproducts in native DNA. Journal of the American Chemical Society, 133(32).
  More info

  The integrity of the genetic information in all living organisms is constantly threatened by a variety of endogenous and environmental insults. To counter this risk, the DNA-damage response is employed for repairing lesions and maintaining genomic integrity. However, an aberrant DNA-damage response can potentially lead to genetic instability and mutagenesis, carcinogenesis, or cell death. To directly monitor DNA damage events in the context of native DNA, we have designed two new sensors utilizing genetically fragmented firefly luciferase (split luciferase). The sensors are comprised of a methyl-CpG binding domain (MBD) attached to one fragment of split luciferase for localizing the sensor to DNA (50-80% of the CpG dinucleotide sites in the genome are symmetrically methylated at cytosines), while a damage-recognition domain is attached to the complementary fragment of luciferase to probe adjacent nucleotides for lesions. Specifically, we utilized oxoguanine glycosylase 1 (OGG1) to detect 8-oxoguanine caused by exposure to reactive oxygen species and employed the damaged-DNA binding protein 2 (DDB2) for detection of pyrimidine dimer photoproducts induced by UVC light. These two sensors were optimized and validated using oligonucleotides, plasmids, and mammalian genomic DNA, as well as HeLa cells that were systematically exposed to a variety of environmental insults, demonstrating that this methodology utilizing MBD-directed DNA localization provides a simple, sensitive, and potentially general approach for the rapid profiling of specific chemical modifications associated with DNA damage and repair.
• Ghosh, I., Furman, J. L., Mok, P., Shen, S., Stains, C. I., & Ghosh, I. -. (2011). A turn-on split-luciferase sensor for the direct detection of poly(ADP-ribose) as a marker for DNA repair and cell death. Chemical communications (Cambridge, England), 47(1).
  More info

  Designed sensors comprising split-firefly luciferase conjugated to tandem poly(ADP-ribose) binding domains allow for the direct solution phase detection of picogram quantities of PAR and for monitoring temporal changes in poly(ADP-ribosyl)ation events in mammalian cells.
• Ghosh, I., Shekhawat, S. S., & Ghosh, I. -. (2011). Split-protein systems: beyond binary protein-protein interactions. Current opinion in chemical biology, 15(6).
  More info

  It has been estimated that 650,000 protein-protein interactions exist in the human interactome (Stumpf et al., 2008), a subset of all possible macromolecular partnerships that dictate life. Thus there is a continued need for the development of sensitive and user-friendly methods for cataloguing biomacromolecules in complex environments and for detecting their interactions, modifications, and cellular location. Such methods also allow for establishing differences in the interactome between a normal and diseased cellular state and for quantifying the outcome of therapeutic intervention. A promising approach for deconvoluting the role of macromolecular partnerships is split-protein reassembly, also called protein fragment complementation. This approach relies on the appropriate fragmentation of protein reporters, such as the green fluorescent protein or firefly luciferase, which when attached to possible interacting partners can reassemble and regain function, thereby confirming the partnership. Split-protein methods have been effectively utilized for detecting protein-protein interactions in cell-free systems, Escherichia coli, yeast, mammalian cells, plants, and live animals. Herein, we present recent advances in engineering split-protein systems that allow for the rapid detection of ternary protein complexes, small molecule inhibitors, as well as a variety of macromolecules including nucleic acids, poly(ADP) ribose, and iron sulfur clusters. We also present advances that combine split-protein systems with chemical inducers of dimerization strategies that allow for regulating the activity of orthogonal split-proteases as well as aid in identifying enzyme inhibitors. Finally, we discuss autoinhibition strategies leading to turn-on sensors as well as future directions in split-protein methodology including possible therapeutic approaches.
• Ghosh, I., Shekhawat, S. S., Campbell, S. T., & Ghosh, I. -. (2011). A comprehensive panel of turn-on caspase biosensors for investigating caspase specificity and caspase activation pathways. Chembiochem : a European journal of chemical biology, 12(15).
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  Caspases play a central role in apoptosis, differentiation, and proliferation, and represent important therapeutic targets for treating cancer and inflammatory disorders. Toward the goal of developing new tools to probe caspase substrate cleavage specificity as well as to systematically interrogate caspase activation pathways, we have constructed and investigated a comprehensive panel of caspase biosensors with a split-luciferase enabled bioluminescent read out. We first interrogated the panel of caspase biosensors for substrate cleavage specificity of caspase 1-10 in widely utilized in vitro translation systems, namely, rabbit reticulocyte lysate (RRL) and wheat germ extract (WGE). Commercial RRL was found to be unsuitable for investigating caspase specificity, owing to surprising levels of endogenous caspase activity, while specificity profiles of the caspase sensors in WGE agree very well with traditional peptide probes. The full panel of biosensors was utilized for studying caspase activation and inhibition in several mammalian cytosolic extracts, clearly demonstrating that they can be utilized to directly monitor activation or inhibition of procaspase 3/7. Furthermore, the complete panel of caspase biosensors also provided new insights into caspase activation pathways wherein we surprisingly discovered the activation of procaspase 3/7 by caspase 4/5.
• Ghosh, I., Shomin, C. D., Restituyo, E., Cox, K. J., & Ghosh, I. -. (2011). Selection of cyclic-peptide inhibitors targeting Aurora kinase A: problems and solutions. Bioorganic & medicinal chemistry, 19(22).
  More info

  The critical role of Aurora kinase in cell cycle progression and its deregulation in cancer has garnered significant interest. As such, numerous Aurora targeted inhibitors have been developed to date, almost all of which target the ATP cleft at the active site. These current inhibitors display polypharmacology; that is, they target multiple kinases, and some are being actively pursued as therapeutics. Currently, there are no general approaches for targeting Aurora at sites remote from the active site, which in the long term may provide new insights regarding the inhibition of Aurora as well as other protein kinases, and provide pharmacological tools for dissecting Aurora kinase biology. Toward this long term goal, we have recently developed a bivalent selection strategy that allows for the identification of cyclic peptides that target the surface of PKA, while the active site is blocked by an ATP-competitive compound. Herein, we extend this approach to Aurora kinase (Aurora A), which required significant optimization of selection conditions to eliminate background peptides that target the streptavidin matrix upon which the kinases are immobilized. Using our optimized selection conditions, we have successfully selected several cyclic peptide ligands against Aurora A. Two of these inhibitors demonstrated IC(50) values of 10 μM and were further interrogated. The CTRPWWLC peptide was shown to display a noncompetitive mode of inhibition suggesting that alternate sites on Aurora beyond the ATP and peptide substrate binding site may be potentially targeted.
• Jester, B. W., Gaj, A., Shomin, C. D., Cox, K. J., & Ghosh, I. (2011). Testing the Promiscuity of Commercial Kinase Inhibitors Against the AGC Kinase Group Using a Split-luciferase Screen. JOURNAL OF MEDICINAL CHEMISTRY, 55(4), 1526-1537.
• Porter, J. R., Lockwood, S. H., Segal, D. J., Ghosh, I., Mackay, J., & Segal, D. (2011). Seeing Genetic and Epigenetic Information Without DNA Denaturation Using Sequence-Enabled Reassembly (SEER). ENGINEERED ZINC FINGER PROTEINS: METHODS AND PROTOCOLS, 649, 365-382.
• Porter, J. R., Stains, C. I., Segal, D. J., & Ghosh, I. (2011). Split beta-lactamase sensor for the sequence-specific detection of DNA methylation. ANALYTICAL CHEMISTRY, 79(17), 6702-6708.
• Shekhawat, S. S., & Ghosh, I. (2011). Split-protein systems: Beyond binary protein-protein interactions. Current Opinion in Chemical Biology, 15(6), 790-797.
  More info

  Abstract: It has been estimated that 650,000 protein-protein interactions exist in the human interactome (Stumpf et al., 2008 [1]), a subset of all possible macromolecular partnerships that dictate life. Thus there is a continued need for the development of sensitive and user-friendly methods for cataloguing biomacromolecules in complex environments and for detecting their interactions, modifications, and cellular location. Such methods also allow for establishing differences in the interactome between a normal and diseased cellular state and for quantifying the outcome of therapeutic intervention. A promising approach for deconvoluting the role of macromolecular partnerships is split-protein reassembly, also called protein fragment complementation. This approach relies on the appropriate fragmentation of protein reporters, such as the green fluorescent protein or firefly luciferase, which when attached to possible interacting partners can reassemble and regain function, thereby confirming the partnership. Split-protein methods have been effectively utilized for detecting protein-protein interactions in cell-free systems, Escherichia coli, yeast, mammalian cells, plants, and live animals. Herein, we present recent advances in engineering split-protein systems that allow for the rapid detection of ternary protein complexes, small molecule inhibitors, as well as a variety of macromolecules including nucleic acids, poly(ADP) ribose, and iron sulfur clusters. We also present advances that combine split-protein systems with chemical inducers of dimerization strategies that allow for regulating the activity of orthogonal split-proteases as well as aid in identifying enzyme inhibitors. Finally, we discuss autoinhibition strategies leading to turn-on sensors as well as future directions in split-protein methodology including possible therapeutic approaches. © 2011 Elsevier Ltd.
• Shekhawat, S. S., & Ghosh, I. (2011). Split-protein systems: beyond binary protein-protein interactions.. Current opinion in chemical biology, 15(6), 789-797.
  More info

  PMID: 22070901;Abstract: It has been estimated that 650,000 protein-protein interactions exist in the human interactome (Stumpf et al., 2008), a subset of all possible macromolecular partnerships that dictate life. Thus there is a continued need for the development of sensitive and user-friendly methods for cataloguing biomacromolecules in complex environments and for detecting their interactions, modifications, and cellular location. Such methods also allow for establishing differences in the interactome between a normal and diseased cellular state and for quantifying the outcome of therapeutic intervention. A promising approach for deconvoluting the role of macromolecular partnerships is split-protein reassembly, also called protein fragment complementation. This approach relies on the appropriate fragmentation of protein reporters, such as the green fluorescent protein or firefly luciferase, which when attached to possible interacting partners can reassemble and regain function, thereby confirming the partnership. Split-protein methods have been effectively utilized for detecting protein-protein interactions in cell-free systems, Escherichia coli, yeast, mammalian cells, plants, and live animals. Herein, we present recent advances in engineering split-protein systems that allow for the rapid detection of ternary protein complexes, small molecule inhibitors, as well as a variety of macromolecules including nucleic acids, poly(ADP) ribose, and iron sulfur clusters. We also present advances that combine split-protein systems with chemical inducers of dimerization strategies that allow for regulating the activity of orthogonal split-proteases as well as aid in identifying enzyme inhibitors. Finally, we discuss autoinhibition strategies leading to turn-on sensors as well as future directions in split-protein methodology including possible therapeutic approaches. Copyright © 2011 Elsevier Ltd. All rights reserved.
• Shekhawat, S. S., Campbell, S. T., & Ghosh, I. (2011). A comprehensive panel of turn-on caspase biosensors for investigating caspase specificity and caspase activation pathways. ChemBioChem, 12(15), 2353-2364.
  More info

  PMID: 21850719;Abstract: Caspases play a central role in apoptosis, differentiation, and proliferation, and represent important therapeutic targets for treating cancer and inflammatory disorders. Toward the goal of developing new tools to probe caspase substrate cleavage specificity as well as to systematically interrogate caspase activation pathways, we have constructed and investigated a comprehensive panel of caspase biosensors with a split-luciferase enabled bioluminescent read out. We first interrogated the panel of caspase biosensors for substrate cleavage specificity of caspase 1-10 in widely utilized in vitro translation systems, namely, rabbit reticulocyte lysate (RRL) and wheat germ extract (WGE). Commercial RRL was found to be unsuitable for investigating caspase specificity, owing to surprising levels of endogenous caspase activity, while specificity profiles of the caspase sensors in WGE agree very well with traditional peptide probes. The full panel of biosensors was utilized for studying caspase activation and inhibition in several mammalian cytosolic extracts, clearly demonstrating that they can be utilized to directly monitor activation or inhibition of procaspase 3/7. Furthermore, the complete panel of caspase biosensors also provided new insights into caspase activation pathways wherein we surprisingly discovered the activation of procaspase 3/7 by caspase 4/5. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
• Shomin, C. D., Restituyo, E., Cox, K. J., & Ghosh, I. (2011). Selection of cyclic-peptide inhibitors targeting Aurora kinase A: Problems and solutions. BIOORGANIC & MEDICINAL CHEMISTRY, 19(22), 6743-6749.
• Shomin, C. D., Restituyo, E., Cox, K. J., & Ghosh, I. (2011). Selection of cyclic-peptide inhibitors targeting Aurora kinase A: Problems and solutions. Bioorganic and Medicinal Chemistry, 19(22), 6743-6749.
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  PMID: 22004849;PMCID: PMC3206593;Abstract: The critical role of Aurora kinase in cell cycle progression and its deregulation in cancer has garnered significant interest. As such, numerous Aurora targeted inhibitors have been developed to date, almost all of which target the ATP cleft at the active site. These current inhibitors display polypharmacology; that is, they target multiple kinases, and some are being actively pursued as therapeutics. Currently, there are no general approaches for targeting Aurora at sites remote from the active site, which in the long term may provide new insights regarding the inhibition of Aurora as well as other protein kinases, and provide pharmacological tools for dissecting Aurora kinase biology. Toward this long term goal, we have recently developed a bivalent selection strategy that allows for the identification of cyclic peptides that target the surface of PKA, while the active site is blocked by an ATP-competitive compound. Herein, we extend this approach to Aurora kinase (Aurora A), which required significant optimization of selection conditions to eliminate background peptides that target the streptavidin matrix upon which the kinases are immobilized. Using our optimized selection conditions, we have successfully selected several cyclic peptide ligands against Aurora A. Two of these inhibitors demonstrated IC 50 values of 10 μM and were further interrogated. The CTRPWWLC peptide was shown to display a noncompetitive mode of inhibition suggesting that alternate sites on Aurora beyond the ATP and peptide substrate binding site may be potentially targeted. © 2011 Elsevier Ltd. All rights reserved.
• Deng, Z., Lie, F. L., Shen, S., Ghosh, I., Mansuripur, M., & Muscat, A. J. (2010). Water-Based Route to Ligand-Selective Synthesis of ZnSe and Cd-Doped ZnSe Quantum Dots with Tunable Ultraviolet A to Blue Photoluminescence. LANGMUIR, 25(1), 434-442.
• Furman, J. L., Badran, A. H., Ajulo, O., Porter, J. R., Stains, C. I., Segal, D. J., & Ghosh, I. (2010). Toward a general approach for RNA-templated hierarchical assembly of split-proteins. Journal of the American Chemical Society, 132(33), 11692-11701.
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  PMID: 20681585;PMCID: PMC2954892;Abstract: The ability to conditionally turn on a signal or induce a function in the presence of a user-defined RNA target has potential applications in medicine and synthetic biology. Although sequence-specific pumilio repeat proteins can target a limited set of ssRNA sequences, there are no general methods for targeting ssRNA with designed proteins. As a first step toward RNA recognition, we utilized the RNA binding domain of argonaute, implicated in RNA interference, for specifically targeting generic 2-nucleotide, 3′ overhangs of any dsRNA. We tested the reassembly of a split-luciferase enzyme guided by argonaute-mediated recognition of newly generated nucleotide overhangs when ssRNA is targeted by a designed complementary guide sequence. This approach was successful when argonaute was utilized in conjunction with a pumilio repeat and expanded the scope of potential ssRNA targets. However, targeting any desired ssRNA remained elusive as two argonaute domains provided minimal reassembled split-luciferase. We next designed and tested a second hierarchical assembly, wherein ssDNA guides are appended to DNA hairpins that serve as a scaffold for high affinity zinc fingers attached to split-luciferase. In the presence of a ssRNA target containing adjacent sequences complementary to the guides, the hairpins are brought into proximity, allowing for zinc finger binding and concomitant reassembly of the fragmented luciferase. The scope of this new approach was validated by specifically targeting RNA encoding VEGF, hDM2, and HER2. These approaches provide potentially general design paradigms for the conditional reassembly of fragmented proteins in the presence of any desired ssRNA target. © 2010 American Chemical Society.
• Furman, J. L., Mok, P., Badran, A. H., & Ghosh, I. (2010). Turn-On DNA Damage Sensors for the Direct Detection of 8-Oxoguanine and Photoproducts in Native DNA. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 133(32), 12518-12527.
• Ghosh, I., Furman, J. L., Badran, A. H., Ajulo, O., Porter, J. R., Stains, C. I., Segal, D. J., & Ghosh, I. -. (2010). Toward a general approach for RNA-templated hierarchical assembly of split-proteins. Journal of the American Chemical Society, 132(33).
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  The ability to conditionally turn on a signal or induce a function in the presence of a user-defined RNA target has potential applications in medicine and synthetic biology. Although sequence-specific pumilio repeat proteins can target a limited set of ssRNA sequences, there are no general methods for targeting ssRNA with designed proteins. As a first step toward RNA recognition, we utilized the RNA binding domain of argonaute, implicated in RNA interference, for specifically targeting generic 2-nucleotide, 3' overhangs of any dsRNA. We tested the reassembly of a split-luciferase enzyme guided by argonaute-mediated recognition of newly generated nucleotide overhangs when ssRNA is targeted by a designed complementary guide sequence. This approach was successful when argonaute was utilized in conjunction with a pumilio repeat and expanded the scope of potential ssRNA targets. However, targeting any desired ssRNA remained elusive as two argonaute domains provided minimal reassembled split-luciferase. We next designed and tested a second hierarchical assembly, wherein ssDNA guides are appended to DNA hairpins that serve as a scaffold for high affinity zinc fingers attached to split-luciferase. In the presence of a ssRNA target containing adjacent sequences complementary to the guides, the hairpins are brought into proximity, allowing for zinc finger binding and concomitant reassembly of the fragmented luciferase. The scope of this new approach was validated by specifically targeting RNA encoding VEGF, hDM2, and HER2. These approaches provide potentially general design paradigms for the conditional reassembly of fragmented proteins in the presence of any desired ssRNA target.
• Ghosh, I., Jester, B. W., Cox, K. J., Gaj, A., Shomin, C. D., Porter, J. R., & Ghosh, I. -. (2010). A coiled-coil enabled split-luciferase three-hybrid system: applied toward profiling inhibitors of protein kinases. Journal of the American Chemical Society, 132(33).
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  The 518 protein kinases encoded in the human genome are exquisitely regulated and their aberrant function(s) are often associated with human disease. Thus, in order to advance therapeutics and to probe signal transduction cascades, there is considerable interest in the development of inhibitors that can selectively target protein kinases. However, identifying specific compounds against such a large array of protein kinases is difficult to routinely achieve utilizing traditional activity assays, where purified protein kinases are necessary. Toward a simple, rapid, and practical method for identifying specific inhibitors, we describe the development and application of a split-protein methodology utilizing a coiled-coil-assisted three-hybrid system. In this approach, a protein kinase of interest is attached to the C-terminal fragment of split-firefly luciferase and the coiled-coil Fos, which is specific for the coiled-coil Jun, is attached to the N-terminal fragment. Upon addition of Jun conjugated to a pan-kinase inhibitor such as staurosporine, a three-hybrid complex is established with concomitant reassembly of the split-luciferase enzyme. An inhibitor can be potentially identified by the commensurate loss in split-luciferase activity by displacement of the modified staurosporine. We demonstrate that this new three-hybrid approach is potentially general by testing protein kinases from the different kinase families. To interrogate whether this method allows for screening inhibitors, we tested six different protein kinases against a library of 80 known protein kinase inhibitors. Finally, we demonstrate that this three-hybrid system can potentially provide a rapid method for structure/function analysis as well as aid in the identification of allosteric inhibitors.
• Ghosh, I., Porter, J. R., Helmers, M. R., Wang, P., Furman, J. L., Joy, S. T., Arora, P. S., & Ghosh, I. -. (2010). Profiling small molecule inhibitors against helix-receptor interactions: the Bcl-2 family inhibitor BH3I-1 potently inhibits p53/hDM2. Chemical communications (Cambridge, England), 46(42).
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  We validate a practical methodology for the rapid profiling of small molecule inhibitors of protein-protein interactions. We find that a well known BH3 family inhibitor can potently inhibit the p53/hDM2 interaction.
• Ghosh, I., Porter, J. R., Lockwood, S. H., Segal, D. J., & Ghosh, I. -. (2010). Seeing genetic and epigenetic information without DNA denaturation using sequence-enabled reassembly (SEER). Methods in molecular biology (Clifton, N.J.), 649.
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  Virtually all methods for reading the sequence of bases in DNA rely on the ability to denature double-stranded DNA into single strands and then use Watson-Crick base-pairing rules to hybridize the strands with high specificity to another DNA primer or probe. However, nature frequently uses an alternative method, reading the sequence information directly from double-stranded DNA using sequence-specific DNA-binding proteins. Here we describe methods for the construction and testing of sequence probes based on engineered zinc finger DNA-binding proteins. Background is reduced using split-reporter molecules, and signal is amplified using enzymatic reporters. The resulting sequence-enabled reassembly (SEER) probes can be configured to detect DNA sequence (genetic) or DNA methylation (epigenetic) information.
• Ghosh, I., Stains, C. I., Furman, J. L., Porter, J. R., Rajagopal, S., Li, Y., Wyatt, R. T., & Ghosh, I. -. (2010). A general approach for receptor and antibody-targeted detection of native proteins utilizing split-luciferase reassembly. ACS chemical biology, 5(10).
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  The direct detection of native proteins in heterogeneous solutions remains a challenging problem. Standard methodologies rely on a separation step to circumvent nonspecific signal generation. We hypothesized that a simple and general method for the detection of native proteins in solution could be achieved through ternary complexation, where the conditional signal generation afforded by split-protein reporters could be married to the specificity afforded by either native receptors or specific antibodies. Toward this goal, we describe a solution phase split-luciferase assay for native protein detection, where we fused fragmented halves of firefly luciferase to separate receptor fragments or single-chain antibodies, allowing for conditional luciferase complementation in the presence of several biologically significant protein targets. To demonstrate the utility of this strategy, we have developed and validated assay platforms for the vascular endothelial growth factor, the gp120 coat protein from HIV-1, and the human epidermal growth factor receptor 2 (HER2), a marker for breast cancer. The specificities of the recognition elements, CD4 and the 17b single-chain antibody, employed in the gp120 sensor allowed us to parse gp120s from different clades. Our rationally designed HER2 sensing platform was capable of discriminating between HER2 expression levels in several tumor cell lines. In addition, luminescence from reassembled luciferase was linear across a panel of cell lines with increasing HER2 expression. We envision that the proof of principle studies presented herein may allow for the potential detection of a broad range of biological analytes utilizing ternary split-protein systems.
• Henchey, L. K., Porter, J. R., Ghosh, I., & Arora, P. S. (2010). High Specificity in Protein Recognition by Hydrogen-Bond-Surrogate alpha-Helices: Selective Inhibition of the p53/MDM2 Complex. CHEMBIOCHEM, 11(15), 2104-2107.
• Henchey, L. K., Porter, J. R., Ghosh, I., & Arora, P. S. (2010). High specificity in protein recognition by hydrogen-bond-surrogate α-helices: Selective inhibition of the p53/MDM2 complex. ChemBioChem, 11(15), 2104-2107.
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  PMID: 20821791;PMCID: PMC3017812;
• Jester, B. W., Cox, K. J., Gaj, A., Shomin, C. D., Porter, J. R., & Ghosh, I. (2010). A Coiled-Coil Enabled Split-Luciferase Three-Hybrid System: Applied Toward Profiling Inhibitors of Protein Kinases. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 132(33), 11727-11735.
• Jester, B. W., Cox, K. J., Gaj, A., Shomin, C. D., Porter, J. R., & Ghosh, I. (2010). A coiled-coil enabled split-luciferase three-hybrid system: Applied toward profiling inhibitors of protein kinases. Journal of the American Chemical Society, 132(33), 11727-11735.
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  PMID: 20669947;PMCID: PMC2966823;Abstract: The 518 protein kinases encoded in the human genome are exquisitely regulated and their aberrant function(s) are often associated with human disease. Thus, in order to advance therapeutics and to probe signal transduction cascades, there is considerable interest in the development of inhibitors that can selectively target protein kinases. However, identifying specific compounds against such a large array of protein kinases is difficult to routinely achieve utilizing traditional activity assays, where purified protein kinases are necessary. Toward a simple, rapid, and practical method for identifying specific inhibitors, we describe the development and application of a split-protein methodology utilizing a coiled-coil-assisted three-hybrid system. In this approach, a protein kinase of interest is attached to the C-terminal fragment of split-firefly luciferase and the coiled-coil Fos, which is specific for the coiled-coil Jun, is attached to the N-terminal fragment. Upon addition of Jun conjugated to a pan-kinase inhibitor such as staurosporine, a three-hybrid complex is established with concomitant reassembly of the split-luciferase enzyme. An inhibitor can be potentially identified by the commensurate loss in split-luciferase activity by displacement of the modified staurosporine. We demonstrate that this new three-hybrid approach is potentially general by testing protein kinases from the different kinase families. To interrogate whether this method allows for screening inhibitors, we tested six different protein kinases against a library of 80 known protein kinase inhibitors. Finally, we demonstrate that this three-hybrid system can potentially provide a rapid method for structure/function analysis as well as aid in the identification of allosteric inhibitors. © 2010 American Chemical Society.
• Livnat, I., Finkelshtein, D., Ghosh, I., Arai, H., & Reiner, O. (2010). PAF-AH catalytic subunits modulate the wnt pathway in developing gabaergic neurons. Frontiers in Cellular Neuroscience, 4(MAY).
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  Abstract: Platelet-activating factor acetylhydrolase 1B (PAF-AH) inactivates the potent phospholipid platelet-activating factor (PAF) and is composed of two catalytic subunits (α1 and α2) and a dimeric regulatory subunit, LIS1. The function of the catalytic subunits in brain development remains unknown. Here we examined their effects on proliferation in the ganglionic eminences and tangential migration. In α1 and α2 catalytic subunits knockout mice we noticed an increase in the size of the ganglionic eminences resulting from increased proliferation of GABAergic neurons. Our results indicate that the catalytic subunits act as negative regulators of the Wnt signaling pathway. Overexpression of each of the PAF-AH catalytic subunits reduced the amount of nuclear beta-catenin and provoked a shift of this protein from the nucleus to the cytoplasm. In the double mutant mice, Wnt signaling increased in the ganglionic eminences and in the dorsal part of the cerebral cortex. In situ hybridization revealed increased and expanded expression of a downstream target of the Wnt pathway (Cyclin D1), and of upstream Wnt components (Tcf4, Tcf3 and Wnt7B). Furthermore, the interneurons in the cerebral cortex were more numerous and in a more advanced position. Transplantation assays revealed a non-cell autonomous component to this phenotype, which may be explained in part by increased and expanded expression of Sdf1 and Netrin-1. Our findings strongly suggest that PAF-AH catalytic subunits modulate the Wnt pathway in restricted areas of the developing cerebral cortex. We hypothesize that modulation of the Wnt pathway is the evolutionary conserved activity of the PAF-AH catalytic subunits. © 2010 Livnat, Finkelshtein, Ghosh, Arai and Reiner.
• Meyer, S. C., & Ghosh, I. (2010). Phage display technology in biosensor development. Recognition Receptors in Biosensors, 723-749.
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  Abstract: The utility of phage display technology in biosensor development lies in the ability to rapidly discover novel peptide and protein modules that serve as molecular detection domains in sensor architectures. Phage display allows for connecting a protein or peptide (phenotype) to the DNA (genotype) that encodes it, allowing users to screen billion member libraries of compounds against targets of interest, while using the genetic information of the virus for both output identification and amplification. This process known as in vitro selection mimics natural selection through the use of selective pressure followed by the amplification of the selected mutant in a bacterial host cell. Because of the robust nature of phage, the output virions from a selection can sometimes be used directly in a biosensor. More commonly, the genetic information contained within the phage can be translated to a peptide, protein, or antibody that serves as the recognition domain in a biosensor. Several modalities of phage display have been utilized for discovering receptors for biosensors, including peptide display (low and high valency), antibody display, and small protein display. These phage display approaches have greatly aided in the development of novel biosensor receptors. © Springer Science+Business Media, LLC 2010.
• Porter, J. R., Helmers, M. R., Wang, P., Furman, J. L., Joy, S. T., Arora, P. S., & Ghosh, I. (2010). Profiling small molecule inhibitors against helix-receptor interactions: The Bcl-2 family inhibitor BH3I-1 potently inhibits p53/hDM2. Chemical Communications, 46(42), 8020-8022.
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  PMID: 20856941;Abstract: We validate a practical methodology for the rapid profiling of small molecule inhibitors of protein-protein interactions. We find that a well known BH3 family inhibitor can potently inhibit the p53/hDM2 interaction. © 2010 The Royal Society of Chemistry.
• Porter, J. R., Lockwood, S. H., Segal, D. J., & Ghosh, I. (2010). Seeing genetic and epigenetic information without DNA denaturation using sequence-enabled reassembly (SEER). Methods in Molecular Biology, 649, 365-382.
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  PMID: 20680847;Abstract: Virtually all methods for reading the sequence of bases in DNA rely on the ability to denature double-stranded DNA into single strands and then use Watson-Crick base-pairing rules to hybridize the strands with high specificity to another DNA primer or probe. However, nature frequently uses an alternative method, reading the sequence information directly from double-stranded DNA using sequence-specific DNA-binding proteins. Here we describe methods for the construction and testing of sequence probes based on engineered zinc finger DNA-binding proteins. Background is reduced using split-reporter molecules, and signal is amplified using enzymatic reporters. The resulting sequence-enabled reassembly (SEER) probes can be configured to detect DNA sequence (genetic) or DNA methylation (epigenetic) information. © 2010 Springer Science+Business Media, LLC.
• Rajagopal, S., Meyer, S. C., Goldman, A., Zhou, M., & Ghosh, I. (2010). A minimalist approach toward protein recognition by epitope transfer from functionally evolved beta-sheet surfaces. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 128(44), 14356-14363.
• Shomin, C. D., Meyer, S. C., & Ghosh, I. (2010). Staurosporine tethered peptide ligands that target cAMP-dependent protein kinase (PKA): Optimization and selectivity profiling. BIOORGANIC & MEDICINAL CHEMISTRY, 17(17), 6196-6202.
• Smith, T. J., Stains, C. I., Meyer, S. C., & Ghosh, I. (2010). Inhibition of beta-amyloid fibrillization by directed evolution of a beta-sheet presenting miniature protein. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 128(45), 14456-14457.
• Stains, C. I., & Ghosh, I. (2010). When conjugated polymers meet amyloid fibrils. ACS CHEMICAL BIOLOGY, 2(8), 525-528.
• Stains, C. I., Furman, J. L., Porter, J. R., Rajagopal, S., Yuxing, L. i., Wyatt, R. T., & Ghosh, I. (2010). A general approach for receptor and antibody-targeted detection of native proteins utilizing split-luciferase reassembly. ACS Chemical Biology, 5(10), 943-952.
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  PMID: 20681584;PMCID: PMC2955838;Abstract: The direct detection of native proteins in heterogeneous solutions remains a challenging problem. Standard methodologies rely on a separation step to circumvent nonspecific signal generation. We hypothesized that a simple and general method for the detection of native proteins in solution could be achieved through ternary complexation, where the conditional signal generation afforded by split-protein reporters could be married to the specificity afforded by either native receptors or specific antibodies. Toward this goal, we describe a solution phase split-luciferase assay for native protein detection, where we fused fragmented halves of firefly luciferase to separate receptor fragments or single-chain antibodies, allowing for conditional luciferase complementation in the presence of several biologically significant protein targets. To demonstrate the utility of this strategy, we have developed and validated assay platforms for the vascular endothelial growth factor, the gp120 coat protein from HIV-1, and the human epidermal growth factor receptor 2 (HER2), a marker for breast cancer. The specificities of the recognition elements, CD4 and the 17b single-chain antibody, employed in the gp120 sensor allowed us to parse gp120s from different clades. Our rationally designed HER2 sensing platform was capable of discriminating between HER2 expression levels in several tumor cell lines. In addition, luminescence from reassembled luciferase was linear across a panel of cell lines with increasing HER2 expression. We envision that the proof of principle studies presented herein may allow for the potential detection of a broad range of biological analytes utilizing ternary split-protein systems. © 2010 American Chemical Society.
• Yinni, Y. u., Blair, S., Gillespie, D., Jensen, R., Myszka, D., Badran, A. H., Ghosh, I., & Chagovetz, A. (2010). Direct DNA methylation profiling using methyl binding domain proteins. Analytical Chemistry, 82(12), 5012-5019.
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  PMID: 20507169;PMCID: PMC2946844;Abstract: Methylation of DNA is responsible for gene silencing by establishing heterochromatin structure that represses transcription, and studies have shown that cytosine methylation of CpG islands in promoter regions acts as a precursor to early cancer development. The naturally occurring methyl binding domain (MBD) proteins from mammals are known to bind to the methylated CpG dinucleotide (mCpG) and subsequently recruit other chromatin-modifying proteins to suppress transcription. Conventional methods of detection for methylated DNA involve bisulfite treatment or immunoprecipitation prior to performing an assay. We focus on proof-of-concept studies for a direct microarray-based assay using surface-bound methylated probes. The recombinant protein 1xMBD-GFP recognizes hemimethylation and symmetric methylation of the CpG sequence of hybridized dsDNA, while displaying greater affinity for the symmetric methylation motif, as evaluated by SPR. From these studies, for symmetric mCpG, the KD for 1xMBD-GFP ranged from 106 to 870 nM, depending upon the proximity of the methylation site to the sensor surface. The KD values for nonsymmetrical methylation motifs were consistently greater (>2 μM), but the binding selectivity between symmetric and hemimethylation motifs ranged from 4 to 30, with reduced selectivity for sites close to the surface or multiple sites in proximity, which we attribute to steric effects. Fitting skew normal probability density functions to our data, we estimate an accuracy of 97.5% for our method in identifying methylated CpG loci, which can be improved through optimization of probe design and surface density. © 2010 American Chemical Society.
• Badran, A. H., Furman, J. L., Ma, A. S., Comi, T. J., Porter, J. R., & Ghosh, I. (2009). Evaluating the Global CpG Methylation Status of Native DNA Utilizing a Bipartite Split-Luciferase Sensor. ANALYTICAL CHEMISTRY, 83(18), 7151-7157.
• Furman, J. L., Badran, A. H., Shen, S., Stains, C. I., Hannallah, J., Segal, D. J., & Ghosh, I. (2009). Systematic evaluation of split-fluorescent proteins for the direct detection of native and methylated DNA. BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 19(14), 3748-3751.
• Furman, J. L., Badran, A. H., Shen, S., Stains, C. I., Hannallah, J., Segal, D. J., & Ghosh, I. (2009). Systematic evaluation of split-fluorescent proteins for the direct detection of native and methylated DNA. Bioorganic and Medicinal Chemistry Letters, 19(14), 3748-3751.
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  PMID: 19457665;PMCID: PMC2709697;Abstract: In order to directly detect nucleic acid polymers, we have designed biosensors comprising sequence-specific DNA binding proteins tethered to split-reporter proteins, which generate signal upon binding a predetermined nucleic acid target, in an approach termed SEquence-Enabled Reassembly (SEER). Herein we demonstrate that spectroscopically distinct split-fluorescent protein variants, GFPuv, EGFP, Venus, and mCherry, function effectively in the SEER system, providing sensitive DNA detection and the ability to simultaneously detect two target oligonucleotides. Additionally, a methylation-specific SEER-Venus system was generated, which was found to clearly distinguish between methylated versus non-methylated target DNA. These results will aid in refinement of the SEER system for the detection of user defined nucleic acid sequences and their chemical modifications as they relate to human disease. © 2009 Elsevier Ltd. All rights reserved.
• Ghosh, I., Furman, J. L., Badran, A. H., Shen, S., Stains, C. I., Hannallah, J., Segal, D. J., & Ghosh, I. -. (2009). Systematic evaluation of split-fluorescent proteins for the direct detection of native and methylated DNA. Bioorganic & medicinal chemistry letters, 19(14).
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  In order to directly detect nucleic acid polymers, we have designed biosensors comprising sequence-specific DNA binding proteins tethered to split-reporter proteins, which generate signal upon binding a predetermined nucleic acid target, in an approach termed SEquence-Enabled Reassembly (SEER). Herein we demonstrate that spectroscopically distinct split-fluorescent protein variants, GFPuv, EGFP, Venus, and mCherry, function effectively in the SEER system, providing sensitive DNA detection and the ability to simultaneously detect two target oligonucleotides. Additionally, a methylation-specific SEER-Venus system was generated, which was found to clearly distinguish between methylated versus non-methylated target DNA. These results will aid in refinement of the SEER system for the detection of user defined nucleic acid sequences and their chemical modifications as they relate to human disease.
• Ghosh, I., Shekhawat, S. S., Porter, J. R., Sriprasad, A., & Ghosh, I. -. (2009). An autoinhibited coiled-coil design strategy for split-protein protease sensors. Journal of the American Chemical Society, 131(42).
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  Proteases are widely studied as they are integral players in cell-cycle control and apoptosis. We report a new approach for the design of a family of genetically encoded turn-on protease biosensors. In our design, an autoinhibited coiled-coil switch is turned on upon proteolytic cleavage, which results in the complementation of split-protein reporters. Utilizing this new autoinhibition design paradigm, we present the rational construction and optimization of three generations of protease biosensors, with the final design providing a 1000-fold increase in bioluminescent signal upon addition of the TEV protease. We demonstrate the generality of the approach utilizing two different split-protein reporters, firefly luciferase and beta-lactamase, while also testing our design in the context of a therapeutically relevant protease, caspase-3. Finally, we present a dual protease sensor geometry that allows for the use of these turn-on sensors as potential AND logic gates. Thus, these studies potentially provide a new method for the design and implementation of genetically encoded turn-on protease sensors while also providing a general autoinhibited coiled-coil strategy for controlling the activity of fragmented proteins.
• Ghosh, I., Shomin, C. D., Meyer, S. C., & Ghosh, I. -. (2009). Staurosporine tethered peptide ligands that target cAMP-dependent protein kinase (PKA): optimization and selectivity profiling. Bioorganic & medicinal chemistry, 17(17).
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  We have recently developed a fragment based selection strategy for targeting kinases, where a small molecule warhead can be non-covalently tethered to a phage-displayed library of peptides. This approach was applied to the conversion of the promiscuous kinase inhibitor, staurosporine, into a potent bivalent ligand for cAMP-dependent protein kinase (PKA). Herein we report a systematic evaluation of this new bivalent ligand (BL); (a) Lineweaver-Burke analysis revealed that the BL, unlike substrate-based bivalent kinase inhibitors, displayed non-competitive inhibition with respect to the peptide substrate, suggesting an allosteric mechanism of action; (b) linker optimization of the BL, afforded one of the most potent, sub-nanomolar, inhibitors of PKA reported to date; (c) the BL was found to be modular, where attachment of active site targeted small molecule warheads in lieu of staurosporine could achieve similar gains in affinity; and (d) profiling studies of both the staurosporine derivative and the BL (amide isostere) against a panel of 90 kinases revealed almost unique enhancement in selectivity against PKA (>5-fold) compared to the starting staurosporine derivative. These combined results provide new insights for BL discovery, which has the potential to provide guidance toward the development of kinase selective reagents while uncovering new allosteric sites on kinases for therapeutic targeting.
• Meyer, S. C., Ghosh, I., & Zourob, M. (2009). Phage Display Technology in Biosensor Development. RECOGNITION RECEPTORS IN BIOSENSORS, 723-749.
• Shekhawat, S. S., Porter, J. R., Sriprasad, A., & Ghosh, I. (2009). An autoinhibited coiled-coil design strategy for split-protein protease sensors. Journal of the American Chemical Society, 131(42), 15284-15290.
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  PMID: 19803505;PMCID: PMC2783329;Abstract: Proteases are widely studied as they are integral players in cell-cycle control and apoptosis. We report a new approach for the design of a family of genetically encoded turn-on protease biosensors. In our design, an autoinhibited coiled-coil switch is turned on upon proteolytic cleavage, which results in the complementation of split-protein reporters. Utilizing this new autoinhibition design paradigm, we present the rational construction and optimization of three generations of protease biosensors, with the final design providing a 1000-fold increase in bioluminescent signal upon addition of the TEV protease. We demonstrate the generality of the approach utilizing two different split-protein reporters, firefly luciferase and β-lactamase, while also testing our design in the context of a therapeutically relevant protease, caspase-3. Finally, we present a dual protease sensor geometry that allows for the use of these turn-on sensors as potential AND logic gates. Thus, these studies potentially provide a new method for the design and implementation of genetically encoded turn-on protease sensors while also providing a general autoinhibited coiled-coil strategy for controlling the activity of fragmented proteins. © 2009 American Chemical Society.
• Shomin, C. D., Meyer, S. C., & Ghosh, I. (2009). Staurosporine tethered peptide ligands that target cAMP-dependent protein kinase (PKA): Optimization and selectivity profiling. Bioorganic and Medicinal Chemistry, 17(17), 6196-6202.
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  PMID: 19674907;PMCID: PMC2749490;Abstract: We have recently developed a fragment based selection strategy for targeting kinases, where a small molecule warhead can be non-covalently tethered to a phage-displayed library of peptides. This approach was applied to the conversion of the promiscuous kinase inhibitor, staurosporine, into a potent bivalent ligand for cAMP-dependent protein kinase (PKA). Herein we report a systematic evaluation of this new bivalent ligand (BL); (a) Lineweaver-Burke analysis revealed that the BL, unlike substrate-based bivalent kinase inhibitors, displayed non-competitive inhibition with respect to the peptide substrate, suggesting an allosteric mechanism of action; (b) linker optimization of the BL, afforded one of the most potent, sub-nanomolar, inhibitors of PKA reported to date; (c) the BL was found to be modular, where attachment of active site targeted small molecule warheads in lieu of staurosporine could achieve similar gains in affinity; and (d) profiling studies of both the staurosporine derivative and the BL (amide isostere) against a panel of 90 kinases revealed almost unique enhancement in selectivity against PKA (>5-fold) compared to the starting staurosporine derivative. These combined results provide new insights for BL discovery, which has the potential to provide guidance toward the development of kinase selective reagents while uncovering new allosteric sites on kinases for therapeutic targeting. © 2009 Elsevier Ltd. All rights reserved.
• Ghosh, I., Porter, J. R., Stains, C. I., Jester, B. W., & Ghosh, I. -. (2008). A general and rapid cell-free approach for the interrogation of protein-protein, protein-DNA, and protein-RNA interactions and their antagonists utilizing split-protein reporters. Journal of the American Chemical Society, 130(20).
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  Split-protein reporters have emerged as a powerful methodology for imaging biomolecular interactions which are of much interest as targets for chemical intervention. Herein we describe a systematic evaluation of split-proteins, specifically the green fluorescent protein, beta-lactamase, and several luciferases, for their ability to function as reporters in completely cell-free systems to allow for the extremely rapid and sensitive determination of a wide range of biomolecular interactions without the requirement for laborious transfection, cell culture, or protein purification (12-48 h). We demonstrate that the cell-free split-luciferase system in particular is amenable for directly interrogating protein-protein, protein-DNA, and protein-RNA interactions in homogeneous assays with very high sensitivity (22-1800 fold) starting from the corresponding mRNA or DNA. Importantly, we show that the cell-free system allows for the rapid (2 h) identification of target-site specificity for protein-nucleic acid interactions and in evaluating antagonists of protein-protein and protein-peptide complexes circumventing protein purification bottlenecks. Moreover, we show that the cell-free split-protein system is adaptable for analysis of both protein-protein and protein-nucleic acid interactions in artificial cell systems comprising water-in-oil emulsions. Thus, this study provides a general and enabling methodology for the rapid interrogation of a wide variety of biomolecular interactions and their antagonists without the limitations imposed by current in vitro and in vivo approaches.
• Meyer, S. C., Gaj, T., & Ghosh, I. (2008). Highly selective cyclic peptide ligands for NeutrAvidin and avidin identified by phage display. CHEMICAL BIOLOGY & DRUG DESIGN, 68(1), 3-10.
• Porter, J. R., Stains, C. I., Jester, B. W., & Ghosh, I. (2008). A general and rapid cell-free approach for the interrogation of protein-protein, protein-DNA, and protein-RNA interactions and their antagonists utilizing split-protein reporters. Journal of the American Chemical Society, 130(20), 6488-6497.
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  PMID: 18444624;Abstract: Split-protein reporters have emerged as a powerful methodology for imaging biomolecular interactions which are of much interest as targets for chemical intervention. Herein we describe a systematic evaluation of split-proteins, specifically the green fluorescent protein, β-lactamase, and several luciferases, for their ability to function as reporters in completely cell-free systems to allow for the extremely rapid and sensitive determination of a wide range of biomolecular interactions without the requirement for laborious transfection, cell culture, or protein purification (12-48 h). We demonstrate that the cell-free split-luciferase system in particular is amenable for directly interrogating protein-protein, protein-DNA, and protein-RNA interactions in homogeneous assays with very high sensitivity (22-1800 fold) starting from the corresponding mRNA or DNA. Importantly, we show that the cell-free system allows for the rapid (2 h) identification of target-site specificity for protein-nucleic acid interactions and in evaluating antagonists of protein-protein and protein-peptide complexes circumventing protein purification bottlenecks. Moreover, we show that the cell-free split-protein system is adaptable for analysis of both protein-protein and protein-nucleic acid interactions in artificial cell systems comprising water-in-oil emulsions. Thus, this study provides a general and enabling methodology for the rapid interrogation of a wide variety of biomolecular interactions and their antagonists without the limitations imposed by current in vitro and in vivo approaches. © 2008 American Chemical Society.
• Restituyo, E., Camacho-Soto, K., Ghosh, I., & Derda, R. (2008). A Fragment-Based Selection Approach for the Discovery of Peptide Macrocycles Targeting Protein Kinases. PEPTIDE LIBRARIES: METHODS AND PROTOCOLS, 1248, 95-104.
• Camacho-Soto, K., Castillo-Montoya, J., Tye, B., & Ghosh, I. (2007). Ligand-Gated Split-Kinases. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 136(10), 3995-4002.
• Gaj, T., Meyer, S. C., & Ghosh, I. (2007). The AviD-tag, a NeutrAvidin/avidin specific peptide affinity tag for the immobilization and purification of recombinant proteins. Protein Expression and Purification, 56(1), 54-61.
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  PMID: 17697784;Abstract: The widespread success of affinity tags throughout the biological sciences has prompted interest in developing new and convenient labeling strategies. Affinity tags are well-established tools for recombinant protein immobilization and purification. More recently these tags have been utilized for selective biological targeting towards multiplexed protein detection in numerous imaging applications as well as for drug-delivery. Recently, we discovered a phage-display selected cyclic peptide motif that was shown to bind selectively to NeutrAvidin and avidin but not to the structurally similar streptavidin. Here, we have exploited this selectivity to develop an affinity tag based on the evolved DRATPY moiety that is orthogonal to known Strep-tag technologies. As proof of principle, the divalent AviD-tag (Avidin-Di-tag) was expressed as a Green Fluorescent Protein variant conjugate and exhibited superior immobilization and elution characteristics to the first generation Strep-tag and a monovalent DRATPY GFP-fusion protein analogue. Additionally, we demonstrate the potential for a peptide based orthogonal labeling strategy involving our divalent AviD-tag in concert with existing streptavidin-based affinity reagents. We believe the AviD-tag and its unique recognition properties will provide researchers with a useful new affinity reagent and tool for a variety of applications in the biological and chemical sciences. © 2007 Elsevier Inc. All rights reserved.
• Ghosh, I., & Wirth, M. J. (2007). Parsing the motion of single molecules: a novel algorithm for deconvoluting the dynamics of individual receptors at the cell surface.. Science's STKE : signal transduction knowledge environment, 2007(388), pe28.
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  PMID: 17536098;Abstract: To truly understand signal transduction, we will ultimately need to understand the dynamics and kinetics of individual proteins as they perform their functions in a single cell. Groundbreaking advances in single-molecule biophysics now allow us to follow the motion of many individual proteins on the cell surface with the use of fluorescent probes, such as quantum dots. However, discriminating the directed movement of single molecules from their natural Brownian motion remains a challenge. A recent paper provides a powerful statistical approach for distinguishing periods of directed motion of individual gamma-aminobutyric acid (GABA) receptors from periods during which they undergo Brownian motion. This new methodology should help single-molecule researchers determine the dynamics of individual proteins participating in signaling cascades.
• Ghosh, I., Gaj, T., Meyer, S. C., & Ghosh, I. -. (2007). The AviD-tag, a NeutrAvidin/avidin specific peptide affinity tag for the immobilization and purification of recombinant proteins. Protein expression and purification, 56(1).
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  The widespread success of affinity tags throughout the biological sciences has prompted interest in developing new and convenient labeling strategies. Affinity tags are well-established tools for recombinant protein immobilization and purification. More recently these tags have been utilized for selective biological targeting towards multiplexed protein detection in numerous imaging applications as well as for drug-delivery. Recently, we discovered a phage-display selected cyclic peptide motif that was shown to bind selectively to NeutrAvidin and avidin but not to the structurally similar streptavidin. Here, we have exploited this selectivity to develop an affinity tag based on the evolved DRATPY moiety that is orthogonal to known Strep-tag technologies. As proof of principle, the divalent AviD-tag (Avidin-Di-tag) was expressed as a Green Fluorescent Protein variant conjugate and exhibited superior immobilization and elution characteristics to the first generation Strep-tag and a monovalent DRATPY GFP-fusion protein analogue. Additionally, we demonstrate the potential for a peptide based orthogonal labeling strategy involving our divalent AviD-tag in concert with existing streptavidin-based affinity reagents. We believe the AviD-tag and its unique recognition properties will provide researchers with a useful new affinity reagent and tool for a variety of applications in the biological and chemical sciences.
• Ghosh, I., Ghosh, I. -., Zhou, M., Nakatani, E., Gronenberg, L. S., Tokimoto, T., Wirth, M. J., Hruby, V. J., Roberts, A., & Lynch, R. M. (2007). Peptide-labeled quantum dots for imaging GPCRs in whole cells and as single molecules. Bioconjugate chemistry, 18(2).
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  We report a robust and practical method for the preparation of water-soluble luminescent quantum dots (QDs) selectively coupled through an amine or thiol linkage to peptide ligands targeted to G-protein coupling receptors (GPCRs) and demonstrate their utility in whole-cell and single-molecule imaging. We utilized a low molecular weight ( approximately 1200 Da) diblock copolymer with acrylic acids as hydrophilic segments and amido-octyl side chains as hydrophobic segments for facile encapsulation of QDs (QD 595 and QD 514) in aqueous solutions. As proof of principle, these QDs were targeted to the human melanocortin receptor (hMCR) by chemoselectively coupling the polymer-coated QDs to either a hexapeptide analog of alpha-melanocyte stimulating hormone or to the highly potent MT-II ligand containing a unique amine. To label QDs with ligands lacking orthogonal amines, the diblock copolymers were readily modified with water-soluble trioxa-tridecanediamine to incorporate freely available amine functionalities. The amine-functionalized QDs underwent facile reaction with the bifunctional linker NHS-maleimide, allowing for covalent coupling to GPCR-targeted ligands modified with unique cysteines. We demonstrate the utility of these maleimide-functionalized QDs by covalent conjugation to a highly potent Deltorphin-II analog that allowed for selective cell-surface and single-molecule imaging of the human delta-opioid receptor (hDOR).
• Ghosh, I., Meyer, S. C., Shomin, C. D., Gaj, T., & Ghosh, I. -. (2007). Tethering small molecules to a phage display library: discovery of a selective bivalent inhibitor of protein kinase A. Journal of the American Chemical Society, 129(45).
• Ghosh, I., Porter, J. R., Stains, C. I., Segal, D. J., & Ghosh, I. -. (2007). Split beta-lactamase sensor for the sequence-specific detection of DNA methylation. Analytical chemistry, 79(17).
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  The methylation pattern of genes at CpG dinucleotide sites is an emerging area in epigenetics. Furthermore, the hypermethylation profiles of tumor suppressor genes are linked to specific tumor types. Thus, new molecular approaches for the rapid determination of the methylation status of these genes could provide a powerful method for early cancer diagnosis as well as insight into mechanisms of epigenetic regulation of genetic information. Toward this end, we have recently reported the first design of a split-protein sensor for the site-specific detection of DNA methylation. In this approach a split green fluorescent protein reporter provided a sequence-specific readout of CpG methylation. In the present work, we describe a sensitive second-generation methylation detection system that utilizes the split enzymatic reporter, TEM-1 beta-lactamase, attached to specific DNA binding elements. This system, termed mCpG-SEER-beta-Lac, shows a greater than 40-fold specificity for methylated versus nonmethylated CpG target sites. Importantly, the resulting signal enhancement afforded by the catalytic activity of split-beta-lactamase allowed for the sensitive detection of 2.5 fmol of methylated target dsDNA in 5 min. Thus, this new sensor geometry represents a 250-fold enhancement in assay time and a 2000-fold enhancement in sensitivity over our first-generation system for the detection of specific sites of DNA methylation.
• Ghosh, I., Stains, C. I., & Ghosh, I. -. (2007). When conjugated polymers meet amyloid fibrils. ACS chemical biology, 2(8).
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  In the early 1900s, Alois Alzheimer diagnosed one of his patients with a devastating neurological impairment, and this form of dementia became known as Alzheimer's disease (AD). Much research over the past century has clearly established that numerous human diseases, ranging from AD and Parkinson's disease to dialysis-related amyloidosis, are best characterized by the abnormal aggregation of specific proteins. However, in the case of AD, the true toxic molecular species is still debated. Thus, the recent development of new diagnostic agents capable of distinguishing between different morphologies of aggregated proteins is of much interest.
• Ghosh, I., Stains, C. I., Mondal, K., & Ghosh, I. -. (2007). Molecules that target beta-amyloid. ChemMedChem, 2(12).
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  The devastating effects of Alzheimer's and related amyloidogenic diseases have inspired the synthesis and evaluation of numerous ligands to understand the molecular mechanism of the aggregation of the beta-amyloid peptide. Our review focuses on the current knowledge in this field with respect to molecules that have been demonstrated to interact with either oligomeric or fibrillar forms of the beta-amyloid peptide. We describe natural proteins, peptides, peptidomimetics, and small molecules that have been found to interfere with beta-amyloid aggregation. We also detail recent efforts in selecting molecules that target beta-amyloid isolated from antibody, protein, and peptide libraries. These new molecules will likely aid in deciphering the details of the aggregation pathway for the beta-amyloid peptide and provide reagents that may stabilize relevant oligomeric intermediates which likely have bearing on the pathophysiology of Alzheimer's disease. Moreover, the described anti-amyloid molecular toolbox will also provide an avenue for designing new diagnostic and therapeutic reagents.
• Ghosh, I., Zhou, M., & Ghosh, I. -. (2007). Quantum dots and peptides: a bright future together. Biopolymers, 88(3).
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  Nanocrystalline semi-conductor materials, called quantum dots (QDs), exhibit unique optical and spectroscopic properties, which include, broad absorption, narrow and tunable emission, resistance to photobleaching, strong luminescence, and long luminescent lifetimes. These remarkable properties of QDs have resulted in their use as an alternative to both small-molecule and protein fluorophores in innumerable biological applications. The overlap of QDs with the rich chemistry and biology that is characteristic of the peptide arena is an emerging research area. Peptides engineered with appropriate cysteines or histidines have served as ligands for producing water soluble QDs as well as for tagging protein ligands and biosensors to QD surfaces. Incorporation of cell-penetrating peptides on QD surfaces has allowed for the translocation of functionalized QDs into cells for intracellular imaging applications. QDs containing fluorescently labeled peptide substrates have shown utility in the development of novel protease assays. Moreover, QDs-labeled peptides that serve as ligands for cellular receptors provide an alternative to antibody mediated imaging at the whole-cell and single molecule level to study receptor distribution and trafficking. This review highlights the overlap between QD and peptide chemistry and speculates on future research directions.
• Meyer, S. C., Shomin, C. D., Gaj, T., & Ghosh, I. (2007). Tethering small molecules to a phage display library: Discovery of a selective bivalent inhibitor of protein kinase A. Journal of the American Chemical Society, 129(45), 13812-13813.
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  PMID: 17944472;Abstract: We report a noncovalent tethering methodology for the fragment-based selection of bivalent ligands targeting protein kinases. In this approach, a small-molecule warhead, staurosporine, directs a phage display cyclic peptide library to the active site of cAMP-dependent protein kinase (PKA), allowing for targeted library enrichment. A cyclic peptide discovered through this selection, when covalently attached to a staurosporine derivative, displayed a 90-fold increase in affinity for PKA. Moreover, the bivalent inhibitor was shown to be significantly more selective than the starting warhead when tested against a small panel of kinases. Thus our general methodology allows for covalent linkage of known small-molecule ligands to biological libraries for discovering potent bivalent inhibitors of biological targets. Copyright © 2007 American Chemical Society.
• Porter, J. R., Helmers, M. R., Wang, P., Furman, J. L., Joy, S. T., Arora, P. S., & Ghosh, I. (2007). Profiling small molecule inhibitors against helix-receptor interactions: the Bcl-2 family inhibitor BH3I-1 potently inhibits p53/hDM2. CHEMICAL COMMUNICATIONS, 46(42), 8020-8022.
• Porter, J. R., Stains, C. I., Segal, D. J., & Ghosh, I. (2007). Split β-lactamase sensor for the sequence-specific detection of DNA methylation. Analytical Chemistry, 79(17), 6702-6708.
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  PMID: 17685552;Abstract: The methylation pattern of genes at CpG dinucleotide sites is an emerging area in epigenetics. Furthermore, the hypermethylation profiles of tumor suppressor genes are linked to specific tumor types. Thus, new molecular approaches for the rapid determination of the methylation status of these genes could provide a powerful method for early cancer diagnosis as well as insight into mechanisms of epigenetic regulation of genetic information. Toward this end, we have recently reported the first design of a split-protein sensor for the site-specific detection of DNA methylation. In this approach a split green fluorescent protein reporter provided a sequence-specific readout of CpG methylation. In the present work, we describe a sensitive second-generation methylation detection system that utilizes the split enzymatic reporter, TEM-1 β-lactamase, attached to specific DNA binding elements. This system, termed mCpG-SEER-β-Lac, shows a greater than 40-fold specificity for methylated versus nonmethylated CpG target sites. Importantly, the resulting signal enhancement afforded by the catalytic activity of split-β-lactamase allowed for the sensitive detection of 2.5 fmol of methylated target dsDNA in 5 min. Thus, this new sensor geometry represents a 250-fold enhancement in assay time and a 2000-fold enhancement in sensitivity over our first-generation system for the detection of specific sites of DNA methylation. © 2007 American Chemical Society.
• Shekhawat, S. S., Porter, J. R., Sriprasad, A., & Ghosh, I. (2007). An Autoinhibited Coiled-Coil Design Strategy for Split-Protein Protease Sensors. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 131(42), 15284-15290.
• Stains, C. I., & Ghosh, I. (2007). When conjugated polymers meet amyloid fibrils. ACS Chemical Biology, 2(8), 525-528.
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  PMID: 17708668;Abstract: In the early 1900s, Alois Alzheimer diagnosed one of his patients with a devastating neurological impairment, and this form of dementia became known as Alzheimer's disease (AD). Much research over the past century has clearly established that numerous human diseases, ranging from AD and Parkinson's disease to dialysis-related amyloidosis, are best characterized by the abnormal aggregation of specific proteins. However, in the case of AD, the true toxic molecular species is still debated. Thus, the recent development of new diagnostic agents capable of distinguishing between different morphologies of aggregated proteins is of much interest. © 2007 American Chemical Society.
• Stains, C. I., Mondal, K., & Ghosh, I. (2007). Molecules that target beta-amyloid. ChemMedChem, 2(12), 1674-1692.
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  PMID: 17952881;Abstract: The devastating effects of Alzheimer's and related amyloidogenic diseases have inspired the synthesis and evaluation of numerous ligands to understand the molecular mechanism of the aggregation of the beta-amyloid peptide. Our review focuses on the current knowledge in this field with respect to molecules that have been demonstrated to interact with either oligomeric or fibrillar forms of the beta-amyloid peptide. We describe natural proteins, peptides, peptidomimetics, and small molecules that have been found to interfere with beta-amyloid aggregation. We also detail recent efforts in selecting molecules that target beta-amyloid isolated from antibody, protein, and peptide libraries. These new molecules will likely aid in deciphering the details of the aggregation pathway for the beta-amyloid peptide and provide reagents that may stabilize relevant oligomeric intermediates which likely have bearing on the pathophysiology of Alzheimer's disease. Moreover, the described anti-amyloid molecular toolbox will also provide an avenue for designing new diagnostic and therapeutic reagents. © 2007 Wiley-VCH Verlag GmbH & Co. KGaA.
• Tokimoto, T., Bethea, T. R., Zhou, M., Ghosh, I., & Wirth, M. J. (2007). Probing orientations of single fluorescent labels on a peptide reversibly binding to the human delta-opioid receptor. APPLIED SPECTROSCOPY, 61(2), 130-137.
• Tokimoto, T., R., T., Zhou, M., Ghosh, I., & Wirth, M. J. (2007). Probing orientations of single fluorescent labels on a peptide reversibly binding to the human delta-opioid receptor. Applied Spectroscopy, 61(2), 130-137.
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  PMID: 17331303;Abstract: We report the first in-depth study of single-molecule polarization behavior of a species that is undergoing reversible binding with its biological receptor. We examine the utility of the information in single-molecule polarization measurements for investigations of binding. The human δ- opioid receptor, which is a G protein-coupled receptor, was incorporated into a supported lipid bilayer. A Cy3 label was covalently attached by a hydrophilic linker to a peptide agonist, Deltorphin II (5,6 Ile-Ile). The fluorescence excitation was alternated between s- and p-polarization using a microscope having the capability of total internal reflectance fluorescence (TIRF) excitation. The polarization behavior reveals that nonspecific binding events for this system give emission that is mostly s-polarized, while binding to the receptor gives emission that has a strong component of p-polarization. The results show that a high signal-to-noise ratio is achievable with single-molecule polarization measurements. The experiment detected 37 binding events of short duration (
• Zhou, M., & Ghosh, I. (2007). Quantum dots and peptides: A bright future together. BIOPOLYMERS, 88(3), 325-339.
• Zhou, M., Nakatani, E., Gronenberg, L. S., Tokimoto, T., Wirth, M. J., Hruby, V. J., Roberts, A., Lynch, R. M., & Ghosh, I. (2007). Peptide-labeled quantum dots for imaging GPCRs in whole cells and as single molecules. BIOCONJUGATE CHEMISTRY, 18(2), 323-332.
• Zhou, M., Zhou, M., Ghosh, I., & Ghosh, I. (2007). Current trends in peptide science quantum dots and peptides: A bright future together. Biopolymers - Peptide Science Section, 88(3), 325-339.
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  Abstract: Nanocrystalline semi-conductor materials, called quantum dots (QDs), exhibit unique optical and spectroscopic properties, which include broad absorption, narrow and tunable emission, resistance to photobleaching, strong luminescence, and long luminescent lifetimes. These remarkable properties of QDs have resulted in their use as an alternative to both small-molecule and protein fluorophores in innumerable biological applications. The overlap of QDs with the rich chemistry and biology that is characteristic of the peptide arena is an emerging research area. Peptides engineered with appropriate cysteines or histidines have served as ligands for producing water soluble QDs as well as for tagging protein ligands and biosensors to QD surfaces. Incorporation of cell-penetrating peptides on QD surfaces has allowed for the translocation of functionalized QDs into cells for intracellular imaging applications. QDs containing fluorescently labeled peptide substrates have shown utility in the development of novel protease assays. Moreover, QD labeled peptides that serve as ligands for cellular receptors provide an alternative to antibody mediated imaging at the whole-cell and single molecule level to study receptor distribution and trafficking. This review highlights the overlap between QD and peptide chemistry and speculates on future research directions. © 2006 Wiley Periodicals, Inc.
• Zhou, M., Zhou, M., Ghosh, I., & Ghosh, I. (2007). Quantum dots and peptides: a bright future together.. Biopolymers, 88(3), 325-339.
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  PMID: 17167795;Abstract: Nanocrystalline semi-conductor materials, called quantum dots (QDs), exhibit unique optical and spectroscopic properties, which include, broad absorption, narrow and tunable emission, resistance to photobleaching, strong luminescence, and long luminescent lifetimes. These remarkable properties of QDs have resulted in their use as an alternative to both small-molecule and protein fluorophores in innumerable biological applications. The overlap of QDs with the rich chemistry and biology that is characteristic of the peptide arena is an emerging research area. Peptides engineered with appropriate cysteines or histidines have served as ligands for producing water soluble QDs as well as for tagging protein ligands and biosensors to QD surfaces. Incorporation of cell-penetrating peptides on QD surfaces has allowed for the translocation of functionalized QDs into cells for intracellular imaging applications. QDs containing fluorescently labeled peptide substrates have shown utility in the development of novel protease assays. Moreover, QDs-labeled peptides that serve as ligands for cellular receptors provide an alternative to antibody mediated imaging at the whole-cell and single molecule level to study receptor distribution and trafficking. This review highlights the overlap between QD and peptide chemistry and speculates on future research directions. Copyright 2006 Wiley Periodicals, Inc.
• Furman, J. L., Badran, A. H., Ajulo, O., Porter, J. R., Stains, C. I., Segal, D. J., & Ghosh, I. (2006). Toward a General Approach for RNA-Templated Hierarchical Assembly of Split-Proteins. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 132(33), 11692-11701.
• Furman, J. L., Mok, P., Shen, S., Stains, C. I., & Ghosh, I. (2006). A turn-on split-luciferase sensor for the direct detection of poly(ADP-ribose) as a marker for DNA repair and cell death. CHEMICAL COMMUNICATIONS, 47(1), 397-399.
• Ghosh, I., Meyer, S. C., Gaj, T., & Ghosh, I. -. (2006). Highly selective cyclic peptide ligands for NeutrAvidin and avidin identified by phage display. Chemical biology & drug design, 68(1).
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  Screening combinatorial libraries of conformationally constrained peptides against macromolecular targets is utilized in identifying novel drug leads and in developing new reagents for chemical biology. In methods such as phage-display selections, biotinylated macromolecular targets are often immobilized on avidin- and streptavidin-functionalized supports. Thus, the characterization of peptides that bind avidin and streptavidin is necessary for accurate interpretation of screening and selection results. Toward this goal, we panned a phage-displayed cyclic peptide library against NeutrAvidin, a chemically deglycosylated version of avidin. The selection produced a highly homologous consensus motif (Asp-Arg/Leu-Ala-Ser/Thr-Pro-Tyr/Trp). Two of these cyclic peptides, CDRATPYC and CDRASPYC, bound both NeutrAvidin and avidin with low-micromolar dissociation constants, whereas their acyclic counterparts had negligible affinity (< 80-fold). Moreover, these cyclic peptides were very specific for their targets and did not bind the structurally and functionally similar protein, streptavidin. Thus, we have identified a new class of cyclic peptides, distinct from the much-studied streptavidin-binding His-Pro-Gln peptide motif. These results will not only allow for discriminating between desired and background cyclic peptide motifs in selections and screens but also provide a new protein/peptide model system and a useful reagent in chemical biology that can have utility in protein immobilization, purification, and chemical tagging.
• Ghosh, I., Rajagopal, S., Meyer, S. C., Goldman, A., Zhou, M., & Ghosh, I. -. (2006). A minimalist approach toward protein recognition by epitope transfer from functionally evolved beta-sheet surfaces. Journal of the American Chemical Society, 128(44).
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  New approaches for identifying small molecules that specifically target protein surfaces as opposed to active site clefts are of much current interest. Toward this goal, we describe a three-step methodology: in step one, we target a protein of interest by directed evolution of a small beta-sheet scaffold; in step two, we identify residues on the scaffold that are implicated in binding; and in step three, we transfer the chemical information from the beta-sheet to a small molecule mimic. As a case study, we targeted the proteolytic enzyme thrombin, involved in blood coagulation, utilizing a library of beta-sheet epitopes displayed on phage that were previously selected for conservation of structure. We found that the thrombin-binding, beta-sheet displaying mini-proteins retained their structure and stability while inhibiting thrombin at low micromolar inhibition constants. A conserved dityrosine recognition motif separated by 9.2 A was found to be common among the mini-protein inhibitors and was further verified by alanine scanning. A molecule containing two tyrosine residues separated by a linker that matched the spacing on the beta-sheet scaffold inhibited thrombin, whereas a similar dityrosine molecule separated by a shorter 6 A linker could not. Moreover, kinetic analysis revealed that both the mini-protein as well as its minimalist mimic with only two functional residues exhibited noncompetitive inhibition of thrombin. Thus, this reductionist approach affords a simple methodology for transferring information from structured protein scaffolds to yield small molecule leads for targeting protein surfaces with novel mechanisms of action.
• Ghosh, I., Smith, T. J., Stains, C. I., Meyer, S. C., & Ghosh, I. -. (2006). Inhibition of beta-amyloid fibrillization by directed evolution of a beta-sheet presenting miniature protein. Journal of the American Chemical Society, 128(45).
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  We describe the directed evolution of a miniature beta-sheet protein for targeting beta-amyloid oligomers implicated in Alzheimer's disease. Circular dichroism spectroscopy, thermal denaturation experiments, and immunoglobulin binding assays established that our beta-amyloid-targeted miniature protein, TJ10, presents a well-folded thermostable beta-sheet. TJ10 was found to prevent beta-amyloid fibrillization at stoichiometric concentrations and was also an effective inhibitor at substoichiometric concentrations. Thus our results provide a new and potent beta-sheet chemical template for effectively targeting beta-amyloid while also demonstrating a general strategy for targeting proteins implicated in other amyloidogenic diseases.
• Ghosh, I., Stains, C. I., Furman, J. L., Segal, D. J., & Ghosh, I. -. (2006). Site-specific detection of DNA methylation utilizing mCpG-SEER. Journal of the American Chemical Society, 128(30).
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  Currently there are no direct methods for the sequence-specific detection of DNA-methylation at CpG dinucleotides, which provide a possible diagnostic marker for cancer. Toward this goal, we present a methodology termed mCpG-SEquence Enabled Reassembly (mCpG-SEER) of proteins utilizing a split green fluorescent protein (GFP) tethered to specific DNA recognition elements. Our system, mCpG-SEER, employs a zinc-finger attached to one-half of GFP to target a specific sequence of dsDNA, while a methyl-CpG binding domain protein attached to the complementary half of GFP targets an adjacent methylated CpG dinucleotide site. We demonstrate that the presence of both DNA sites is necessary for the reassembly and concomitant fluorescence of the reassembled GFP. We further show that the GFP-dependent fluorescence reaches a maximum when the methyl-CpG and zinc-finger sites are separated by two base pairs and the fluorescence signal is linear to 5 pmol of methylated target DNA. Finally, the specificity of this reporter system, mCpG-SEER, was found to be >40-fold between a methylated versus a nonmethylated CpG target site.
• Ghosh, I., Stains, C. I., Ooi, A. T., & Segal, D. J. (2006). Direct detection of double-stranded DNA: Molecular methods and applications for DNA diagnostics. Molecular BioSystems, 2(11), 551-560.
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  PMID: 17216036;Abstract: Methodologies to detect DNA sequences with high sensitivity and specificity have tremendous potential as molecular diagnostic agents. Most current methods exploit the ability of single-stranded DNA (ssDNA) to base pair with high specificity to a complementary molecule. However, recent advances in robust techniques for recognition of DNA in the major and minor groove have made possible the direct detection of double-stranded DNA (dsDNA), without the need for denaturation, renaturation, or hybridization. This review will describe the progress in adapting polyamides, triplex DNA, and engineered zinc finger DNA-binding proteins as dsDNA diagnostic systems. In particular, the sequence-enabled reassembly (SEER) method, involving the use of custom zinc finger proteins, offers the potential for direct detection of dsDNA in cells, with implications for cell-based diagnostics and therapeutics. © The Royal Society of Chemistry 2006.
• Meyer, S. C., Gaj, T., & Ghosh, I. (2006). Highly selective cyclic peptide ligands for neutravidin and avidin identified by phage display. Chemical Biology and Drug Design, 68(1), 3-10.
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  PMID: 16923020;Abstract: Screening combinatorial libraries of conformationally constrained peptides against macromolecular targets is utilized in identifying novel drug leads and in developing new reagents for chemical biology. In methods such as phage-display selections, biotinylated macromolecular targets are often immobilized on avidin- and streptavidin-functionalized supports. Thus, the characterization of peptides that bind avidin and streptavidin is necessary for accurate interpretation of screening and selection results. Toward this goal, we panned a phage-displayed cyclic peptide library against NeutrAvidin, a chemically deglycosylated version of avidin. The selection produced a highly homologous consensus motif (Asp-Arg/Leu-Ala-Ser/Thr-Pro-Tyr/Trp). Two of these cyclic peptides, CDRATPYC and CDRASPYC, bound both NeutrAvidin and avidin with low-micromolar dissociation constants, whereas their acyclic counterparts had negligible affinity (
• Ogunleye, L. O., Jester, B. W., Riemen, A. J., Badran, A. H., Wang, P., & Ghosh, I. (2006). When tight is too tight: Dasatinib and its lower affinity analogue for profiling kinase inhibitors in a three-hybrid split-luciferase system. MEDCHEMCOMM, 5(3), 328-332.
• Ooi, A. T., Stains, C. I., Ghosh, I., & Segal, D. J. (2006). Sequence-enabled reassembly of β-lactamase (SEER-LAC): A sensitive method for the detection of double-stranded DNA. Biochemistry, 45(11), 3620-3625.
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  PMID: 16533044;PMCID: PMC2688710;Abstract: This work describes the development of a new methodology for the detection of specific double-stranded DNA sequences. We previously showed that two inactive fragments of green fluorescent protein, each coupled to engineered zinc finger DNA-binding proteins, were able to reassemble an active reporter complex in the presence of a predefined DNA sequence. This system, designated sequence-enabled reassembly (SEER), was demonstrated in vitro to produce a DNA-concentration-dependent signal. Here we endow the SEER system with catalytic capability using the reporter enzyme TEM-1 β-lacatamase. This system could distinguish target DNA from nontarget DNA in less than 5 min, representing a more than 1000-fold improvement over our previous SEER design. A single base-pair substitution in the DNA binding sequence reduced the signal to nearly background levels. Substitution of a different custom zinc finger DNA-binding domain produced a signal only on the new cognate target. Signal intensity was not affected by genomic DNA when present in equal mass to the target DNA. These results present SEER as a rapid and sensitive method for the detection of double-stranded DNA sequences. © 2006 American Chemical Society.
• Rajagopal, S., Meyer, S. C., Goldman, A., Zhou, M., & Ghosh, I. (2006). A minimalist approach toward protein recognition by epitope transfer from functionally evolved β-sheet surfaces. Journal of the American Chemical Society, 128(44), 14356-14363.
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  PMID: 17076509;Abstract: New approaches for identifying small molecules that specifically target protein surfaces as opposed to active site clefts are of much current interest. Toward this goal, we describe a three-step methodology: in step one, we target a protein of interest by directed evolution of a small β-sheet scaffold; in step two, we identify residues on the scaffold that are implicated in binding; and in step three, we transfer the chemical information from the β-sheet to a small molecule mimic. As a case study, we targeted the proteolytic enzyme thrombin, involved in blood coagulation, utilizing a library of β-sheet epitopes displayed on phage that were previously selected for conservation of structure. We found that the thrombin-binding, β-sheet displaying mini-proteins retained their structure and stability while inhibiting thrombin at low micromolar inhibition constants. A conserved dityrosine recognition motif separated by 9.2 Å was found to be common among the mini-protein inhibitors and was further verified by alanine scanning. A molecule containing two tyrosine residues separated by a linker that matched the spacing on the β-sheet scaffold inhibited thrombin, whereas a similar dityrosine molecule separated by a shorter 6 Å linker could not. Moreover, kinetic analysis revealed that both the mini-protein as well as its minimalist mimic with only two functional residues exhibited noncompetitive inhibition of thrombin. Thus, this reductionist approach affords a simple methodology for transferring information from structured protein scaffolds to yield small molecule leads for targeting protein surfaces with novel mechanisms of action. © 2006 American Chemical Society.
• Shekhawat, S. S., Campbell, S. T., & Ghosh, I. (2006). A Comprehensive Panel of Turn-On Caspase Biosensors for Investigating Caspase Specificity and Caspase Activation Pathways. CHEMBIOCHEM, 12(15), 2353-2364.
• Smith, T. J., Stains, C. I., Meyer, S. C., & Ghosh, I. (2006). Inhibition of β-amyloid fibrillization by directed evolution of a β-sheet presenting miniature protein. Journal of the American Chemical Society, 128(45), 14456-14457.
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  PMID: 17090018;Abstract: We describe the directed evolution of a miniature β-sheet protein for targeting β-amyloid oligomers implicated in Alzheimer's disease. Circular dichroism spectroscopy, thermal denaturation experiments, and immunoglobulin binding assays established that our β-amyloid-targeted miniature protein, TJ10, presents a well-folded thermostable β-sheet. TJ10 was found to prevent β-amyloid fibrillization at stoichiometric concentrations and was also an effective inhibitor at substoichiometric concentrations. Thus our results provide a new and potent β-sheet chemical template for effectively targeting β-amyloid while also demonstrating a general strategy for targeting proteins implicated in other amyloidogenic diseases. Copyright © 2006 American Chemical Society.
• Stains, C. I., Furman, J. L., Porter, J. R., Rajagopal, S., Li, Y., Wyatt, R. T., & Ghosh, I. (2006). A General Approach for Receptor and Antibody-Targeted Detection of Native Proteins Utilizing Split-Luciferase Reassembly. ACS CHEMICAL BIOLOGY, 5(10), 943-952.
• Stains, C. I., Furman, J. L., Segal, D. J., & Ghosh, I. (2006). Site-specific detection of DNA methylation utilizing mCpG-SEER. Journal of the American Chemical Society, 128(30), 9761-9765.
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  PMID: 16866532;Abstract: Currently there are no direct methods for the sequence-specific detection of DNA-methylation at CpG dinucleotides, which provide a possible diagnostic marker for cancer. Toward this goal, we present a methodology termed mCpG-SEquence Enabled Reassembly (mCpG-SEER) of proteins utilizing a split green fluorescent protein (GFP) tethered to specific DNA recognition elements. Our system, mCpG-SEER, employs a zinc-finger attached to one-half of GFP to target a specific sequence of dsDNA, while a methyl-CpG binding domain protein attached to the complementary half of GFP targets an adjacent methylated CpG dinucleotide site. We demonstrate that the presence of both DNA sites is necessary for the reassembly and concomitant fluorescence of the reassembled GFP. We further show that the GFP-dependent fluorescence reaches a maximum when the methyl-CpG and zinc-finger sites are separated by two base pairs and the fluorescence signal is linear to 5 pmol of methylated target DNA. Finally, the specificity of this reporter system, mCpG-SEER, was found to be >40-fold between a methylated versus a nonmethylated CpG target site. © 2006 American Chemical Society.
• Ghosh, I., Meyer, S. C., Huerta, C., & Ghosh, I. -. (2005). Single-site mutations in a hyperthermophilic variant of the B1 domain of protein G result in self-assembled oligomers. Biochemistry, 44(7).
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  We have characterized two homologous, single-point core mutants of a 57-residue, hyperthermophilic variant of the B1 domain of protein G (HTB1). These single-point mutations in HTB1 replace a Phe residue in the hydrophobic core with either a Glu or Asp residue. Both of these homologous core-variant mutants undergo significant structural rearrangement from the native, monomeric fold and exist as stable soluble oligomeric species of 5 and 30 nm in diameter. Gel-filtration, dynamic light scattering, circular dichroism spectroscopy, fluorescence spectroscopy, along with Congo Red and Thioflavin T binding clearly demonstrated that these core-variants undergo significant structural rearrangement from the native, monomeric ubiquitin fold. The two oligomeric species did not equilibrate over extended periods of time and displayed distinct secondary structures. The larger of the two species was found to possess structural features that are reminiscent of an emerging class of protein assemblies prone to beta-sheet-mediated aggregation. These results are significant as there are very few examples of extensive conformational or oligomerization switching brought about by single-point mutations in a stable protein-fold.
• Ghosh, I., Stains, C. I., Porter, J. R., Ooi, A. T., Segal, D. J., & Ghosh, I. -. (2005). DNA sequence-enabled reassembly of the green fluorescent protein. Journal of the American Chemical Society, 127(31).
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  We describe a general methodology for the direct detection of DNA by the design of a split-protein system that reassembles to form an active complex only in the presence of a targeted DNA sequence. This approach, called SEquence Enabled Reassembly (SEER) of proteins, combines the ability to rationally dissect proteins to construct oligomerization-dependent protein reassembly systems and the availability of DNA binding Cys2-His2 zinc-finger motifs for the recognition of specific DNA sequences. We demonstrate the feasibility of the SEER approach utilizing the split green fluorescent protein appended to appropriate zinc fingers, such that chromophore formation is only catalyzed in the presence of DNA sequences that incorporate binding sites for both zinc fingers.
• Magliery, T. J., G., C., Pan, W., Mishler, D., Ghosh, I., Hamilton, A. D., & Regan, L. (2005). Detecting protein-protein interactions with a green fluorescent protein fragment reassembly trap: Scope and mechanism. Journal of the American Chemical Society, 127(1), 146-157.
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  PMID: 15631464;Abstract: Identification of protein binding partners is one of the key challenges of proteomics. We recently introduced a screen for detecting protein-protein interactions based on reassembly of dissected fragments of green fluorescent protein fused to interacting peptides. Here, we present a set of comaintained Escherichia coli plasmids for the facile subcloning of fusions to the green fluorescent protein fragments. Using a library of antiparallel leucine zippers, we have shown that the screen can detect very weak interactions (KD ≈ 1 mM). In vitro kinetics show that the reassembly reaction is essentially irreversible, suggesting that the screen may be useful for detecting transient interactions. Finally, we used the screen to discriminate cognate from noncognate protein-ligand interactions for tetratricopeptide repeat domains. These experiments demonstrate the general utility of the screen for larger proteins and elucidate mechanistic details to guide the further use of this screen in proteomic analysis. Additionally, this work gives insight into the positional inequivalence of stabilizing interactions in antiparallel coiled coils.
• Meyer, S. C., Huerta, C., & Ghosh, I. (2005). Single-site mutations in a hyperthermophilic variant of the B1 domain of protein G result in self-assembled oligomers. Biochemistry, 44(7), 2360-2368.
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  PMID: 15709748;Abstract: We have characterized two homologous, single-point core mutants of a 57-residue, hyperthermophilic variant of the B1 domain of protein G (HTB1). These single-point mutations in HTB1 replace a Phe residue in the hydrophobic core with either a Glu or Asp residue. Both of these homologous core-variant mutants undergo significant structural rearrangement from the native, monomeric fold and exist as stable soluble oligomeric species of 5 and 30 nm in diameter. Gel-filtration, dynamic light scattering, circular dichroism spectroscopy, fluorescence spectroscopy, along with Congo Red and Thioflavin T binding clearly demonstrated that these core-variants undergo significant structural rearrangement from the native, monomeric ubiquitin fold. The two oligomeric species did not equilibrate over extended periods of time and displayed distinct secondary structures. The larger of the two species was found to possess structural features that are reminiscent of an emerging class of protein assemblies prone to β-sheet-mediated aggregation. These results are significant as there are very few examples of extensive conformational or oligomerization switching brought about by single-point mutations in a stable protein-fold.
• Ooi, A., Stains, C., Ghosh, ., & Segal, D. (2005). Sequence-enabled reassembly of beta-lactamase (SEER-LAC): A sensitive method for the detection of double-stranded DNA. BIOCHEMISTRY, 45(11), 3620-3625.
• Rajagopal, S., Meza-Romero, R., & Ghosh, . (2005). Dual surface selection methodology for the identification of thrombin binding epitopes from hotspot biased phage-display libraries. BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 14(6), 1389-1393.
• Stains, C. I., Porter, J. R., Ooi, A. T., Segal, D. J., & Ghosh, I. (2005). DNA sequence-enabled reassembly of the green fluorescent protein. Journal of the American Chemical Society, 127(31), 10782-10783.
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  PMID: 16076155;Abstract: We describe a general methodology for the direct detection of DNA by the design of a split-protein system that reassembles to form an active complex only in the presence of a targeted DNA sequence. This approach, called SEquence Enabled Reassembly (SEER) of proteins, combines the ability to rationally dissect proteins to construct oligomerization-dependent protein reassembly systems and the availability of DNA binding Cys2-His2 zinc-finger motifs for the recognition of specific DNA sequences. We demonstrate the feasibility of the SEER approach utilizing the split green fluorescent protein appended to appropriate zinc fingers, such that chromophore formation is only catalyzed in the presence of DNA sequences that incorporate binding sites for both zinc fingers. Copyright © 2005 American Chemical Society.
• Stains, C., Porter, ., Ooi, A., Segal, D., & Ghosh, . (2005). DNA sequence-enabled reassembly of the green fluorescent protein. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 127(31), 10782-10783.
• Zhou, M., & Ghosh, . (2005). Noncovalent multivalent assembly of Jun peptides on a leucine Zipper dendrimer displaying Fos peptides. ORGANIC LETTERS, 6(20), 3561-3564.
• Zhou, M., Bentley, D., & Ghosh, . (2005). Helical supramolecules and fibers utilizing leucine zipper-displaying dendrimers. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 126(3), 734-735.
• Gdalyahu, A., Ghosh, I., Levy, T., Sapir, T., Sapoznik, S., Fishler, Y., Azoulai, D., & Reiner, O. (2004). DCX, a new mediator of the JNK pathway. EMBO Journal, 23(4), 823-832.
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  PMID: 14765123;PMCID: PMC380994;Abstract: Mutations in the X-linked gene DCX result in lissencephaly in males, and abnormal neuronal positioning in females, suggesting a role for this gene product during neuronal migration. In spite of several known protein interactions, the involvement of DCX in a signaling pathway is still elusive. Here we demonstrate that DCX is a substrate of JNK and interacts with both c-Jun N-terminal kinase (JNK) and JNK interacting protein (JIP). The localization of this signaling module in the developing brain suggests its functionality in migrating neurons. The localization of DCX at neurite tips is determined by its interaction with JIP and by the interaction of the latter with kinesin. DCX is phosphorylated by JNK in growth cones. DCX mutated in sites phosphorylated by JNK affected neurite outgrowth, and the velocity and relative pause time of migrating neurons. We hypothesize that during neuronal migration, there is a need to regulate molecular motors that are working in the cell in opposite directions: kinesin (a plus-end directed molecular motor) versus dynein (a minus-end directed molecular motor).
• Ghosh, I., & Chmielewski, J. (2004). Peptide self-assembly as a model of proteins in the pre-genomic world. Current Opinion in Chemical Biology, 8(6), 640-644.
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  PMID: 15556409;Abstract: Excellent catalytic efficiency has been obtained within a series of self replicating peptides, and nucleobase inclusion into a salt-switchable self replicating peptide is found to override the switch. Interestingly, cross-catalytic formation of an RNA aptamer is reported with a cationic peptide, and novel, amide-based biopolymers have been designed to self assemble. © 2004 Elsevier Ltd. All rights reserved.
• Ghosh, I., Rajagopal, S., Meza-Romero, R., & Ghosh, I. -. (2004). Dual surface selection methodology for the identification of thrombin binding epitopes from hotspot biased phage-display libraries. Bioorganic & medicinal chemistry letters, 14(6).
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  Protein libraries biased towards amino-acid residues found at so-called 'hotspots' were incorporated into the beta-sheet region of the thermostable variant (HTB1) of the B1 domain of the immunoglobulin (IgG) binding protein G and expressed as gene 3 fusions on M13 bacteriophage. The HTB1 library (2.2 x 10(9)) variants with a minimal 12 amino acid basis set were selected for binding IgG, to ensure structural conservation, and subsequently to thrombin to evolve a thrombin-binding function. We believe that this dual surface selection strategy will have great utility in evolving new bi-functional proteins without compromising structure. Furthermore the discrete beta-sheet epitopes identified by our methodology will lend itself to small-molecule mimicry of beta-sheets.
• Ghosh, I., Zhou, M., & Ghosh, I. -. (2004). Noncovalent multivalent assembly of jun peptides on a leucine zipper dendrimer displaying fos peptides. Organic letters, 6(20).
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  [reaction: see text] The synthesis and characterization of a new leucine-zipper dendrimer (LZD) is reported that displays four copies of the peptide corresponding to the coiled-coiled dimerization domain of Fos. Circular dichroism spectroscopy, fluorescence titration, and sedimentation equilibrium experiments demonstrate that Fos-LZD can noncovalently assemble four copies of the peptide corresponding to the coiled-coil domain of Jun. This work provides the basis for the future construction of noncovalently assembled multivalent protein assemblies displaying any protein of interest.
• Ghosh, I., Zhou, M., Bentley, D., & Ghosh, I. -. (2004). Helical supramolecules and fibers utilizing leucine zipper-displaying dendrimers. Journal of the American Chemical Society, 126(3).
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  We describe a new family of discrete supramolecules comprising leucine-zipper peptides noncovalently assembled upon cognate leucine zippers fused to a dendrimer core. Circular dichroism and sedimentation equilibrium experiments clearly demonstrate that each leucine-zipper dendrimer (D-EZ4 or D-KZ4) can noncovalently display four leucine zippers on their surface that can be utilized for the multivalent display of protein cargo. Furthermore, we show that matched leucine-zipper dendrimers (D-EZ4/D-KZ4) can self-organize into fibers at neutral pH, providing a new scaffold for nanotechnology.
• Meyer, S., Huerta, C., & Ghosh, . (2004). Single-site mutations in a hyperthermophilic variant of the B1 domain of protein G result in self-assembled oligomers. BIOCHEMISTRY, 44(7), 2360-2368.
• Rajagopal, S., Meza-Romero, R., & Ghosh, I. (2004). Dual surface selection methodology for the identification of thrombin binding epitopes from hotspot biased phage-display libraries. Bioorganic and Medicinal Chemistry Letters, 14(6), 1389-1393.
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  PMID: 15006368;Abstract: Protein libraries biased towards amino-acid residues found at so-called 'hotspots' were incorporated into the beta-sheet region of the thermostable variant (HTB1) of the B1 domain of the immunoglobulin (IgG) binding protein G and expressed as gene 3 fusions on M13 bacteriophage. The HTB1 library (2.2×109) variants with a minimal 12 amino acid basis set were selected for binding IgG, to ensure structural conservation, and subsequently to thrombin to evolve a thrombin-binding function. We believe that this dual surface selection strategy will have great utility in evolving new bi-functional proteins without compromising structure. Furthermore the discrete beta-sheet epitopes identified by our methodology will lend itself to small-molecule mimicry of beta-sheets. © 2004 Elsevier Ltd. All rights reserved.
• Reiner, O., Gdalyahu, A., Ghosh, I., Levy, T., Sapoznik, S., Nir, R., & Sapir, T. (2004). DCX's phosphorylation by not just another kinase (JNK). Cell Cycle, 3(6), 747-751.
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  PMID: 15118415;Abstract: The mammalian cortex is generally subdivided into six organized layers, which are formed during development in an organized fashion. This organized cortical layering is disrupted in case of mutations in the doublecortin (DCX) gene. DCX is a Microtubule Associated Protein (MAP). However, besides stabilization of microtubules, it may be involved in additional functions. The participation of this molecule in signal transduction is beginning to emerge via discovery of interacting molecules and its regulation by phosphorylation using several different kinases. We raise the hypothesis, that the combinatorial phosphorylation of DCX by different kinases and at different, sites may be a molecular regulatory switch in the transition of a migrating neuron through multiple phases of migration. Our recent research has suggested the involvement of DCX in the JNK (Jun-N-terminal Kinase) pathway. The JNK pathway is linked to the reelin pathway, known to regulate cortical layering. Positioning of DCX in this signaling pathway opens up additional possibilities of understanding how migrating neurons are controlled.
• Zhou, M., & Ghosh, I. (2004). Noncovalent multivalent assembly of jun peptides on a leucine zipper dendrimer displaying fos peptides. Organic Letters, 6(20), 3561-3564.
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  PMID: 15387548;Abstract: (Chemical Equation Presented) The synthesis and characterization of a new leucine-zipper dendrimer (LZD) is reported that displays four copies of the peptide corresponding to the coiled-coiled dimerization domain of Fos. Circular dichroism spectroscopy, fluorescence titration, and sedimentation equilibrium experiments demonstrate that Fos-LZD can noncovalently assemble four copies of the peptide corresponding to the coiled-coil domain of Jun. This work provides the basis for the future construction of noncovalently assembled multivalent protein assemblies displaying any protein of interest.
• Zhou, M., Bentley, D., & Ghosh, I. (2004). Helical Supramolecules and Fibers Utilizing Leucine Zipper-Displaying Dendrimers. Journal of the American Chemical Society, 126(3), 734-735.
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  PMID: 14733545;Abstract: We describe a new family of discrete supramolecules comprising leucine-zipper peptides noncovalently assembled upon cognate leucine zippers fused to a dendrimer core. Circular dichroism and sedimentation equilibrium experiments clearly demonstrate that each leucine-zipper dendrimer (D-EZ4 or D-KZ4) can noncovalently display four leucine zippers on their surface that can be utilized for the multivalent display of protein cargo. Furthermore, we show that matched leucine-zipper dendrimers (D-EZ4/D-KZ4) can self-organize into fibers at neutral pH, providing a new scaffold for nanotechnology. Copyright © 2004 American Chemical Society.
• Ghosh, I., Bishop, P., & Chmielewski, J. (2001). DNA binding properties of basic helix-loop-helix fusion proteins of Tal and E47. Journal of Peptide Research, 57(5), 354-360.
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  PMID: 11350595;Abstract: The basic helix-loop-helix (bHLH) transcription factor Tal has been shown to form heterodimers with the ubiquitously expressed bHLH transcription factor E47 and thereby modulate gene expression. The absence of homodimeric Tal-DNA complexes had been attributed to the inability of Tal to homodimerize, but subsequent studies have shown that the bHLH region of Tal does homodimerize. In order to correlate the contributions of both the basic region and the helix-loop-helix (HLH) domain to the lack of DNA binding by Tal homodimers, mutant and fusion proteins based on Tal and E47 were designed and synthesized. Size-exclusion chromatography established that all mutant and fusion proteins were dimeric. Point mutations were made within the basic region of Tal based on residues within E47 that are essential for DNA binding, but an affinity for DNA was not observed. Even complete replacement of the basic region in Tal with the basic region of E47, in an E47-Tal fusion protein, did not confer DNA binding upon the protein. However, when the dimerization domain in Tal was replaced with its E47 counterpart, in a Tal-E47 fusion protein, sequence specific DNA binding was observed with an apparent dissociation constant of 3.6×10-9 M2. Furthermore, circular dichroism studies showed that the basic region of Tal in the Tal-E47 fusion protein underwent a random coil to helix transition in the presence of a specific DNA probe. These experimental observations indicate that the inability of Tal homodimers to recognize DNA stems from a misalignment of its basic region with respect to the HLH domain, rather than an intrinsic inability of the Tal basic region to bind DNA.
• Ghosh, I., Hamilton, A. D., & Regan, L. (2000). Antiparallel leucine zipper-directed protein reassembly: Application to the green fluorescent protein [12]. Journal of the American Chemical Society, 122(23), 5658-5659.
• Ghosh, I., Issac, R., & Chmielewski, J. (1999). Structure-function relationship in a β-sheet peptide Inhibitor of E47 dimerization and DNA binding. Bioorganic and Medicinal Chemistry, 7(1), 61-66.
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  PMID: 10199656;Abstract: A β-sheet peptide inhibitor, 2H10, has been developed that inhibits the dimerization of the transcription factor E47. Inhibition of E47 dimerization has been demonstrated to also inhibit the DNA binding of this transcription factor. Truncated peptides based on 2H10 have demonstrated that the β-sheet content of these peptides directly correlates with their inhibitory properties. Individual residues within 2H10 were identified that were responsible for the β-sheet secondary structure by employing an alanine replacement strategy. The β-sheet character of the alanine mutants also correlated well with their inhibition of E47 DNA binding. These results provide further evidence that interactions between the interfacial peptide inhibitors of E47 and the transcription factor itself are mediated by a β-sheet structure. Copyright (C) 1999 Elsevier Science Ltd.
• Yongsheng, M. a., Cunningham, M. E., Wang, X., Ghosh, I., Regan, L., & Longley, B. J. (1999). Inhibition of spontaneous receptor phosphorylation by residues in a putative α-helix in the KIT intracellular juxtamembrane region. Journal of Biological Chemistry, 274(19), 13399-13402.
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  PMID: 10224103;Abstract: KIT receptor kinase activity is repressed, prior to stem cell factor binding, by unknown structural constraints. Using site-directed mutagenesis, we examined the role of KIT intracellular juxtamembrane residues Met-552 through Ile-563 in controlling receptor autophosphorylation. Alanine substitution for Tyr-553, Trp557, Val-559, or Val-560, all sitting along the hydrophobic side of an amphipathic α-helix (Tyr-553-Ile-563) predicted by the Chou-Fasman algorithm, resulted in substantially increased spontaneous receptor phosphorylation, revealing inhibitory roles for these residues. Alanine substitution for other residues, most of which are on the hydrophilic side of the helix, caused no or slightly increased basal receptor phosphorylation. Converting Tyr-553 or Trp-557 to phenylalanine generated slight or no elevation, respectively, in basal KIT phosphorylation, indicating that the phenyl ring of Tyr-553 and the hydrophobicity of Trp-557 are critical for the inhibition. Although alanine substitution for Lys-558 had no effect on receptor phosphorylation, its substitution with proline produced high spontaneous receptor phosphorylation, suggesting that the predicted α-helical conformation is involved in the inhibition. A synthetic peptide comprising Tyr-553 through Ile-563 showed circular dichroism spectra characteristic of α-helix, supporting the structural prediction. Thus, the KIT intracellular juxtamembrane region contains important residues which, in a putative α-helical conformation, exert inhibitory control on the kinase activity of ligand-unoccupied receptor.
• Ghosh, I., & Chmielewski, J. (1998). A β-sheet peptide inhibitor of E47 dimerization and DNA binding. Chemistry and Biology, 5(8), 439-445.
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  PMID: 9710566;Abstract: Background: Many transcription factors are active only in their dimeric form, including the basic-helix-loop-helix (bHLH) family of transcription factors. The disruption of the dimer therefore presents a means of inhibiting the biological functions of such transcription factors. E47 is a homodimeric bHLH transcription factor with a four-helix bundle dimerization interface. Here, we investigate the concept of dimerization inhibition using peptides derived from the dimerization domain of E47. Results: We have synthesized several peptides corresponding to the E47 dimerization interface that inhibit E47 DNA-binding activity with IC50 values in the range of 3.6-120 mM. Interestingly, helix II, a peptide corresponding to the carboxy-terminal helix of the E47 dimerization interface, adopted a β-sheet structure in solution, as shown using circular dichroism (CD), and inhibited the binding of E47 to DNA at equimolar concentrations. Size-exclusion chromatography, analytical ultracentrifugation and cross-linking experiments verified that this peptide prevented E47 dimerization. Furthermore, CD experiments provided evidence that helix II could induce a β-sheet secondary structure upon the highly α-helical E47 bHLH domain. Conclusions: This study is the first demonstration of dissociative inhibition in the bHLH class of transcription factors and also provides an example of β-sheet induction in an α-helical protein. Future experiments will probe the structural determinants of the β-sheet secondary structure in helix II and investigate the generality of the dissociative strategy in other transcription factor families.
• Yao, S., Ghosh, I., Zutshi, R., & Chmielewski, J. (1998). A self-replicating peptide under ionic control. Angewandte Chemie - International Edition in English, 37(4), 478-481.
• Yao, S., Ghosh, I., Zutshi, R., & Chmielewski, J. (1998). Selective amplification by auto- and cross-catalysis in a replicating peptide system. Nature, 396(6710), 447-450.
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  PMID: 9853750;Abstract: Self-replication has been demonstrated in synthetic chemical systems based on oligonucleotides, peptides and complementary molecules without natural analogues. However, within a living cell virtually no molecule catalyses its own formation, and the search for chemical systems in which both auto- and cross-catalysis can occur has therefore attracted wide interest. One such system, consisting of two self-replicating peptides that catalyse each other's production, has been reported. Here we describe a four- component peptide system that is capable of auto- and cross-catalysis and allows for the selective amplification of one or more of the products by changing the reaction conditions. The ability of this system selectively to amplify one or more molecules in response to changes in environmental conditions such as pH or salt concentration supports the suggestion that self-replicating peptides may have played a role in the origin of life.
• Yao, S., Ghosh, I., Zutshi, R., & Chmielewski, J. (1997). A pH-modulated, self-replicating peptide. Journal of the American Chemical Society, 119(43), 10559-10560.
• Bishop, P., Ghosh, I., Jones, C., & Chmielewski, J. (1995). Basic-helix-loop-helix region of tal: Evaluation of structure and DNA affinity. Journal of the American Chemical Society, 117(31), 8283-8284.
• Bishop, P., Jones, C., Ghosh, I., & Chmielewski, J. (1995). Synthesis of the basic-helix-loop-helix region of the immunoglobulin enhancer binding protein E47 and evaluation of its structural and DNA binding properties. International Journal of Peptide and Protein Research, 46(2), 149-154.
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  PMID: 8567169;Abstract: The basic-helix-loop-helix (bHLH) region of the immunoglobulin enhancer binding protein E47 (IEB E47) was prepared in high yield by a solid-phase peptide synthesis methodology. Size-exclusion chromatography, sedimentation equilibrium and cross-linking data showed that the synthetic bHLH protein, 1, was dimeric, and higher-order aggregates of trimer and tetramer were also observed. The circular dichroism spectrum of 1 showed a high helical content, which increased upon addition of DNA. containing the κE2 sequence. Gel mobility shift experiments showed that protein 1 bound sequence specifically to the κE2 sequence with a binding constant of 10-10 M2, and had an affinity for other E box sequences as well. Comparisons between the co-crystal structure of IEB E47 with DNA and structural studies in solution showed lower helical contents in solution as would have been predicted from the crystal structure.

Proceedings Publications

• Ooi, A. T., Stains, C. I., Porter, J. R., Ghosh, I., Segal, D. J., & Blondelle, S. (2008, 2006). Sequence-enabled reassembly (SEER) peptides for the detection of DNA sequences. In Understanding Biology Using Peptides, 214-215.

Reviews

• Cox, K. J., Shomin, C. D., & Ghosh, I. (2011. Tinkering outside the kinase ATP box: allosteric (type IV) and bivalent (type V) inhibitors of protein kinases(pp 29-43).
• Ghosh, I., Stains, C. I., Ooi, A. T., & Segal, D. J. (2011. Direct detection of double-stranded DNA: molecular methods and applications for DNA diagnostics(pp 551-560).
• Lamba, V., & Ghosh, I. (2007. New Directions in Targeting Protein Kinases: Focusing Upon True Allosteric and Bivalent Inhibitors(pp 2936-2945).
• Stains, C. I., Mondal, K., & Ghosh, I. (2007. Molecules that target beta-amyloid(pp 1674-1692).
• Shekhawat, S. S., & Ghosh, I. (2006. Split-protein systems: beyond binary protein-protein interactions(pp 789-797).
• Ghosh, ., & Chrnielewski, J. (2004. Peptide self-assembly as a model of proteins in the pre-genomic world(pp 640-644).

Others

• Bhakta, M., Brayer, K., Lockwood, S., Ooi, A., Ghosh, I., Brilliant, M., Camenisch, T., & Segal, D. J. (2010, DEC). Editing and imaging genomes using engineering zinc finger proteins. TRANSGENIC RESEARCH.
• Ooi, A. T., Stains, C. I., Ghosh, I., & Segal, D. J. (2006, MAY). SEquence-Enabled Reassembly of beta-Lactamase (SEER-LAC): A Sensitive Method for the Detection of Double-Stranded DNA. MOLECULAR THERAPY.
• Ghosh, . (2005, SEP). Celebrating Victor Hruby 65th birthday: An odyssey in chemistry and biology. MEDICINAL RESEARCH REVIEWS.