Gregory Thatcher

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• Xiong, R., Thatcher, G. R., Shen, Z., Rong, L., Ratia, K., Li, Y., Lee, H., Kwon, Y., Kong, D., Huang, F., Dubrovskyi, O., Cooper, L., & Alqarni, S. (2022). Design of SARS-CoV-2 PLpro Inhibitors for COVID-19 Antiviral Therapy Leveraging Binding Cooperativity.. Journal of medicinal chemistry, 65(4), 2940-2955. doi:10.1021/acs.jmedchem.1c01307
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  Antiviral agents that complement vaccination are urgently needed to end the COVID-19 pandemic. The SARS-CoV-2 papain-like protease (PLpro), one of only two essential cysteine proteases that regulate viral replication, also dysregulates host immune sensing by binding and deubiquitination of host protein substrates. PLpro is a promising therapeutic target, albeit challenging owing to featureless P1 and P2 sites recognizing glycine. To overcome this challenge, we leveraged the cooperativity of multiple shallow binding sites on the PLpro surface, yielding novel 2-phenylthiophenes with nanomolar inhibitory potency. New cocrystal structures confirmed that ligand binding induces new interactions with PLpro: by closing of the BL2 loop of PLpro forming a novel "BL2 groove" and by mimicking the binding interaction of ubiquitin with Glu167 of PLpro. Together, this binding cooperativity translates to the most potent PLpro inhibitors reported to date, with slow off-rates, improved binding affinities, and low micromolar antiviral potency in SARS-CoV-2-infected human cells.
• Abderrahman, B., Maximov, P. Y., Curpan, R. F., Fanning, S. W., Hanspal, J. S., Fan, P., Foulds, C. E., Chen, Y., Malovannaya, A., Jain, A., Xiong, R., Greene, G. L., Tonetti, D. A., Thatcher, G. R., & Jordan, V. C. (2021). Rapid Induction of the Unfolded Protein Response and Apoptosis by Estrogen Mimic TTC-352 for the Treatment of Endocrine-Resistant Breast Cancer. Molecular cancer therapeutics, 20(1), 11-25.
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  Patients with long-term estrogen-deprived breast cancer, after resistance to tamoxifen or aromatase inhibitors develops, can experience tumor regression when treated with estrogens. Estrogen's antitumor effect is attributed to apoptosis via the estrogen receptor (ER). Estrogen treatment can have unpleasant gynecologic and nongynecologic adverse events; thus, the development of safer estrogenic agents remains a clinical priority. Here, we study synthetic selective estrogen mimics (SEM) BMI-135 and TTC-352, and the naturally occurring estrogen estetrol (E), which are proposed as safer estrogenic agents compared with 17β-estradiol (E), for the treatment of endocrine-resistant breast cancer. TTC-352 and E are being evaluated in breast cancer clinical trials. Cell viability assays, real-time PCR, immunoblotting, DNA pulldowns, mass spectrometry, X-ray crystallography, docking and molecular dynamic simulations, live cell imaging, and Annexin V staining were conducted in 11 biologically different breast cancer models. Results were compared with the potent full agonist E, less potent full agonist E, the benchmark partial agonist triphenylethylene bisphenol (BPTPE), and antagonists 4-hydroxytamoxifen and endoxifen. We report ERα's regulation and coregulators' binding profiles with SEMs and E We describe TTC-352's pharmacology as a weak full agonist and antitumor molecular mechanisms. This study highlights TTC-352's benzothiophene scaffold that yields an H-bond with Glu353, which allows Asp351-to-helix 12 (H12) interaction, sealing ERα's ligand-binding domain, recruiting E-enriched coactivators, and triggering rapid ERα-induced unfolded protein response (UPR) and apoptosis, as the basis of its anticancer properties. BPTPE's phenolic OH yields an H-Bond with Thr347, which disrupts Asp351-to-H12 interaction, delaying UPR and apoptosis and increasing clonal evolution risk.
• Fata, F., Silvestri, I., Ardini, M., Ippoliti, R., Di Leandro, L., Demitri, N., Polentarutti, M., Di Matteo, A., Lyu, H., Thatcher, G. R., Petukhov, P. A., Williams, D. L., & Angelucci, F. (2021). Probing the Surface of a Parasite Drug Target Thioredoxin Glutathione Reductase Using Small Molecule Fragments. ACS infectious diseases, 7(7), 1932-1944.
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  Fragment screening is a powerful drug discovery approach particularly useful for enzymes difficult to inhibit selectively, such as the thiol/selenol-dependent thioredoxin reductases (TrxRs), which are essential and druggable in several infectious diseases. Several known inhibitors are reactive electrophiles targeting the selenocysteine-containing C-terminus and thus often suffering from off-target reactivity . The lack of structural information on the interaction modalities of the C-terminus-targeting inhibitors, due to the high mobility of this domain and the lack of alternative druggable sites, prevents the development of selective inhibitors for TrxRs. In this work, fragments selected from actives identified in a large screen carried out against Thioredoxin Glutathione Reductase from (SmTGR) were probed by X-ray crystallography. SmTGR is one of the most promising drug targets for schistosomiasis, a devastating, neglected disease. Utilizing a multicrystal method to analyze electron density maps, structural analysis, and functional studies, three binding sites were characterized in SmTGR: two sites are close to or partially superposable with the NADPH binding site, while the third one is found between two symmetry related SmTGR subunits of the crystal lattice. Surprisingly, one compound bound to this latter site stabilizes, through allosteric effects mediated by the so-called guiding bar residues, the crucial redox active C-terminus of SmTGR, making it finally visible at high resolution. These results further promote fragments as small molecule probes for investigating functional aspects of the target protein, exemplified by the allosteric effect on the C-terminus, and providing fundamental chemical information exploitable in drug discovery.
• Gaisina, I. N., Hushpulian, D. M., Gaisin, A. M., Kazakov, E. H., Ammal Kaidery, N., Ahuja, M., Poloznikov, A. A., Gazaryan, I. G., Thatcher, G. R., & Thomas, B. (2021). Identification of a potent Nrf2 displacement activator among aspirin-containing prodrugs. Neurochemistry international, 149, 105148.
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  Aspirin is a desired leaving group in prodrugs aimed at treatment of neurodegeneration and other conditions. A library of aspirin derivatives of various scaffolds potentially activating Nrf2 has been tested in Neh2-luc reporter assay which screens for direct Nrf2 protein stabilizers working via disruption of Nrf2-Keap1 interaction. Most aspirin prodrugs had a pro-alkylating or pro-oxidant motif in the structure and, therefore, were toxic at high concentrations. However, among the active compounds, we identified a molecule resembling a well-known Nrf2 displacement activator, bis-1,4-(4-methoxybenzenesulfonamidyl) naphthalene (NMBSA). The direct comparison of the newly identified compound with NMBSA and its improved analog in the reporter assay showed no quenching with N-acetyl cysteine, thus pointing to Nrf2 stabilization mechanism without cysteine alkylation. The potency of the newly identified compound in the reporter assay was much stronger than NMBSA, despite its inhibitory action in the commercial fluorescence polarization assay was observed only in the millimolar range. Molecular docking predicted that mono-deacetylation of the novel prodrug should generate a potent displacement activator. The time-course of reporter activation with the novel prodrug had a pronounced lag-period pointing to a plausible intracellular transformation leading to an active product. Treatment of the novel prodrug with blood plasma or cell lysate demonstrated stepwise deacetylation as judge by liquid chromatography-mass spectrometry (LC-MS). Hence, the esterase-catalyzed hydrolysis of the prodrug liberates only acetyl groups from aspirin moiety and generates a potent Nrf2 activator. The discovered mechanism of prodrug activation makes the newly identified compound a promising lead for future optimization studies.
• Scheinman, S. B., Zaldua, S., Dada, A., Krochmaliuk, K., Dye, K., Marottoli, F. M., Thatcher, G. R., & Tai, L. M. (2021). Systemic Candesartan Treatment Modulates Behavior, Synaptic Protein Levels, and Neuroinflammation in Female Mice That Express Human. Frontiers in neuroscience, 15, 628403.
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  Evidence suggests that angiotensin receptor blockers (ARBs) could be beneficial for Alzheimer's disease (AD) patients independent of any effects on hypertension. However, studies in rodent models directly testing the activity of ARB treatment on behavior and AD-relevent pathology including neuroinflammation, Aβ levels, and cerebrovascular function, have produced mixed results. is a major genetic risk factor for AD and has been linked to many of the same functions as those purported to be modulated by ARB treatment. Therefore, evaluating the effects of ARB treatment on behavior and AD-relevant pathology in mice that express human could provide important information on whether to further develop ARBs for AD therapy. In this study, we treated female and male mice that express the human gene in the absence (E4FAD-) or presence (E4FAD+) of high Aβ levels with the ARB prodrug candesartan cilexetil for a duration of 4 months. Compared to vehicle, candesartan treatment resulted in greater memory-relevant behavior and higher hippocampal presynaptic protein levels in female, but not male, E4FAD- and E4FAD+ mice. The beneficial effects of candesartan in female E4FAD- and E4FAD+ mice occurred in tandem with lower GFAP and Iba1 levels in the hippocampus, whereas there were no effects on markers of cerebrovascular function and Aβ levels. Collectively, these data imply that the effects of ARBs on AD-relevant pathology may be modulated in part by the interaction between genotype and biological sex. Thus, the further development of ARBs could provide therapeutic options for targeting neuroinflammation in female carriers.
• Shen, Z., Ratia, K., Cooper, L., Kong, D., Lee, H., Kwon, Y., Li, Y., Alqarni, S., Huang, F., Dubrovskyi, O., Rong, L., Thatcher, G. R., & Xiong, R. (2021). Design of SARS-CoV-2 PLpro Inhibitors for COVID-19 Antiviral Therapy Leveraging Binding Cooperativity. Journal of medicinal chemistry.
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  Antiviral agents that complement vaccination are urgently needed to end the COVID-19 pandemic. The SARS-CoV-2 papain-like protease (PLpro), one of only two essential cysteine proteases that regulate viral replication, also dysregulates host immune sensing by binding and deubiquitination of host protein substrates. PLpro is a promising therapeutic target, albeit challenging owing to featureless P1 and P2 sites recognizing glycine. To overcome this challenge, we leveraged the cooperativity of multiple shallow binding sites on the PLpro surface, yielding novel 2-phenylthiophenes with nanomolar inhibitory potency. New cocrystal structures confirmed that ligand binding induces new interactions with PLpro: by closing of the BL2 loop of PLpro forming a novel "BL2 groove" and by mimicking the binding interaction of ubiquitin with Glu167 of PLpro. Together, this binding cooperativity translates to the most potent PLpro inhibitors reported to date, with slow off-rates, improved binding affinities, and low micromolar antiviral potency in SARS-CoV-2-infected human cells.
• Shen, Z., Ratia, K., Cooper, L., Kong, D., Lee, H., Kwon, Y., Li, Y., Alqarni, S., Huang, F., Dubrovskyi, O., Rong, L., Thatcher, G. R., & Xiong, R. (2021). Potent, Novel SARS-CoV-2 PLpro Inhibitors Block Viral Replication in Monkey and Human Cell Cultures. bioRxiv : the preprint server for biology.
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  Antiviral agents blocking SARS-CoV-2 viral replication are desperately needed to complement vaccination to end the COVID-19 pandemic. Viral replication and assembly are entirely dependent on two viral cysteine proteases: 3C-like protease (3CLpro) and the papain-like protease (PLpro). PLpro also has deubiquitinase (DUB) activity, removing ubiquitin (Ub) and Ub-like modifications from host proteins, disrupting the host immune response. 3CLpro is inhibited by many known cysteine protease inhibitors, whereas PLpro is a relatively unusual cysteine protease, being resistant to blockade by such inhibitors. A high-throughput screen of biased and unbiased libraries gave a low hit rate, identifying only CPI-169 and the positive control, GRL0617, as inhibitors with good potency (IC50 < 10 lower case Greek μM). Analogues of both inhibitors were designed to develop structure-activity relationships; however, without a co-crystal structure of the CPI-169 series, we focused on GRL0617 as a starting point for structure-based drug design, obtaining several co-crystal structures to guide optimization. A series of novel 2-phenylthiophene-based non-covalent SARS-CoV-2 PLpro inhibitors were obtained, culminating in low nanomolar potency. The high potency and slow inhibitor off-rate were rationalized by newly identified ligand interactions with a 'BL2 groove' that is distal from the active site cysteine. Trapping of the conformationally flexible BL2 loop by these inhibitors blocks binding of viral and host protein substrates; however, until now it has not been demonstrated that this mechanism can induce potent and efficacious antiviral activity. In this study, we report that novel PLpro inhibitors have excellent antiviral efficacy and potency against infectious SARS-CoV-2 replication in cell cultures. Together, our data provide structural insights into the design of potent PLpro inhibitors and the first validation that non-covalent inhibitors of SARS-CoV-2 PLpro can block infection of human cells with low micromolar potency.
• Singh, S. K., Kumar, S., Viswakarma, N., Principe, D. R., Das, S., Sondarva, G., Nair, R. S., Srivastava, P., Sinha, S. C., Grippo, P. J., Thatcher, G. R., Rana, B., & Rana, A. (2021). MAP4K4 promotes pancreatic tumorigenesis via phosphorylation and activation of mixed lineage kinase 3. Oncogene, 40(43), 6153-6165.
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  MAP4K4 is a Ste20 member and reported to play important roles in various pathologies, including in cancer. However, the mechanism by which MAP4K4 promotes pancreatic cancer is not fully understood. It is suggested that MAP4K4 might function as a cancer promoter via specific downstream target(s) in an organ-specific manner. Here we identified MLK3 as a direct downstream target of MAP4K4. The MAP4K4 and MLK3 associates with each other, and MAP4K4 phosphorylates MLK3 on Thr738 and increases MLK3 kinase activity and downstream signaling. The phosphorylation of MLK3 by MAP4K4 promotes pancreatic cancer cell proliferation, migration, and colony formation. Moreover, MAP4K4 is overexpressed in human pancreatic tumors and directly correlates with the disease progression. The MAP4K4-specific pharmacological inhibitor, GNE-495, impedes pancreatic cancer cell growth, migration, induces cell death, and arrests cell cycle progression. Additionally, the GNE-495 reduced the tumor burden and extended survival of the KPC mice with pancreatic cancer. The MAP4K4 inhibitor also reduced MAP4K4 protein expression, tumor stroma, and induced cell death in murine pancreatic tumors. These findings collectively suggest that MLK3 phosphorylation by MAP4K4 promotes pancreatic cancer, and therefore therapies targeting MAP4K4 might alleviate the pancreatic cancer tumor burden in patients.
• Thatcher, G. (2021). Discovery of Nonlipogenic ABCA1 Inducing Compounds with Potential in Alzheimer’s Disease and Type 2 Diabetes. ACS Pharmacology & Translational Science.
• Thatcher, G. (2021). Evidence for distinct mechanisms of small molecule inhibitors of filovirus entry. PLOS Pathogens.
• Thatcher, G. (2021). Extending the Calpain–Cathepsin Hypothesis to the Neurovasculature: Protection of Brain Endothelial Cells and Mice from Neurotrauma. ACS Pharmacology & Translational Science.
• Thatcher, G. (2021). Metabolomic analysis of a selective ABCA1 inducer in obesogenic challenge provides a rationale for therapeutic development. EBioMedicine.
• Thatcher, G. R., Xiong, R., Abderrahman, B., Maximov, P. Y., Curpan, R. F., Fanning, S. W., Hanspal, J. S., Fan, P., Foulds, C. E., Chen, Y., Malovannaya, A., Jain, A., Greene, G. L., Tonetti, D. A., & Jordan, V. C. (2021). Rapid Induction of the Unfolded Protein Response and Apoptosis by Estrogen Mimic TTC-352 for the Treatment of Endocrine-Resistant Breast Cancer. Molecular Cancer Therapeutics, 20(1), 11-25. doi:10.1158/1535-7163.mct-20-0563