Timothy Marlowe

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Journals/Publications

• Feng, A., Caro, Y. M., Gardner, C., Grischo, G., Liang, Y., Wickremasinghe, P. D., Polmann, M., Kala, M., Marlowe, T., Black, S. M., Knox, K. S., & Wang, T. (2023). PTK2-associated gene signature could predict the prognosis of IPF. Respiratory research, 24(1), 304.
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  Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease with a poor prognosis. Current/available clinical prediction tools have limited sensitivity and accuracy when evaluating clinical outcomes of IPF. Research has shown that focal adhesion kinase (FAK), produced by the protein tyrosine kinase 2 (PTK2) gene, is crucial in IPF development. FAK activation is a characteristic of lesional fibroblasts; Thus, FAK may be a valuable therapeutic target or prognostic biomarker for IPF. This study aimed to create a gene signature based on PTK2-associated genes and microarray data from blood cells to predict disease prognosis in patients with IPF. PTK2 levels were found to be higher in lung tissues of IPF patients compared to healthy controls, and PTK2 inhibitor Defactinib was found to reduce TGFβ-induced FAK activation and increase α-smooth muscle actin. Although the blood PTK2 levels were higher in IPF patients, blood PTK level alone could not predict IPF prognosis. From 196 PTK2-associated genes, 11 genes were prioritized to create a gene signature (PTK2 molecular signature) and a risk score system using univariate and multivariate Cox regression analysis. Patients were divided into high-risk and low-risk groups using PTK2 molecular signature. Patients in the high-risk group experienced decreased survival rates compared to patients in the low-risk group across all discovery and validation cohorts. Further functional enrichment and immune cell proportion analyses revealed that the PTK2 molecular signature strongly reflected the activation levels of immune pathways and immune cells. These findings suggested that PTK2 is a molecular target of IPF and the PTK2 molecular signature is an effective IPF prognostic biomarker.
• Stahl, E., Nott, R., Koessel, K., Cance, W., & Marlowe, T. (2020). Computational-based discovery of FAK FERM domain chemical probes that inhibit HER2-FAK cancer signaling. Chemical biology & drug design, 95(6), 584-599.
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  The N-terminal FERM domain of focal adhesion kinase (FAK) contributes to FAK scaffolding and interacts with HER2, an oncogene and receptor tyrosine kinase. The interaction between HER2 and FAK drives resistance to FAK-kinase domain inhibitors through FAK Y397 transphosphorylation and FAK re-activation upon inhibition. As such, FAK FERM remains an attractive drug discovery target. In this report, we detail an alternative approach to targeting FAK through virtual screening-based discovery of chemical probes that target FAK FERM. We validated the binding interface between HER2 and FAK using site-directed mutagenesis and GST pull-down experiments. We assessed the ligandability of key-binding residues of HER2 and FAK utilizing computational tools. We developed a virtual screening method to screen ~200,000 compounds against the FAK FERM domain, identifying 20 virtual chemical probes. We performed GST pull-down screening on these compounds, discovering two hits, VS4 and VS14, with nanomolar IC s in disrupting HER2-FAK. We performed further testing, including molecular docking, immunofluorescence, phosphorylation, and cellular invasion assays to evaluate the compounds' biological effects. One probe, VS14, was identified with the ability to block both auto- and transphosphorylation of Y397. In all, these studies identify two new probes that target FAK FERM, enabling future investigation of this domain.
• Alvarado, C., Stahl, E., Koessel, K., Rivera, A., Cherry, B. R., Pulavarti, S. V., Szyperski, T., Cance, W., & Marlowe, T. (2019). Development of a Fragment-Based Screening Assay for the Focal Adhesion Targeting Domain Using SPR and NMR. Molecules, 24(18).
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  The Focal Adhesion Targeting (FAT) domain of Focal Adhesion Kinase (FAK) is a promising drug target since FAK is overexpressed in many malignancies and promotes cancer cell metastasis. The FAT domain serves as a scaffolding protein, and its interaction with the protein paxillin localizes FAK to focal adhesions. Various studies have highlighted the importance of FAT-paxillin binding in tumor growth, cell invasion, and metastasis. Targeting this interaction through high-throughput screening (HTS) provides a challenge due to the large and complex binding interface. In this report, we describe a novel approach to targeting FAT through fragment-based drug discovery (FBDD). We developed two fragment-based screening assays-a primary SPR assay and a secondary heteronuclear single quantum coherence nuclear magnetic resonance (HSQC-NMR) assay. For SPR, we designed an AviTag construct, optimized SPR buffer conditions, and created mutant controls. For NMR, resonance backbone assignments of the human FAT domain were obtained for the HSQC assay. A 189-compound fragment library from Enamine was screened through our primary SPR assay to demonstrate the feasibility of a FAT-FBDD pipeline, with 19 initial hit compounds. A final total of 11 validated hits were identified after secondary screening on NMR. This screening pipeline is the first FBDD screen of the FAT domain reported and represents a valid method for further drug discovery efforts on this difficult target.
• Marlowe, T., Alvarado, C., Rivera, A., Lenzo, F., Nott, R., Bondugji, D., Montoya, J., Hurley, A., Kaplan, M., Capaldi, A., & Cance, W. (2019). Development of a High-Throughput Fluorescence Polarization Assay to Detect Inhibitors of the FAK-Paxillin Interaction. SLAS discovery : advancing life sciences R & D, 25(1), 21-32.
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  Focal adhesion kinase (FAK) is a promising cancer drug target due to its massive overexpression in multiple solid tumors and its critical role in the integration of signals that control proliferation, invasion, apoptosis, and metastasis. Previous FAK drug discovery and high-throughput screening have exclusively focused on the identification of inhibitors that target the kinase domain of FAK. Because FAK is both a kinase and scaffolding protein, the development of novel screening assays that detect inhibitors of FAK protein-protein interactions remains a critical need. In this report, we describe the development of a high-throughput fluorescence polarization (FP) screening assay that measures the interactions between FAK and paxillin, a focal adhesion-associated protein. We designed a tetramethylrhodamine (TAMRA)-tagged paxillin peptide based on the paxillin LD2 motif that binds to the focal adhesion targeting (FAT) domain with significant dynamic range, specificity, variability, stability, and a Z'-factor suitable for high-throughput screening. In addition, we performed a pilot screen of 1593 compounds using this FP assay, showing its feasibility for high-throughput drug screening. Finally, we identified three compounds that show dose-dependent competition of FAT-paxillin binding. This assay represents the first described high-throughput screening assay for FAK scaffold inhibitors and can accelerate drug discovery efforts for this promising drug target.
• Marlowe, T., Dementiev, A., Figel, S., Rivera, A., Flavin, M., & Cance, W. (2019). High resolution crystal structure of the FAK FERM domain reveals new insights on the Druggability of tyrosine 397 and the Src SH3 binding site. BMC molecular and cell biology, 20(1), 10.
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  Focal Adhesion Kinase (FAK) is a major cancer drug target that is involved in numerous aspects of tumor progression and survival. While multiple research groups have developed ATP-competitive small molecule inhibitors that target the kinase enzyme, recent attention has been focused on the FAK FERM (Band 4.1, Ezrin, Radixin, Moesin) domain that contains key residue Y397 and contributes to many protein-protein interactions. Previous x-ray crystal structures of the FAK FERM domain gave conflicting results on the structure of the Y397 region and therefore the overall druggability.
• Martelly, W., Fellows, B., Senior, K., Marlowe, T., & Sharma, S. (2019). Identification of a noncanonical RNA binding domain in the U2 snRNP protein SF3A1. RNA (New York, N.Y.), 25(11), 1509-1521.
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  During splicing of pre-mRNA, 5' and 3' splice sites are brought within proximity by interactions between the pre-mRNA bound U1 and U2 snRNPs, followed by recruitment of the tri-snRNP for assembly of the mature spliceosome. Previously, we identified an interaction between the U2 snRNP-specific protein SF3A1 and the stem-loop 4 (SL4) of the U1 snRNA that occurs during the early steps of spliceosome assembly. Although harboring many annotated domains, SF3A1 lacks a canonical RNA binding domain. To identify the U1-SL4 binding region in SF3A1, we expressed amino- and carboxy-terminal deletion constructs using a HeLa cell-based cell free expression system. UV-crosslinking of the truncated proteins with P-U1-SL4 RNA identified the carboxy-terminal ubiquitin-like (UBL) domain of SF3A1 as the RNA binding region. Characterization of the interaction between SF3A1-UBL and U1-SL4 by electrophoretic mobility shift assay and surface plasmon resonance determined high binding affinity ( = ∼97 nM), and revealed the double-stranded G-C rich stem of U1-SL4 as an important feature for binding to the UBL domain. Further, mutations of two conserved tyrosine residues, Y772 and Y773, were found to cause a two- and fivefold decrease in the binding affinity for U1-SL4, respectively. Finally, we found that SF3A1-UBL can specifically pull down the U1 snRNP from HeLa nuclear extract, demonstrating its capacity to bind U1-SL4 in the context of the intact snRNP. Thus, the data show that the UBL domain of SF3A1 can function as an RNA binding domain and that mutations in this region may interfere with U1-SL4 binding.

Proceedings Publications

• Marlowe, T. (2022). FN-2012, a Small Peptide Inhibitor of the Focal Adhesion Kinase (FAK)-Paxillin Interaction Attenuates Hepatic Stellate Cell Activation and Liver Fibrosis. In American Association for the Study of Liver Diseases.
• Marlowe, T. (2022). Novel Non-Catalytic FAK Inhibition Downregulates Lung Fibroblast Matrix Production. In American Thoracic Society.
• Marlowe, T. (2022). Structure-Based Discovery Stapled Peptide PPI Inhibitors of Focal Adhesion Kinase (FAK) Scaffolding with a Novel Mechanism of Action and Murine Anticancer Efficacy. In American Chemical Society.
• Marlowe, T. (2020, Jan). Abstract 1032-C: Development of screening platforms to target the “undruggable” focal adhesion targeting (FAT) domain. In Society for Laboratory Automation & Screening (SLAS) 2020 Annual Conference.

Presentations

• Marlowe, T. (2022). Development of Peptidic Inhibitors of Focal Adhesion Kinase (FAK) for Use in Melanoma and Beyond. Center Cancer Biology Graduate Interdisciplinary Program (GIDP).
• Marlowe, T. (2022). TLA AD Project: Novel Peptidic Inhibitors of Focal Adhesion Kinase (FAK). Tech Launch Arizona Asset Development Event.
• Marlowe, T. (2022). Translating Therapeutics against Focal Adhesion Kinase (FAK). UA Cancer Center Annual Retreat.
• Marlowe, T. (2022). Cancer Drug Discovery Research. Sun City Community Presentation.

Poster Presentations

• Marlowe, T. (2022). Drug Discovery Capabilities of the Molecular Discovery Core (MDC). Arizona Center for Drug Discovery Summit.
• Marlowe, T. (2021). Analyzing the sensitivity of FAK-FAT domain inhibition in NRAS and BRAF mutant melanoma. UACC Scientific Retreat. Tucson, AZ.
• Marlowe, T. (2021). Discovery of Novel FAK-FAT Domain Inhibitor and its Evaluation in Melanoma. ABRC-Flinn Research Conference.
• Marlowe, T. (2021). The key role of Focal Adhesion Kinase in Idiopathic Pulmonary Fibrosis. Re-Imagine Health Research Symposium.
• Marlowe, T. (2020, Feb). Therapeutic use of FAK inhibition against KRAS-driven lung cancer. 1. 5th Annual Arizona Biomedical Research Centre-Flinn Foundation Research Conference.. Phoenix, AZ: ABRC-Flinn.
• Marlowe, T. (2020, Nov). Discovery of high potency FAK FAT domain inhibitors for cancer therapy. UACC Annual Scientific Retreat. Tucson, AZ: UA Cancer Center.
• Nott, R., Buchberger, A., Rivera, A., Weiner, W., Stephanopoulos, N., Marlowe, T., & Cance, W. (2019, March 2019). Synthesis and Characterization of Stapled LD2 Peptide Analogs to Inhibit Focal Adhesion Kinase in Cancer. ASU SOLUR Research Symposium. ASU.