Development of Peptide Inhibitors of Cancer Cell Signaling Targets, STAT3 and LC3B
Cerulli, Robert
2020
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Despite decades of research and considerable advancements in the field of novel cancer therapeutics, there remains a high demand for new strategies and new molecules to combat aggressive, chemoresistant malignancies. Two cell signaling pathways that have been studied for several decades and have been the subject of inhibitor development, are STAT3 signaling and the autophagy pathway. There is ... read moreconsiderable evidence to suggest that both pathways contribute to cancer progression and chemoresistance, yet to date, neither pathway has been granted an FDA-approved inhibitor for use in the treatment of cancer. As such, the manuscripts of this work describe our efforts to develop peptide-based inhibitors of these pathways. For STAT3, these efforts consist of peptides targeting the Src Homology 2 (SH2) domain, conjugated to highly cytosolically efficient cell-penetrating peptides (CPPs). We studied the binding affinity of STAT3-targeted peptides incorporating hydrolytically stable phosphotyrosine (pTyr) analogs. Further, we studied the cell penetration of these CPP-conjugated peptides, observing impressive cell penetration for CPP-conjugated anionic peptides. We also studied serum and lysate stability of CPP-conjugated peptides, noting not only resistance to dephosphorylation but additional resistance to proteolytic degradation with use of pTyr isosteres. This work provides several insights into the balance of affinity, stability, and cell penetration of SH2-targeted inhibitor development. The autophagy-aimed efforts of this work focused on peptide inhibitors of the central autophagy protein, LC3B. In this work, we focused largely on structure-activity relationship efforts, studying the binding of the LC3-Interacting Region (LIR) motif of the adapter protein FYCO1. We studied the FYCO1 LIR motif binding to LC3B, determining the contributions to affinity of residues in the N- and C-termini, as well as the seven negatively charged residues, and the hydrophobic pocket-targeted residues. Further we incorporated numerous artificial amino acids and ultimately perform diversity-oriented peptide stapling to generate optimized LC3B inhibitors with up to 2.4-fold improvement in binding affinity while maintaining LC3B paralog selectivity and improving lysate stability by about 2-fold. Both our STAT3- and LC3B-aimed efforts provide useful insights into the development of selective inhibitors of these cancer-relevant protein targets.
Thesis (Ph.D.)--Tufts University, 2020.
Submitted to the Dept. of Cell, Molecular & Developmental Biology.
Advisor: Joshua Kritzer.
Committee: James Baleja, Philip Hinds, Rebecca Scheck, and Loren Walensky.
Keyword: Molecular biology.read less - ID:
- 6q1830966
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