Applications of Covalent Ligand Discovery and Targeted Protein Degradation

Abstract

Small molecules have powerful applications in drug discovery, target validation (or invalidation), and the study of biological mechanisms. However, the potency, selectivity, and cellular target engagement of chemical probes must be sufficiently characterized to meaningfully interrogate biological systems. The use of non-selective and/or poorly annotated small molecules can convolute the scientific literature. Here, we employ covalent inhibition and targeted protein degradation to yield high quality chemical probes for the peptidyl-prolyl isomerase, Pin1, and the tyrosine kinase, Wee1. Pin1 regulates the function and stability of phosphoproteins by catalyzing the cis/trans isomerization of phosphorylated Ser/Thr-Pro motifs. While Pin1 is dispensable for viability in mice, it is required for activated Ras to induce tumorigenesis and is frequently overexpressed in cancer, including pancreatic ductal adenocarcinoma (PDAC). In Chapter 2, we report the rational design of a peptide inhibitor, BJP-06-005-3, that selectively and covalently targets Cys113, a conserved cysteine in the Pin1 active site. In parallel to inhibitor development, we employed genetic and chemical-genetic strategies to evaluate the consequences of Pin1 loss in human PDAC cell lines. We demonstrate that Pin1 overexpression cooperates with mutant KRAS to promote transformation, and that Pin1 loss impairs cell viability in PDAC cells over time. While BJP-06-005-3 is a potent, selective, and cell-penetrant Pin1 inhibitor, it is not suitable for evaluation in vivo. Therefore, in Chapter 3 we present the development and characterization of a second, structurally distinct Pin1 inhibitor series, identified by screening an electrophilic fragment library for covalent binding to Pin1’s Cys113. The resulting lead inhibitor, Pin1-3, is potent, selective, and orally bioavailable in mice. Mirroring the effects of BJP-06-005-3, Pin1-3 only impacts cancer cell viability after prolonged treatments. Despite this mild phenotype in monolayer cell culture, Pin1-3 effectively prevents the development of MYCN-driven neuroblastoma in zebrafish, with no evident toxicity. Chapter 4 describes the synthesis and characterization of the first selective small molecule Wee1 degrader. Wee1 regulates mitotic entry by phosphorylating and inactivating cyclin-dependent kinase 1 (CDK1). Given that the majority of human cancers are characterized by G1/S checkpoint defects, abrogating the G2/M checkpoint by targeting Wee1 can selectively sensitize cancer cells to DNA-damaging agents. Informed by molecular docking, we conjugated the clinical candidate Wee1 inhibitor, AZD1775, to the Cereblon-binding ligand, pomalidomide, to generate ZNL-02-096 as a potent and selective Wee1 degrader. ZNL-02-096 induces premature mitotic entry at 10-fold lower concentrations than AZD1775, and potently synergizes with Olaparib in human ovarian cancer cell lines. Ultimately, this work showcases a well-characterized set of tool compounds that can be used to study Pin1 and Wee1 biology, and to evaluate the therapeutic significance of these targets in human disease.