Identification and Optimization of Small Molecule Binders for ALK2, USP5, and DCAF1
This thesis describes the optimization of small molecule binders targeting three proteins involved in the development of various cancers: Activin receptor-like kinase 2 (ALK2), ubiquitin-specific protease 5 (USP5), and DDB1-cullin4 associated factor 1 (DCAF1). For ALK2, a kinase involved in the development of diffuse intrinsic pontine glioma, we report the design, synthesis, and evaluation of a first-in-class set of 5- to 7-membered ether-linked and 7-membered amine-linked constrained inhibitors of ALK2. We explored novel scaffold modifications using M4K2009 as the lead compound to optimize potency against ALK2 and increase selectivity over important off-targets such as ALK5. Our rigidification strategy led us to generate compounds M4K2308, M4K2281 and M4K2304 which are the most potent inhibitors of ALK2 reported to date (IC50 ≤ 3 nM). Additionally, compounds M4K2304 and M4K2306 exhibited high levels of selectivity for ALK2 over ALK5, surpassing levels exhibited by the reference compound. For USP5, a deubiquitinase overexpressed in most cancers, we conducted structure-activity relationship (SAR) studies to optimize UBTR008295a. Our optimization studies led us to the discovery of compound 22, which exhibited single-digit micromolar affinity for USP5's ZnF-UBD and was >30-fold selective over nine structurally similar ZnF-UBDs. For DCAF1, an E3 ligase involved in protein degradation and a potential drug target for diverse cancers, we report a series of SAR studies to optimize compound ZI391232269. Here we report the discovery of compound 19, a DCAF1 ligand that binds to its WDR domain with a KD of 38 nM and that exhibits cellular target engagement with an EC50 of 10 µM by cellular thermal shift assay. Moreover, we described a second SAR effort to optimize compound 19 with the intent of generating a chemical probe to investigate the biology of DCAF1 in cells. Lastly, we outlined the optimization of an alternative chemical scaffold targeting the WDR domain of DCAF1. This thesis has enabled major advances in developing high quality binders for ALK2, USP5, and DCAF1, providing valuable compounds for investigating the biological roles of these proteins in cancer and offering promising leads for drug development.
ALK2, Cancer Research, DCAF1, Diffuse Intrinsic Pontine Glioma, Drug Discovery, USP5
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