Structure-Based Design of Selective RIPK1 and RIPK2 Inhibitors

Kinases regulate various biological functions by post-translational phosphorylation of proteins. Kinase dysfunction is associated with many pathological conditions. Therefore, kinase inhibitors have become an important class of drugs and chemical biology probes for mechanistic studies of diseases. This dissertation focuses on a structural-based design of selective RIPK1 and RIPK2 inhibitors using various approaches. Chapter 1 presents an introduction to protein kinases and a review of kinase inhibitor types for understanding the rationale and design of selective RIPK1 and RIPK2 inhibitors. Chapter 2 describes two design strategies to potent and selective RIPK1 inhibition. The first strategy involves modifications of ponatinib, a type II Abl kinase inhibitor, exploiting differences in the steric and hydrophilic characteristics of Abl and RIPK1 gatekeeper residues. An introduction of tert-butyl on the central phenyl (CS5) caused unfavorable interactions with Abl’s gatekeeper and resulted in significantly improved selectivity for RIPK1 versus Abl/RIPK2/RIPK3. To improve cellular activity, a hybridization strategy linking ponatinib and Nec-1, a type III RIPK1 inhibitor, was pursued. PN10 displayed selectivity for RIPK1 inhibition and better RIPK1 cellular activity than either Nec-1 or ponatinib. In Chapter 3, strategies to develop selective type II RIPK2 inhibitors based on regorafenib, a VEGFR inhibitor, are presented. The first strategy is based on structural differences between RIPK2 and VEGFR around allosteric hydrophobic pocket and gatekeeper residues. The second strategy targeted the activation loop, a region of kinases with diverse amino acid sequences. CSR35 was identified and shown to form an interaction with the activation loop. A RIPK2CSR35 co-crystal structure revealed a resolved activation loop with an ionic interaction between the carboxylic acid installed in CSR35 and Lys169. Chapter 4 presents a hybridization strategy between ALK2 type I inhibitor LDN-214117 and B-Raf type I½ inhibitor PLX4032 as an approach to conformational distinct αC-helix-displacing RIPK2 inhibitors. Potent and selective RIPK2 inhibitors (CSLP43 and CSLP37) were achieved through modifications of the trimethoxyphenyl in LDN-214117 that occupies the hydrophobic pocket in RIPK2 next to the αC-helix. RIPK2inhibitor co-crystal structures of several derivatives suggested that they bind in a type I mode. However, further analysis of CSLP43 and CSLP37 is warranted since they display greater cellular potency.