Optimization of Receptor-Interacting Protein Kinase 2 (RIPK2) Inhibitors and Development of Mixed Lineage Kinase Domain-Like (MLKL) Activators

Protein kinases and pseudokinases play pivotal roles in complex cellular signaling and regulatory pathways. Protein kinases’ catalytic activity is critical for controlling a myriad of cellular events and functions. Despite a lack of catalytic activity, pseudokinases have merged as nuanced modulators, scaffolds, and molecular switches in diverse signaling pathways. Chapter one presents an introduction to kinases and pseudokinases, delving deeper into their structural features, functions, and their role in disease pathways. Chapter two focuses on receptor-interacting protein kinase 2 (RIPK2) as a modulator of innate and adaptive immune responses and explores the therapeutic potential of its recently developed inhibitor, termed CSLP series. This chapter addresses the challenges of developing a potent and selective RIPK2 inhibitor for targeting inflammatory conditions. Implementing structure-activity relationship studies, the lead inhibitor CSLP37 was modified to increase its brain permeability and cellular potency for developing a probe to investigate neuroinflammatory conditions such as multiple sclerosis. Several areas of the structure were altered, aiming to reduce molecular weight, total polar surface area, and hydrogen bond donors. Enhancing brain permeability proved challenging, and despite many attempts, dramatic changes in the inhibitor’s scaffold resulted in loss of cellular activity. Nonetheless, our exploration led to the development of an inhibitor with enhanced in vivo characteristics whilst maintaining cellular potency. Moreover, we designed the first RIPK2 inhibitor with efficacy in the experimental encephalomyelitis model of multiple sclerosis. Lastly, chapter three describes the development of the first small molecule activator of mixed lineage kinase domain-like (MLKL) pseudokinase as the final effector of the necroptosis cell death pathway. Previously, our lab discovered compound UH15-22 capable of binding MLKL and inducing necroptosis. However, the compound displayed off-target activity and low binding affinity. Therefore, we aimed to modify UH15-22 to enhance its selectivity and binding affinity for MLKL. Utilizing structure-activity relationship studies, three areas of the compound were altered to increase the interaction of the activator with MLKL, thereby improving potency and binding affinity. However, improved binding affinity did not necessarily translate to improved necroptotic activity, thus making it challenging to find a balance between the two issues. So far, we have successfully developed an MLKL activator with improved necroptotic activity and removed the off-target activity. Even though the binding affinity has improved by three-fold, there is still a need for further improvement to have a therapeutic effect.