Small molecule engagement of kinase complexes in cells
Authors:   , Robers Matthew Brian 
Published: November 2024

Drug action is predicated on target engagement in cells and tissues. Historically, pure proteins or protein fragments have been used to query engagement and guide medicinal chemistry efforts toward optimized small molecule inhibitors. However, pure protein assays may fail to comprise the protein complexes and conformations relevant to the disease state. Thus, drug pharmacology observed in a defined, isolated setting may not correlate with pharmacology observed using a biomarker of target activity in the milieu of a living cell. Assessments of target vulnerability are ideally evaluated in live cells, in a microenvironment representing where drug action would occur. Kinases are among the most diverse classes of intracellular enzymes and are implicated in the pathophysiology of numerous diseases. With over 80 FDA approved kinase inhibitors, many kinases are intrinsically vulnerable to enzymatic inhibition via small molecule engagement in the ATP co-substrate binding pocket. Although such vulnerability is readily observed in an isolated setting, achieving selective intracellular engagement without collateral engagement to off-target kinases can be a major challenge. In cells, kinase engagement can be impacted by a number of factors, including ATP competition, multi-protein complexes, target activation state, and drug partitioning. Such factors can be difficult to simulate in a defined system, and methods to interrogate critical binding parameters in cells represents an unmet need. Therefore, intracellular engagement data and selectivity profiles for novel chemical matter is not generally reported in the literature, even for clinical-stage inhibitors. For many kinase inhibitors, cell- free data represents a vast over-estimate of the polypharmacology occurring in a native setting. Thus, providing a more physiologically-accurate assessment of compound selectivity and affinity would enable chemical probe initiatives and pharmaceutical discovery efforts. The RAS/RAF/MEK/ERK pathway (so called MAPK pathway) is among the most highly studied kinase-related pathways in human disease. These MAPK signaling components naturally function in a multi-protein complex (i.e., the RAS signalosome). Dysregulation in this pathway is known driver of tumorigenesis, and mutations in RAS or RAF are observed in >30% of human cancers. For example, RAF kinase is commonly mutated in melanoma, and the GTPase KRAS is the most mutated proto- oncogene in solid tumors. Despite ongoing multidisciplinary drug discovery efforts, this signaling complex has proven undruggable for several decades. As this multiprotein complex cannot currently be reconstituted in a biochemical setting, demonstrating vulnerability at protomers within the RAS/RAF complex has represented a significant challenge. Although potent drug binding or enzyme inhibition can be observed using isolated fragments of RAS or RAF kinase, the measured potencies are generally not commensurate with pathway inhibition in disease-relevant cells. For instance, despite sub-nM potency observed with clinical-stage RAF kinase inhibitors in enzymatic assays, potency of intracellular inhibition of MAPK signaling may be >100-1000-fold weaker. This opens the possibility that underappreciated drug escape mechanisms may be evident in cells that are not observed using purified components. Such observations cast doubt on the assumed mechanism of action of such clinical-stage drugs. Thus, new methods are needed to quantify drug engagement at these critical signaling components to support more accurate assessments of drug vulnerability. Herein are reported advancements in the Bioluminescence Resonance Energy Transfer (BRET) method to accurately query target engagement at monomeric and multiprotein complexes in cells. The prototype target engagement method leveraged an intracellular reporter system comprising NanoLuc (NLuc) tagged proteins in a BRET complex with cell-permeable fluorescent drug tracers. Engagement of unmodified compounds results in competitive displacement of the BRET complex in live cells. When the BRET tracers are applied at concentrations approximating their apparent affinity, the resulting IC50 of the test compound is within 2-fold of a constant value. Among all reported target engagement methods, BRET is the only method that offers quantitation in live cells. This quantitative accuracy allows for assessments of selectivity within target families. Furthermore, since the method operates with intact live cells, both affinity and binding kinetics can be measured. Using broad-spectrum BRET tracers, entire target families can be evaluated in a single experiment. Herein, a novel workflow is described, to enable improved broad-spectrum kinome profiling in live cells against nearly 200 full length protein kinases.