ABSTRACT
Protein kinases are known for their highly conserved adenosine triphosphate (ATP)-binding site, rendering the discovery of selective inhibitors a major challenge. In theory, allosteric inhibitors can achieve high selectivity by targeting less conserved regions of the kinases, often with an added benefit of retaining efficacy under high physiological ATP concentration. Although often overlooked in favor of ATP-site directed approaches, performing a screen at high ATP concentration or stringent hit triaging with high ATP concentration offers conceptually simple methods of identifying inhibitors that bind outside the ATP pocket. Here, we applied the latter approach to the With-No-Lysine (K) (WNK) kinases to discover lead molecules for a next-generation antihypertensive that requires a stringent safety profile. This strategy yielded several ATP noncompetitive WNK1-4 kinase inhibitors, the optimization of which enabled cocrystallization with WNK1, revealing an allosteric binding mode consistent with the observed exquisite specificity for WNK1-4 kinases. The optimized compound inhibited rubidium uptake by sodium chloride cotransporter 1 (NKCC1) in HT29 cells, consistent with the reported physiology of WNK kinases in renal electrolyte handling.
Subject(s)
Allosteric Regulation/drug effects , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Adenosine Triphosphate/metabolism , Drug Discovery , HEK293 Cells , HT29 Cells , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/metabolism , Minor Histocompatibility Antigens/chemistry , Minor Histocompatibility Antigens/metabolism , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/metabolism , Solute Carrier Family 12, Member 2/metabolism , WNK Lysine-Deficient Protein Kinase 1ABSTRACT
Translation initiation is a fine-tuned process that plays a critical role in tumorigenesis. The use of small molecules that modulate mRNA translation provides tool compounds to explore the mechanism of translational initiation and to further validate protein synthesis as a potential pharmaceutical target for cancer therapeutics. This report describes the development and use of a click beetle, dual luciferase cell-based assay multiplexed with a measure of compound toxicity using resazurin to evaluate the differential effect of natural products on cap-dependent or internal ribosome entry site (IRES)-mediated translation initiation and cell viability. This screen identified a series of cardiac glycosides as inhibitors of IRES-mediated translation using, in particular, the oncogene mRNA c-Myc IRES. Treatment of c-Myc-dependent cancer cells with these compounds showed a decrease in c-Myc protein associated with a significant modulation of cell viability. These findings suggest that inhibition of IRES-mediated translation initiation may be a strategy to inhibit c-Myc-driven tumorigenesis.
