Expanding the Arsenal: Development of G-Protein Receptor Kinase 5 Inhibitors Utilizing a Covalent Strategy

Abstract

Many cellular events are regulated by G-protein coupled receptors (GPCRs). In order to maintain homeostasis, GPCR kinases (GRKs) selectively recognize and phosphorylate activated GPCRs for internalization. There is growing evidence that both GRK2 and GRK5 have roles in pathological heart failure. Previous work has demonstrated that targeting GRK2 can reduce the amount of receptor desensitization that occurs in cardiomyocytes and increase cardiac output. However, the close homologue GRK5 is equally well expressed in cardiac tissue. Herein, we describe our efforts toward developing selective GRK5 inhibitors using covalent engagement of a non-conserved cysteine to elucidate the role of GRK5 in cardiovascular diseases. Initially, we had identified a lead compound with a pyrrolopyrimidine scaffold (GSK2163632) from the GSK Published Kinase Inhibitor Set that had modest potency for GRK5 and GRK2. Due to the similarity of the hinge region and binding pockets of GRK2 and GRK5, an approach towards building out GRK2 activity had to be based upon non-conserved residues such as Cys474. We used this approach to build covalent, and thereby selective, GRK5 inhibitors. Our initial designs allowed us to identify one strongly covalent inhibitor based on the pyrrolopyrimidine scaffold (CCG-265328) which features a weak electrophile and has 90-fold selectivity for GRK5 over GRK2. Previously, engagement of Cys474 was confirmed through tandem MS/MS, suggesting that this covalent interaction is driving selectivity. Structure-activity relationships (SAR) also revealed that linker length and degrees of freedom had less of an effect on covalent engagement than the reactivity of the electrophilic warhead. Additionally, SAR revealed that without a warhead, the modified pyrrolopyrimidine scaffold (CCG-264561) had only modest potency. We tried to improve potency through exploration of two separate scaffolds mined from a virtual screen and the literature respectively. From a virtual screen an aminopyridine compound (Chembl-1607632A) was independently synthesized and a few analogues were created. We discovered that this scaffold was unviable due to both poor potency (> 1 µM) and untransferable SAR. However, the indolinone scaffold derived from the literature proved a much more tractable option, with low nanomolar potency for the independently synthesized reference compound (CCG-271421). Interestingly, the warhead SAR developed in the pyrrolopyrimidine scaffold proved to be intractable. Indeed, the most reactive warheads, the haloketones (CCG-273220, CCG-273463) were considerably more potent and selective than the pyrrolopyrimidines (CCG-265328). In fact, the haloketones (CCG-273441) proved to be the most potent and selective covalent GRK5 inhibitors (IC50 = 3.8 nM, 1300-fold selective over GRK2) to date. Thus, we have demonstrated through development of the pyrrolopyrimidine series (CCG-265328) that Cys474 can serve as a covalent handle to convey high levels of selectivity through covalent engagement with a warhead and that this covalent strategy is transferable to different scaffolds known in kinase drug discovery, provided these scaffolds have sufficient inherent potency.