Protease-activated receptor 2 exacerbates cisplatin-induced nephrotoxicity

Protease-activated receptors (PARs) are a family of G-protein-coupled receptors (GPCRs) that are irreversibly activated by proteolytic cleavage of the N terminus, which unmasks a tethered peptide ligand that binds and activates the transmembrane receptor domain, eliciting a cellular cascade in response to inflammatory signals and other stimuli. PARs are implicated in a wide range of diseases, such as cancer and inflammation. PARs have been the subject of major pharmaceutical research efforts but the discovery of small-molecule antagonists that effectively bind them has proved challenging. The only marketed drug targeting a PAR is vorapaxar, a selective antagonist of PAR1 used to prevent thrombosis. The structure of PAR1 in complex with vorapaxar has been reported previously. Despite sequence homology across the PAR isoforms, discovery of PAR2 antagonists has been less successful, although GB88 has been described as a weak antagonist. Here we report crystal structures of PAR2 in complex with two distinct antagonists and a blocking antibody. The antagonist AZ8838 binds in a fully occluded pocket near the extracellular surface. Functional and binding studies reveal that AZ8838 exhibits slow binding kinetics, which is an attractive feature for a PAR2 antagonist competing against a tethered ligand. Antagonist AZ3451 binds to a remote allosteric site outside the helical bundle. We propose that antagonist binding prevents structural rearrangements required for receptor activation and signalling. We also show that a blocking antibody antigen-binding fragment binds to the extracellular surface of PAR2, preventing access of the tethered ligand to the peptide-binding site. These structures provide a basis for the development of selective PAR2 antagonists for a range of therapeutic uses.

Nephrotoxicity induced by cisplatin involves tubular cell necrosis and apoptosis; the latter of which may be initiated by multiple mechanisms including activation of the intrinsic mitochondrial pathway. In cultured tubular epithelial cells, cisplatin can activate the proapoptotic protein Bax resulting in cytochrome c release, caspase activation, and apoptosis. Definitive evidence for the involvement of Bax in cisplatin nephrotoxicity in vivo, however, is lacking. We analyzed Bax regulation during cisplatin nephrotoxicity in wild-type mice and determined the pathological role of Bax using mice in which this gene was knocked out. In wild-type mice, cisplatin induced Bax in renal tubular cells which became active, accumulated in the mitochondria, and was accompanied by acute kidney injury. Compared with the wild-type mice renal function, as measured by blood urea nitrogen and serum creatinine, was partially but significantly preserved in Bax knockout mice. The number of apoptotic cells was decreased as was general tissue damage. Additionally, cisplatin-induced cytochrome c release was attenuated in the Bax-deficient mice. This significant decrease in apoptosis and in cytochrome c release was also mirrored in primary cultures of proximal tubular cells prepared from Bax knockout animals. Collectively, our results provide compelling evidence for a role of Bax and its related apoptotic pathway in cisplatin nephrotoxicity.