Lansoprazole inhibits the cysteine protease legumain by binding to the active site

Abstract

Proton pump inhibitors (PPIs) are prodrugs used in the treatment of peptic ulcer diseases. Once activated by acidic pH, the PPIs subsequently inhibit the secretion of gastric acid by covalently forming disulphide bonds with the SH groups of the parietal proton pump, that is the H+/K+‐ATPase. Long‐term use of PPIs has been associated with numerous adverse effects, including bone fractures. Considering the mechanism of activation, PPIs could also be active in acidic micro‐environments such as in lysosomes, tumours and bone resorption sites. We suggested that the SH group in the active site of cysteine proteases could be susceptible for inhibition by PPIs. In this study, the inhibition by lansoprazole was shown on the cysteine proteases legumain and cathepsin B by incubating purified proteases or cell lysates with lansoprazole at different concentrations and pH conditions. The mechanism of legumain inhibition was shown to be a direct interaction of lansoprazole with the SH group in the active site, and thus blocking binding of the legumain‐selective activity‐based probe MP‐L01. Lansoprazole was also shown to inhibit both legumain and cathepsin B in various cell models like HEK293, monoclonal legumain over‐expressing HEK293 cells (M38L) and RAW264.7 macrophages, but not in human bone marrow‐derived skeletal (mesenchymal) stem cells (hBMSC‐TERT). During hBMSC‐TERT differentiation to osteoblasts, lansoprazole inhibited legumain secretion, alkaline phosphatase activity, but had no effects on in vitro mineralization capacity. In conclusion, lansoprazole acts as a direct covalent inhibitor of cysteine proteases via disulphide bonds with the SH group in the protease active site. Such inhibition of cysteine proteases could explain some of the off‐target effects of PPIs.

A cell-based high-throughput screen identifies drugs that cause bleeding disorders by off-targeting the vitamin K cycle

“…Lansoprazole is a proton pump inhibitor that inhibits (H 1 /K 1 )-ATPase by forming disulfide bonds with the sulfhydryl groups of the parietal proton pump. [60][61][62] This inhibition can be reversed by using reducing agents, such as dithiothreitol.…”

The role of cystatin C as a proteasome inhibitor in multiple myeloma

“…In 1999, Sun et al established that human legumain was identical to the clone screened in the cDNA expression library of osteoclast inhibitors [45]. Legumain, also named asparaginyl endopeptidase (AEP) or vacuolar processing enzyme (VPE), significantly inhibits bone resorption and the formation of osteoclasts induced by 1,25-dihydroxyvitamin D 3 (1,25-(OH) 2 D 3 ) and parathyroid hormone-related protein (PTHrP) [46]. Cystatin C is one of the important endogenous cysteine protease inhibitors of papain-like protease, including mammalian legumain [47].…”

Proton pump inhibitors: New mechanisms of action

“…Importantly, the biochemical findings reported by Bosnjak and colleagues may have many clinical implications, particularly for patients with disease conditions. Here, we will briefly explore some aspects related to cardiovascular diseases.…”

“…Given the known interaction of PPIs with cysteines in proton pumps, Bosnjak and colleagues designed a study to examine the possibility that the PPI lansoprazole directly inhibits legumain and cathepsin B, which are cysteine proteases involved in various biological functions. Interestingly, they found that lansoprazole interacts directly with thiol groups of legumain inhibiting its activity.…”

“…Interestingly, they found that lansoprazole interacts directly with thiol groups of legumain inhibiting its activity. They provided strong evidence that lansoprazole inhibits both cysteine proteases legumain and cathepsin B in cell lysates using various cell models . While their study has not included in vivo experiments, their results may have many clinical and functional implications.…”