Inhibition of the
serine protease enteropeptidase (EP) opens a new avenue to the discovery of chemotherapeutics for the treatment of
metabolic diseases.
Camostat has been used clinically for treating
chronic pancreatitis in Japan; however, the mechanistic basis of the observed clinical efficacy has not been fully elucidated. We demonstrate that
camostat is a potent reversible covalent inhibitor of EP, with an inhibition potency (k inact/KI) of 1.5 × 104 M-1s-1 High-resolution liquid chromatography-mass spectrometry (LC-MS) showed addition of 161.6 Da to EP after the reaction with
camostat, consistent with insertion of the carboxyphenylguanidine moiety of
camostat. Covalent inhibition of EP by
camostat is reversible, with an
enzyme reactivation half-life of 14.3 hours. Formation of a covalent adduct was further supported by a crystal structure resolved to 2.19 Å, showing modification of the catalytic
serine of EP by a close analog of
camostat, leading to formation of the carboxyphenylguanidine acyl
enzyme identical to that expected for the reaction with
camostat. Of particular note, minor structural modifications of
camostat led to changes in the mechanism of inhibition. We observed from other studies that sustained inhibition of EP is required to effect a reduction in cumulative food intake and
body weight, with concomitant improved
blood glucose levels in obese and diabetic
leptin-deficient mice. Thus, the structure-activity relationship needs to be driven by not only the inhibition potency but also the mechanistic and kinetic characterization. Our findings support EP as a target for the treatment of
metabolic diseases and demonstrate that reversible covalent EP inhibitors show clinically relevant efficacy. SIGNIFICANCE STATEMENT: Interest in targeted covalent drugs has expanded in recent years, particularly so for
kinase targets, but also more broadly. This study demonstrates that reversible covalent inhibition of the
serine protease enteropeptidase is a therapeutically viable approach to the treatment of
metabolic diseases and that mechanistic details of inhibition are relevant to clinical efficacy. Our mechanistic and kinetic studies outline a framework for detailed inhibitor characterization that is proving essential in guiding discovery efforts in this area.