Allosteric Inhibition of the Neuropeptidase Neurolysin

Christina S Hines,Kallol Ray,Jack J Schmidt,Fei Xiong,Rolf W Feenstra,Mia Pras-Raves,Jan Peter De Moes,Jos H.M Lange,Manana Melikishvili,Michael G Fried,Paul Mortenson,Michael Charlton,Yogendra Patel,Stephen M Courtney,Chris G Kruse,David W Rodgers

doi:10.1074/jbc.m114.620930

Abstract

Neuropeptidases specialize in the hydrolysis of the small bioactive peptides that play a variety of signaling roles in the nervous and endocrine systems. One neuropeptidase, neurolysin, helps control the levels of the dopaminergic circuit modulator neurotensin and is a member of a fold group that includes the antihypertensive target angiotensin converting enzyme. We report the discovery of a potent inhibitor that, unexpectedly, binds away from the enzyme catalytic site. The location of the bound inhibitor suggests it disrupts activity by preventing a hinge-like motion associated with substrate binding and catalysis. In support of this model, the inhibition kinetics are mixed, with both noncompetitive and competitive components, and fluorescence polarization shows directly that the inhibitor reverses a substrate-associated conformational change. This new type of inhibition may have widespread utility in targeting neuropeptidases.

Highlights

We report here the characterization of an inhibitor of neurolysin that binds remotely from the catalytic site and reduces enzyme activity by an allosteric mechanism
Because R2 binds near the predicted hinge axis for this motion, we considered the possibility that it inhibits neurolysin and thimet oligopeptidase (TOP) by locking the enzyme in the open, inactive conformation
We have identified and characterized an unusual inhibitor of the metallopeptidase neurolysin that binds remotely from the catalytic site

Summary

Background

A key function of ACE and other members of the specialized group of metallopeptidases termed neuropeptidases is to regulate and metabolize the hundreds of known bioactive peptides that act as signaling molecules in the central nervous system and periphery [5] These enzymes are restricted to oligopeptide substrates, and they can be either exo- or endopeptidases. Several relatively selective inhibitors of neurolysin and TOP, such as phosphodiepryl 08, have been developed (29 –31) These compounds, like most known metallopeptidase inhibitors, bind at the catalytic site and coordinate the zinc ion cofactor. We report here the characterization of an inhibitor of neurolysin that binds remotely from the catalytic site and reduces enzyme activity by an allosteric mechanism This and related compounds represent a promising new class of inhibitors that should prove useful with other members of this zinc metallopeptidase group

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION