Allosteric Tuning of Caspase-7: Establishing the Nexus of Structure and Catalytic Power.

Caspase-7 (C7), a cysteine protease involved in apoptosis, is a valuable drug target for its role in human diseases (e. g., Parkinson's, Alzheimer's, sepsis). The C7 allosteric site has great potential for small-molecule targeting, but numerous drug discovery efforts have identified precious few allosteric inhibitors. Here we present the first selective, drug-like inhibitor of C7 along with several other improved inhibitors based on our previous fragment hit. We also provide a rational basis for the impact of allosteric binding on the C7 catalytic cycle by using an integrated approach including X-ray crystallography, stopped-flow kinetics, and molecular dynamics simulations. Our findings suggest allosteric binding disrupts C7 pre-acylation by neutralization of the catalytic dyad, displacement of substrate from the oxyanion hole, and altered dynamics of substrate binding loops. This work advances drug targeting efforts and bolsters our understanding of allosteric structure-activity relationships (ASARs).

Elucidating the Catalytic Power of Glutamate Racemase by Investigating a Series of Covalent Inhibitors.

The application of covalent inhibitors has experienced a renaissance within drug discovery programs in the last decade. To leverage the superior potency and drug target residence time of covalent inhibitors, there have been extensive efforts to develop highly specific covalent modifications to decrease off-target liabilities. Herein, we present a series of covalent inhibitors of an antimicrobial drug target, glutamate racemase, discovered through structure-based virtual screening. A combination of enzyme kinetics, mass spectrometry, and surface-plasmon resonance experiments details a highly specific 1,4-conjugate addition of a small-molecule inhibitor with a catalytic cysteine of glutamate racemase. Molecular dynamics simulations and quantum mechanics-molecular mechanics geometry optimizations reveal the chemistry of the conjugate addition. Two compounds from this series of inhibitors display antimicrobial potency similar to ß-lactam antibiotics, with significant activity against methicillin-resistant S. aureus strains. This study elucidates a detailed chemical rationale for covalent inhibition and provides a platform for the development of antimicrobials with a novel mechanism of action against a target in the cell wall biosynthesis pathway.