Inhibitor Fingerprinting of Matrix Metalloproteases Using a Combinatorial Peptide Hydroxamate Library

We report the inhibitor fingerprints of seven matrix metalloproteases, representing all five
established families of this important class of enzymes, against a highly diversified small-molecule library.
A total of 1400 peptide hydroxamates were individually prepared by systematically permuting both natural
and unnatural amino acids across the P1‘, P2‘, and P3‘ positions, thereby generating an inhibitor library
with three-pronged structural diversity. High-throughput screenings were efficiently conducted in microtiter
plate format, providing a rapid and quantitative determination of inhibitor potency across the panel of
enzymes. Despite similarities in substrate preferences and structural homologies within this class of enzymes,
our findings revealed distinct patterns of inhibition for each MMP against varied chemical scaffolds. The
resulting inhibitor fingerprints readily facilitated the identification of inhibitors with good potency as well as
desirable selectivity, potentially minimizing adverse effects when developing such leads into candidate
drugs. The strategy also offers a novel method for the functional classification of matrix metalloproteases,
on the basis of the characteristic profiles obtained using the diverse set of inhibitors. This approach thus
paves the way forward in lead identification by providing a rapid and quantitative method for selectivity
screening at the outset of the drug discovery process.