Protease inhibitor development and substrate specificity profiling

Parag Pinakin Shah, University of Pennsylvania


Through an extensive high-throughput screening campaign, a rigorous chemistry program, and molecular modeling, we have developed a novel small molecule inhibitor for the cysteine protease human cathepsin L that exhibits strong potencies against Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) and Ebola virus pseudotype infections. Following a 57,821 compound high-throughput screen of the NIH Molecular Libraries Small Molecule Repository, a unique thiocarbazate chemotype for cathepsin L inhibition was identified through chemical synthesis and structure elucidation. Kinetic analysis of this compound, PubChem substance identifier (SID) 26681509, reveals it is a competitive, slow-binding, slowly reversible, and selective inhibitor of human cathepsin L with Ki = 890 pM. Synthesis of analogs to this thiocarbazate probe established structure-activity relationships that resulted in synthesis of SID 46493575. SID 46493575 exhibits similar slow-binding inhibition behavior as its precursor with Ki = 150 pM. In SID 46493575, the thiocarbazate sulfur of SID 26681509 was replaced by oxygen and the hydrophobicity of the P1' substructure was improved from 2-ethylphenyl anilide to tetrahydroquinoline anilide. Molecular docking studies demonstrate both compounds form hydrophobic interactions within the S2 and S1' subsites as well as hydrogen bonding interactions with five key residues (Gln19, Cys25, Gly66, Asp158, and Trp177) in the active site of papain. SID 46493575 forms an additional hydrogen bond with His159, which could explain its increased biological potency. Both molecules were non-toxic to human aortic endothelial cells and zebrafish. Tetrahydroquinoline probe SID 46493575 prevented SARS-CoV pseudotype entry with IC50 = 273 nM and Ebola virus pseudotype entry with IC50 = 193 nM. These results are extremely encouraging and open the door for testing against live SARS-CoV and Ebola virus in murine models of the disease. Following a 63,332 compound high-throughput screen, we report the characterization of substituted pyrazole esters as alternate substrates, rather than inhibitors, for human cathepsin B. Dithiothreitol and cysteine used in assay buffers confounded results due to their nucleophilic potential against substrates containing electrophilic functionality. Additionally, substrate specificity and kinetics of four cathepsins and three mannan-binding lectin associated serine proteases were investigated using a 361-member fluorogenic peptide library to identify preferred P2 and P3 substrate residues.

Subject Area

Pharmacology|Pharmacy sciences|Chemical engineering|Bioinformatics

Recommended Citation

Shah, Parag Pinakin, "Protease inhibitor development and substrate specificity profiling" (2008). Dissertations available from ProQuest. AAI3309506.