Michael C. Myers

HTS Identifies Novel and Specific Uncompetitive Inhibitors of the Two-Component NS2B-NS3 Proteinase of West Nile Virus

West Nile virus (WNV), a member of the Flavividae family, is a mosquito-borne, emerging pathogen. In addition to WNV, the family includes dengue, yellow fever, and Japanese encephalitis viruses, which affect millions of individuals worldwide. Because countermeasures are currently unavailable, flaviviral therapy is urgently required. The flaviviral two-component nonstructural NS2B-NS3 proteinase (protease [pro]) is essential for viral life cycle and, consequently, is a promising drug target. We report here the results of the miniaturization of an NS2B-NS3pro activity assay, followed by high-throughput screening of the National Institutes of Healths 65,000 compound library and identification of novel, uncompetitive inhibitors of WNV NS2B-NS3pro that appear to interfere with the productive interactions of the NS2B cofactor with the NS3pro domain. We anticipate that following structure optimization, the identified probes could form the foundation for the design of novel and specific therapeutics for WNV infection. We also provide the structural basis for additional species-selective allosteric inhibitors of flaviviruses.

A Small-Molecule Oxocarbazate Inhibitor of Human Cathepsin L Blocks Severe Acute Respiratory Syndrome and Ebola Pseudotype Virus Infection into Human Embryonic Kidney 293T cells

A tetrahydroquinoline oxocarbazate (PubChem CID 23631927) was tested as an inhibitor of human cathepsin L (EC 3.4.22.15) and as an entry blocker of severe acute respiratory syndrome (SARS) coronavirus and Ebola pseudotype virus. In the cathepsin L inhibition assay, the oxocarbazate caused a time-dependent 17-fold drop in IC50 from 6.9 nM (no preincubation) to 0.4 nM (4-h preincubation). Slowly reversible inhibition was demonstrated in a dilution assay. A transient kinetic analysis using a single-step competitive inhibition model provided rate constants of kon = 153,000 M−1s−1 and koff = 4.40 × 10−5 s−1 (Ki = 0.29 nM). The compound also displayed cathepsin L/B selectivity of >700-fold and was nontoxic to human aortic endothelial cells at 100 μM. The oxocarbazate and a related thiocarbazate (PubChem CID 16725315) were tested in a SARS coronavirus (CoV) and Ebola virus-pseudotype infection assay with the oxocarbazate but not the thiocarbazate, demonstrating activity in blocking both SARS-CoV (IC50 = 273 ± 49 nM) and Ebola virus (IC50 = 193 ± 39 nM) entry into human embryonic kidney 293T cells. To trace the intracellular action of the inhibitors with intracellular cathepsin L, the activity-based probe biotin-Lys-C5 alkyl linker-Tyr-Leu-epoxide (DCG-04) was used to label the active site of cysteine proteases in 293T lysates. The reduction in active cathepsin L in inhibitor-treated cells correlated well with the observed potency of inhibitors observed in the virus pseudotype infection assay. Overall, the oxocarbazate CID 23631927 was a subnanomolar, slow-binding, reversible inhibitor of human cathepsin L that blocked SARS-CoV and Ebola pseudotype virus entry in human cells.

Identification of Novel Inhibitors of Dietary Lipid Absorption Using Zebrafish

Pharmacological inhibition of dietary lipid absorption induces favorable changes in serum lipoprotein levels in patients that are at risk for cardiovascular disease and is considered an adjuvant or alternative treatment with HMG-CoA reductase inhibitors (statins). Here we demonstrate the feasibility of identifying novel inhibitors of intestinal lipid absorption using the zebrafish system. A pilot screen of an unbiased chemical library identified novel compounds that inhibited processing of fluorescent lipid analogues in live zebrafish larvae. Secondary assays identified those compounds suitable for testing in mammals and provided insight into mechanism of action, which for several compounds could be distinguished from ezetimibe, a drug used to inhibit cholesterol absorption in humans that broadly inhibited lipid absorption in zebrafish larvae. These findings support the utility of zebrafish screening assays to identify novel compounds that target complex physiological processes.

KINETIC CHARACTERIZATION AND MOLECULAR DOCKING OF A NOVEL, POTENT, AND SELECTIVE SLOW-BINDING INHIBITOR OF HUMAN CATHEPSIN L

A novel small molecule thiocarbazate (PubChem SID 26681509), a potent inhibitor of human cathepsin L (EC 3.4.22.15) with an IC50 of 56 nM, was developed after a 57,821-compound screen of the National Institutes of Health Molecular Libraries Small Molecule Repository. After a 4-h preincubation with cathepsin L, this compound became even more potent, demonstrating an IC50 of 1.0 nM. The thiocarbazate was determined to be a slow-binding and slowly reversible competitive inhibitor. Through a transient kinetic analysis for single-step reversibility, inhibition rate constants were kon = 24,000 M-1s-1 and koff = 2.2 × 10-5 s-1 (Ki = 0.89 nM). Molecular docking studies were undertaken using the experimentally derived X-ray crystal structure of papain/CLIK-148 (1cvz. pdb). These studies revealed critical hydrogen bonding patterns of the thiocarbazate with key active site residues in papain. The thiocarbazate displayed 7- to 151-fold greater selectivity toward cathepsin L than papain and cathepsins B, K, V, and S with no activity against cathepsin G. The inhibitor demonstrated a lack of toxicity in human aortic endothelial cells and zebrafish. In addition, the thiocarbazate inhibited in vitro propagation of malaria parasite Plasmodium falciparum with an IC50 of 15.4 μM and inhibited Leishmania major with an IC50 of 12.5 μM.

Design, synthesis and evaluation of inhibitors of cathepsin L: exploiting a unique thiocarbazate chemotype

Recently, we identified a thiocarbazate that exhibits potent inhibitory activity against human cathepsin L. Since this structure represents a novel chemotype with potential for activity against the entire cysteine protease family, we designed, synthesized, and assayed a series of analogs to probe the mechanism of action, as well as the structural requirements for cathepsin L activity. Molecular docking studies using coordinates of a papain-inhibitor complex as a model for cathepsin L provided useful insights.

Design, Synthesis and Biological Evaluation of a Library of Thiocarbazates and Their Activity as Cysteine Protease Inhibitors

Recently, we identified a novel class of potent cathepsin L inhibitors, characterized by a thiocarbazate warhead. Given the potential of these compounds to inhibit other cysteine proteases, we designed and synthesized a library of thiocarbazates containing diversity elements at three positions. Biological characterization of this library for activity against a panel of proteases indicated a significant preference for members of the papain family of cysteine proteases over serine, metallo-, and certain classes of cysteine proteases, such as caspases. Several potent inhibitors of cathepsin L and S were identified. The SAR data were employed in docking studies in an effort to understand the structural elements required for cathepsin S inhibition. This study provides the basis for the design of highly potent and selective inhibitors of the papain family of cysteine proteases.