William C. Groutas

Broad-Spectrum Antivirals against 3C or 3C-Like Proteases of Picornaviruses, Noroviruses, and Coronaviruses

ABSTRACT Phylogenetic analysis has demonstrated that some positive-sense RNA viruses can be classified into the picornavirus-like supercluster, which includes picornaviruses, caliciviruses, and coronaviruses. These viruses possess 3C or 3C-like proteases (3Cpro or 3CLpro, respectively), which contain a typical chymotrypsin-like fold and a catalytic triad (or dyad) with a Cys residue as a nucleophile. The conserved key sites of 3Cpro or 3CLpro may serve as attractive targets for the design of broad-spectrum antivirals for multiple viruses in the supercluster. We previously reported the structure-based design and synthesis of potent protease inhibitors of Norwalk virus (NV), a member of the Caliciviridae family. We report herein the broad-spectrum antiviral activities of three compounds possessing a common dipeptidyl residue with different warheads, i.e., an aldehyde (GC373), a bisulfite adduct (GC376), and an α-ketoamide (GC375), against viruses that belong to the supercluster. All compounds were highly effective against the majority of tested viruses, with half-maximal inhibitory concentrations in the high nanomolar or low micromolar range in enzyme- and/or cell-based assays and with high therapeutic indices. We also report the high-resolution X-ray cocrystal structures of NV 3CLpro-, poliovirus 3Cpro-, and transmissible gastroenteritis virus 3CLpro- GC376 inhibitor complexes, which show the compound covalently bound to a nucleophilic Cys residue in the catalytic site of the corresponding protease. We conclude that these compounds have the potential to be developed as antiviral therapeutics aimed at a single virus or multiple viruses in the picornavirus-like supercluster by targeting 3Cpro or 3CLpro.

Neutrophil Elastase Inhibitors

Introduction: Chronic obstructive pulmonary disease (COPD) constitutes a worldwide health problem. There is currently an urgent and unmet need for the development of small molecule therapeutics capable of blocking and/or reversing the progression of the disorder. Recent studies have greatly illuminated our understanding of the multiple pathogenic processes associated with COPD. Of paramount importance is the key role played by proteases, oxidative stress, apoptosis and inflammation. Insights gained from these studies have made possible the exploration of new therapeutic approaches. Areas covered: An overview of major developments in COPD research with emphasis on low-molecular mass neutrophil elastase inhibitors is described in this review. Expert opinion: Great strides have been made toward our understanding of the biochemical and cellular events associated with COPD. However, our knowledge regarding the inter-relationships among the multiple pathogenic mechanisms and their mediators involved is still limited. The problem is further compounded by the unavailability of suitable validated biomarkers for assessing the efficacy of potential therapeutic interventions. The complexity of COPD suggests that effective therapeutic interventions may require the administration of more than one agent such as a human neutrophil elastase or MMP-12 inhibitor with an anti-inflammatory agent such as a PDE4 inhibitor or a dual function agent capable of disrupting the cycle of proteolysis, apoptosis, inflammation and oxidative stress.

Inhibition of Dengue virus and West Nile virus proteases by click chemistry-derived benz[d]isothiazol-3(2H)-one derivatives.

Two click chemistry-derived focused libraries based on the benz[d]isothiazol-3(2H)-one scaffold were synthesized and screened against Dengue virus and West Nile virus NS2B-NS3 proteases. Several compounds (4l, 7j-n) displayed noteworthy inhibitory activity toward Dengue virus NS2B-NS3 protease in the absence and presence of added detergent. These compounds could potentially serve as a launching pad for a hit-to-lead optimization campaign.

Human neutrophil proteinase 3: Mapping of the substrate binding site using peptidyl thiobenzyl esters

A series of peptidyl thiobenzyl esters was used to map the active site of human leukocyte proteinase 3. The steady-state kinetics parameters reveal the following features regarding the substrate specificity of proteinase 3 and its putative active site: (a) the preferred P1 residue is a small hydrophobic amino acid such as aminobutyric acid, norvaline, valine or alanine (in decreasing order of preference); (b) the enzyme has an extended active site; and (c) its active site is similar to that of the related serine proteinases leukocyte elastase and leukocyte cathepsin G.

Design, synthesis and characterization of novel 1,2-benzisothiazol-3(2H)-one and 1,3,4-oxadiazole hybrid derivatives: potent inhibitors of Dengue and West Nile virus NS2B/NS3 proteases.

1,2-Benzisothiazol-3(2H)-ones and 1,3,4-oxadiazoles individually have recently attracted considerable interest in drug discovery, including as antibacterial and antifungal agents. In this study, a series of functionalized 1,2-benzisothiazol-3(2H)-one-1,3,4-oxadiazole hybrid derivatives were synthesized and subsequently screened against Dengue and West Nile virus proteases. Ten out of twenty-four compounds showed greater than 50% inhibition against DENV2 and WNV proteases ([I] = 10 μM). The IC(50) values of compound 7n against DENV2 and WNV NS2B/NS3 were found to be 3.75 ± 0.06 and 4.22 ± 0.07 μM, respectively. The kinetics data support a competitive mode of inhibition by compound 7n. Molecular modeling studies were performed to delineate the putative binding mode of this series of compounds. This study reveals that the hybrid series arising from the linking of the two scaffolds provides a suitable platform for conducting a hit-to-lead optimization campaign via iterative structure-activity relationship studies, in vitro screening and X-ray crystallography.

Design, synthesis, and evaluation of inhibitors of Norwalk virus 3C protease.

The first series of peptidyl aldehyde inhibitors that incorporate in their structure a glutamine surrogate has been designed and synthesized based on the known substrate specificity of Norwalk virus 3C protease. The inhibitory activity of the compounds with the protease and with a norovirus cell-based replicon system was investigated. Members of this class of compounds exhibited noteworthy activity both in vitro and in a cell-based replicon system.

Design, synthesis, and in vitro inhibitory activity toward human leukocyte elastase, cathepsin G, and proteinase 3 of saccharin-derived sulfones and congeners

The inhibitory activity toward human leukocyte elastase (HLE), cathepsin G (Cat G), and proteinase 3 (PR 3) of a series of saccharin derivatives having a sulfinate leaving group was investigated. The results of this study revealed that (a) inhibitory activity is dependent on the nature and pKa of the leaving group, and (b) the synthesized saccharin derivatives exhibit selective inhibition toward HLE and PR 3, with low or no activity toward cathepsin G. The results of exploratory biochemical, HPLC and high-field 13C NMR studies are also described.

Inhibitors of Dengue Virus and West Nile Virus Proteases Based on the Aminobenzamide Scaffold

Dengue and West Nile viruses (WNV) are mosquito-borne members of flaviviruses that cause significant morbidity and mortality. There is no approved vaccine or antiviral drugs for human use to date. In this study, a series of functionalized meta and para aminobenzamide derivatives were synthesized and subsequently screened in vitro against Dengue virus and West Nile virus proteases. Four active compounds were identified which showed comparable activity toward the two proteases and shared in common a meta or para(phenoxy)phenyl group. The inhibition constants (K(i)) for the most potent compound 7n against Dengue and West Nile virus proteases were 8.77 and 5.55 μM, respectively. The kinetics data support a competitive mode of inhibition of both proteases by compound 7n. This conclusion is further supported by molecular modeling. This study reveals a new chemical scaffold which is amenable to further optimization to yield potent inhibitors of the viral proteases via the combined utilization of iterative medicinal chemistry/structure-activity relationship studies and in vitro screening.

Design, synthesis, and bioevaluation of viral 3C and 3C-like protease inhibitors.

Abstract A class of tripeptidyl transition state inhibitors containing a P1 glutamine surrogate, a P2 leucine, and a P3 arylalanines, was found to potently inhibit Norwalk virus replication in enzyme and cell based assays. An array of warheads, including aldehyde, α-ketoamide, bisulfite adduct, and α-hydroxyphosphonate transition state mimic, was also investigated. Tripeptidyls 2 and 6 possess antiviral activities against noroviruses, human rhinovirus, severe acute respiratory syndrome coronavirus, and coronavirus 229E, suggesting a broad range of antiviral activities.

Use of the 1,2,5-thiadiazolidin-3-one 1,1 dioxide and isothiazolidin-3-one 1,1 dioxide scaffolds in the design of potent inhibitors of serine proteinases

The attachment of a phosphate leaving group to the 1,2,5-thiadiazolidin-3-one 1,1 dioxide and isothiazolidin-3-one 1,1 dioxide scaffolds was found to yield highly potent, time-dependent inhibitors of human leukocyte elastase (HLE).