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

Prior, Allan M.; Kim, Yun-Jeong; Weerasekara, Sahani; Moroze, Meghan; Alliston, Kevin R.; Uy, Roxanne Adeline Z.; Groutas, William C.; Chang, Kyeong‐Ok; Hua, Duy H.

doi:10.1016/j.bmcl.2013.09.070

Cited by 57 publications

(71 citation statements)

References 17 publications

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“…The crystal structure of NV Pro shows that it has a two-domain structure similar to those of other viral 3C-like cysteine proteases, with a catalytic triad composed of His-30, Glu-54, and Cys-139 (23,24). NV Pro cleaves at a Q/G or E/G scissile bond, with a preferred substrate specificity for an FxLQ/GP sequence in the P 4 -P 1 /P 1 =P 2 = position, where x is H, Q, N, or A. Small-molecule inhibitors of NV Pro, either designed based on its substrate specificity or screened from compound libraries, have been reported (16,(19)(20)(21)(22). However, except for those that have been tested against an NV replicon system (19,20,22), many of the NV Pro inhibitors were developed by in vitro protease assays and remain to be evaluated using a cell-based system.…”

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confidence: 99%

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Development of a Gaussia Luciferase-Based Human Norovirus Protease Reporter System: Cell Type-Specific Profile of Norwalk Virus Protease Precursors and Evaluation of Inhibitors

Qu¹,

Vongpunsawad²,

Atmar³

et al. 2014

J Virol

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Norwalk virus (NV) is the prototype strain of human noroviruses (HuNoVs), a group of positive-strand RNA viruses in the Caliciviridae family and the leading cause of epidemic gastroenteritis worldwide. Investigation of HuNoV replication and development of antiviral therapeutics in cell culture remain challenging tasks. Here, we present NoroGLuc, a HuNoV protease reporter system based on a fusion of NV p41 protein with a naturally secreted Gaussia luciferase (GLuc), linked by the p41/p22 cleavage site for NV protease (Pro). trans cleavage of NoroGLuc by NV Pro or Pro precursors results in release and secretion of an active GLuc. Using this system, we observed a cell type-specific activity profile of NV Pro and Pro precursors, suggesting that the activity of NV Pro is modulated by other viral proteins in the precursor forms and strongly influenced by cellular factors. NoroGLuc was also cleaved by Pro and Pro precursors generated from replication of NV stool RNA in transfected cells, resulting in a measurable increase of secreted GLuc. Truncation analysis revealed that the N-terminal membrane association domain of NV p41 is critical for NoroGLuc activity. Although designed for NV, a genogroup GI.1 norovirus, NoroGLuc also efficiently detects Pro activities from GII.3 and GII.4 noroviruses. At noncytotoxic concentrations, protease inhibitors ZnCl 2 and N␣-p-tosyl-L-lysine chloromethyl ketone (TLCK) exhibited dose-dependent inhibitory effects on a GII.4 Pro by NoroGLuc assay. These results establish NoroGLuc as a pan-genogroup HuNoV protease reporter system that can be used for the study of HuNoV proteases and precursors, monitoring of viral RNA replication, and evaluation of antiviral agents. IMPORTANCEHuman noroviruses are the leading cause of epidemic gastroenteritis worldwide. Currently, there are no vaccines or antiviral drugs available to counter these highly contagious viruses. These viruses are currently noncultivatable in cell culture. Here, we report the development of a novel cell-based reporter system called NoroGLuc that can be used for studying norovirus replication and also for screening/evaluation of antiviral agents. This system is based on the fusion between viral protein p41 and a naturally secreted Gaussia luciferase (GLuc) with a cleavage site that can be recognized by the viral protease. Cleavage of this fusion protein by the viral protease results in the release and secretion of an active GLuc. Using NoroGLuc, we demonstrated a cell typespecific activity profile of the viral protease and its precursors and dose-dependent inhibitory effects of two protease inhibitors. This novel reporter system should be useful in probing norovirus replication and evaluating antiviral agents.

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confidence: 99%

“…Two of the NV ORF1-encoded enzymes, Pro and Pol, have been extensively studied and also targeted for antiviral therapeutic development due to their essential roles in viral replication (16)(17)(18)(19)(20)(21)(22). The NV Pro is a 3C-like cysteine protease classified in the chymotrypsin-like serine protease superfamily.…”

mentioning

confidence: 99%

Development of a Gaussia Luciferase-Based Human Norovirus Protease Reporter System: Cell Type-Specific Profile of Norwalk Virus Protease Precursors and Evaluation of Inhibitors

Qu¹,

Vongpunsawad²,

Atmar³

et al. 2014

J Virol

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“…Anti-protease inhibitors approved for viral infection treatment are interesting examples in this connection. Recently, these inhibitors are screened for their capability to inhibit Mpro and treat SARS infection using in vivo, in vitro, and in silico experiments (34)(35)(36)(37)(38)(39). The clinical trial of LPV showed a significant decrease in virus titer, reduced rate of death, and improved clinical recovery (40)(41)(42).…”

Section: Discussionmentioning

confidence: 99%

“…The first class includes a peptide chain with an ending reactive group. This kind of inhibitor fits the catalytic site of the enzyme by making a covalent link with Cys145 via its reactive group and therefore it blocks the substrate entry to the active site (37,38).…”

Section: Discussionmentioning

confidence: 99%

Lopinavir; A Potent Drug against Coronavirus Infection: Insight from Molecular Docking Study

Dayer

Taleb-Gassabi

Dayer

2017

Arch Clin Infect Dis

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Background: The severe acute respiratory syndrome (SARS) is a life threatening viral infection caused by a positive, single stranded RNA virus from the enveloped coronaviruse family. Associated with fever, cough, and respiratory complications, the illness causes more than 15% mortality worldwide. So far, there is no remedy for the illness except supportive treatments. However, the main viral proteinase has recently been regarded as a suitable target for drug design against SARS infection due to its vital role in polyproteins processing necessary for coronavirus reproduction. Objectives: The present in silico study was designed to evaluate the effects of anti HIV-1 proteases inhibitors, approved for clinical applications by US FDA, on SARS proteinase inhibition. Methods: In the present study, docking and molecular dynamic experiments were applied to examine the effect of inhibitors on coronavirus proteinase under physiological conditions of similar pH, temperature, and pressure in aqueous solution. Hex software version 5.1 and GROMACS 4.5.5 were used for docking analysis throughout this work. Results:The calculated parameters such as RMSD, RMSF, MSD, dipole moment, diffusion coefficient, binding energy, and binding site similarity indicated effective binding of inhibitors to SARS proteinase resulting in their structural changes, which coincide with proteinase inhibition. Conclusions:The inhibitory potency of HIV 1 protease inhibitors to cronovirus proteinase was as follows: LPV > RTV > APV > TPV > SQV. Lopinavir and Saquinavir were the most and the least powerful inhibitors of cronovirus proteinase, respectively.

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“…Most recently, the same group of investigators has described a class of molecules, generally termed dipeptidyl or tripeptidyl transition state inhibitors, which display potent, broad-spectrum activity in enzymatic and cell culture-based assays against several 3C-like proteases of viruses from the families Picornaviridae, Coronaviridae , and Caliciviridae (including NV), which share conserved features in the protease catalytic site (Kim et al, 2012; Mandadapu et al, 2013; Prior et al, 2013; Takahashi et al, 2013). Mechanistically, these molecules have been shown to be covalently bound to the nucleophilic cysteine residue in the catalytic sites of the proteases of NV and poliovirus in co-crystallography studies (Kim et al, 2012).…”

Section: Current State Of Anti-norovirus Drug Discoverymentioning

confidence: 99%

Treatment of norovirus infections: Moving antivirals from the bench to the bedside

2014

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Noroviruses (NV) are the most common cause of acute gastrointestinal illness in the United States and worldwide. The development of specific antiviral countermeasures has lagged behind that of other viral pathogens, primarily because norovirus disease has been perceived as brief and self-limiting and robust assays suitable for drug discovery have been lacking. The increasing recognition that NV illness can be life-threatening, especially in immunocompromised patients who often require prolonged hospitalization and intensive supportive care, has stimulated new research to develop an effective antiviral therapy. Here, we propose a path forward for evaluating drug therapy in norovirus-infected immunocompromised individuals, a population at high risk for serious and prolonged illness. The clinical and laboratory features of norovirus illness in immunocompromised patients are reviewed, and potential markers of drug efficacy are defined. We discuss the potential design of clinical trials in these patients and how an anti-viral therapy that proves effective in immunocompromised patients might also be used in the setting of acute outbreaks, especially in confined settings such as nursing homes, to block the spread of infection and reduce the severity of illness. We conclude by reviewing the current status of approved and experimental compounds that might be evaluated in a hospital setting.

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

Cited by 57 publications

References 17 publications

Development of a Gaussia Luciferase-Based Human Norovirus Protease Reporter System: Cell Type-Specific Profile of Norwalk Virus Protease Precursors and Evaluation of Inhibitors

Development of a Gaussia Luciferase-Based Human Norovirus Protease Reporter System: Cell Type-Specific Profile of Norwalk Virus Protease Precursors and Evaluation of Inhibitors

Lopinavir; A Potent Drug against Coronavirus Infection: Insight from Molecular Docking Study

Treatment of norovirus infections: Moving antivirals from the bench to the bedside

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