Dataset Information

Discovery of a novel compound with anti-venezuelan equine encephalitis virus activity that targets the nonstructural protein 2.

ABSTRACT: Alphaviruses present serious health threats as emerging and re-emerging viruses. Venezuelan equine encephalitis virus (VEEV), a New World alphavirus, can cause encephalitis in humans and horses, but there are no therapeutics for treatment. To date, compounds reported as anti-VEEV or anti-alphavirus inhibitors have shown moderate activity. To discover new classes of anti-VEEV inhibitors with novel viral targets, we used a high-throughput screen based on the measurement of cell protection from live VEEV TC-83-induced cytopathic effect to screen a 340,000 compound library. Of those, we identified five novel anti-VEEV compounds and chose a quinazolinone compound, CID15997213 (IC50?=?0.84 µM), for further characterization. The antiviral effect of CID15997213 was alphavirus-specific, inhibiting VEEV and Western equine encephalitis virus, but not Eastern equine encephalitis virus. In vitro assays confirmed inhibition of viral RNA, protein, and progeny synthesis. No antiviral activity was detected against a select group of RNA viruses. We found mutations conferring the resistance to the compound in the N-terminal domain of nsP2 and confirmed the target residues using a reverse genetic approach. Time of addition studies showed that the compound inhibits the middle stage of replication when viral genome replication is most active. In mice, the compound showed complete protection from lethal VEEV disease at 50 mg/kg/day. Collectively, these results reveal a potent anti-VEEV compound that uniquely targets the viral nsP2 N-terminal domain. While the function of nsP2 has yet to be characterized, our studies suggest that the protein might play a critical role in viral replication, and further, may represent an innovative opportunity to develop therapeutic interventions for alphavirus infection.

SUBMITTER: Chung DH

PROVIDER: S-EPMC4072787 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Discovery of a novel compound with anti-venezuelan equine encephalitis virus activity that targets the nonstructural protein 2.

Chung Dong-Hoon DH Jonsson Colleen B CB Tower Nichole A NA Chu Yong-Kyu YK Sahin Ergin E Golden Jennifer E JE Noah James W JW Schroeder Chad E CE Sotsky Julie B JB Sosa Melinda I MI Cramer Daniel E DE McKellip Sara N SN Rasmussen Lynn L White E Lucile EL Schmaljohn Connie S CS Julander Justin G JG Smith Jeffrey M JM Filone Claire Marie CM Connor John H JH Sakurai Yasuteru Y Davey Robert A RA

PLoS pathogens 20140626 6

Alphaviruses present serious health threats as emerging and re-emerging viruses. Venezuelan equine encephalitis virus (VEEV), a New World alphavirus, can cause encephalitis in humans and horses, but there are no therapeutics for treatment. To date, compounds reported as anti-VEEV or anti-alphavirus inhibitors have shown moderate activity. To discover new classes of anti-VEEV inhibitors with novel viral targets, we used a high-throughput screen based on the measurement of cell protection from liv ...[more]

PMID: 24967809

Similar Datasets

Project description:The Venezuelan equine encephalitis virus (VEEV) nonstructural protein 2 (nsP2) cysteine protease (EC 3.4.22.-) is essential for viral replication and is involved in the cytopathic effects (CPE) of the virus. The VEEV nsP2 protease is a member of MEROPS Clan CN and characteristically contains a papain-like protease linked to an S-adenosyl-l-methionine-dependent RNA methyltransferase (SAM MTase) domain. The protease contains an alternative active site motif, (475)NVCWAK(480), which differs from papain's (CGS(25)CWAFS), and the enzyme lacks a transition state-stabilizing residue homologous to Gln-19 in papain. To understand the roles of conserved residues in catalysis, we determined the structure of the free enzyme and the first structure of an inhibitor-bound alphaviral protease. The peptide-like E64d inhibitor was found to bind beneath a β-hairpin at the interface of the SAM MTase and protease domains. His-546 adopted a conformation that differed from that found in the free enzyme; one or both of the conformers may assist in leaving group departure of either the amine or Cys thiolate during the catalytic cycle. Interestingly, E64c (200 μM), the carboxylic acid form of the E64d ester, did not inhibit the nsP2 protease. To identify key residues involved in substrate binding, a number of mutants were analyzed. Mutation of the motif residue, N475A, led to a 24-fold reduction in kcat/Km, and the conformation of this residue did not change after inhibition. N475 forms a hydrogen bond with R662 in the SAM MTase domain, and the R662A and R662K mutations both led to 16-fold decreases in kcat/Km. N475 forms the base of the P1 binding site and likely orients the substrate for nucleophilic attack or plays a role in product release. An Asn homologous to N475 is similarly found in coronaviral papain-like proteases (PLpro) of the Severe Acute Respiratory Syndrome (SARS) virus and Middle East Respiratory Syndrome (MERS) virus. Mutation of another motif residue, K480A, led to a 9-fold decrease in kcat and kcat/Km. K480 likely enhances the nucleophilicity of the Cys. Consistent with our substrate-bound models, the SAM MTase domain K706A mutation increased Km 4.5-fold to 500 μM. Within the β-hairpin, the N545A mutation slightly but not significantly increased kcat and Km. The structures and identified active site residues may facilitate the discovery of protease inhibitors with antiviral activity.

Dataset Information

Discovery of a novel compound with anti-venezuelan equine encephalitis virus activity that targets the nonstructural protein 2.

Publications

Discovery of a novel compound with anti-venezuelan equine encephalitis virus activity that targets the nonstructural protein 2.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets