Identification and Characterization of a Small -Molecule Rabies Virus Entry Inhibitor

被引:5
作者
Du Pont, Venice [1 ]
Wirblich, Christoph [2 ]
Yoon, Jeong-Joong [1 ]
Cox, Robert M. [1 ]
Schnell, Matthias J. [2 ]
Plemper, Richard K. [1 ]
机构
[1] Georgia State Univ, Inst Biomed Sci, Atlanta, GA 30303 USA
[2] Thomas Jefferson Univ, Dept Microbiol & Immunol, Sidney Kimmel Med Coll, Philadelphia, PA 19107 USA
关键词
DNA-binding proteins; antiviral; drug discovery; entry inhibitor; lyssavirus; rabies; rhabdovirus; RESPIRATORY SYNCYTIAL VIRUS; RSV FUSION INHIBITOR; ANTIGENIC SITE-III; LOW-PH; CONFORMATIONAL-CHANGES; INFLUENZA-VIRUS; PREFUSION FORM; GLYCOPROTEIN; RECEPTOR; MECHANISM;
D O I
10.1128/JVI.00321-20
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
Rabies virus (RABV) causes a severe and fatal neurological disease, but morbidity is vaccine preventable and treatable prior to the onset of clinical symptoms. However, immunoglobulin (IgG)-based rabies postexposure prophylaxis (PEP) is expensive, restricting access to life-saving treatment, especially for patients in low-income countries where the clinical need is greatest, and does not confer cross-protection against newly emerging phylogroup II lyssaviruses. Toward identifying a cost-effective replacement for the IgG component of rabies PEP, we developed and implemented a high-throughput screening protocol utilizing a single-cycle RABV re-porter strain. A large-scale screen and subsequent direct and orthogonal counter-screens identified a first-in-class direct-acting RABV inhibitor, GRP-60367, with a specificity index (SI) of >100,000. Mechanistic characterization through time-of-addition studies, transient cell-to-cell fusion assays, and chimeric vesicular stomatitis virus (VSV) recombinants expressing the RABV glycoprotein (G) demonstrated that GRP-60367 inhibits entry of a subset of RABV strains. Resistance profiling of the chemotype revealed hot spots in conserved hydrophobic positions of the RABV G protein fusion loop that were confirmed in transient cell-to-cell fusion assays. Transfer of RABV G genes with signature resistance mutations into a recombinant VSV backbone resulted in the recovery of replication-competent virions with low susceptibility to the inhibitor. This work outlines a tangible strategy for mechanistic characterization and resistance profiling of RABV drug candidates and identified a novel, well-behaved molecular probe chemotype that specifically targets the RABV G protein and prevents G-mediated viral entry. IMPORTANCE Rabies PEP depends on anti-RABV IgG, which is expensive and in limited supply in geographical areas with the highest disease burden. Replacing the IgG component with a cost-effective and shelf-stable small-molecule antiviral could ad-dress this unmet clinical need by expanding access to life-saving medication. This study has established a robust protocol for high-throughput anti-RABV drug screens and identified a chemically well-behaved, first-in-class hit with nanomolar anti-RABV potency that blocks RABV G protein-mediated viral entry. Resistance mapping revealed a druggable site formed by the G protein fusion loops that has not previously emerged as a target for neutralizing antibodies. Discovery of this RABV en-try inhibitor establishes a new molecular probe to advance further mechanistic and structural characterization of RABV G that may aid in the design of a next-generation clinical candidate against RABV.
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页数:17
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