Inhibitors of the viral nucleoprotein-polymerase co-factor interaction for human RSV and MPV therapy

Period of Performance: 08/01/2016 - 07/31/2017

$300K

Phase 1 STTR

Recipient Firm

Microbiotix, Inc.
WORCESTER, MA 01605
Principal Investigator

Abstract

Human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV) are non-segmented negative-strand viruses (NNSV) and are the leading causes of acute respiratory tract infections in infants worldwide. Inaddition, hRSV is a significant cause of disease in elderly populations and can often be fatal for patients withcompromised immune systems. Currently no vaccines are available, and existing therapeutics (e.g., ribavirin,immunoglobulin, or anti-hRSV monoclonal, Synagis®) exhibit poor efficacy and present safety concerns. Thedevelopment of safer more effective therapeutics is a major unmet medical need. The goal of this project is toaddress this need by discovering and developing inhibitors of hRSV and hMPV RNA synthesis for therapeuticuse by targeting the interaction between the viral nucleoprotein (N) and the viral P protein, a cofactor for theviral polymerase (L). This interaction is critical for viral RNA synthesis; in cells infected with NNSVs, an L-Ncomplex is required for replication, and P mediates interactions between L and the N-RNA template. Thestrategy is to build and apply biochemical screens for inhibitors of the hRSV and hMPV N/P interaction basedon fluorescence polarization. This approach is based on a successful anti-Ebola virus screening effort carriedout by this team to identify inhibitors of the interaction between the Ebola nucleoprotein (eNP) and the Ebola Pprotein equivalent, known as eVP35. Development and application of a primary fluorescence polarizationassay (FPA) followed by secondary assays including a counter-screen FPA based on an unrelated interactionresulted in the discovery of six specific eVP35/eNP interaction inhibitors with IC50 values ranging from 1 µM to35 µM. Two of these compounds inhibited Ebola RNA synthesis in a cell based assay known as aminigenome replication assay. In Phase I, these efforts will be extended to target this conserved viralinteraction by focusing on hRSV and hMPV, which are of broad clinical importance. Primary FPA screens forinhibitors of the hRSV and hMPV N-protein interactions with fluorophore-labeled peptides from thecorresponding P-proteins will be developed. In addition, biochemical (e.g., biolayer interferometry, BLI) andcellular (e.g., split luciferase) secondary assays with orthogonal read-outs will be constructed to validate initialhits and to assess cellular permeability and mechanism of action. The primary and secondary assays will beapplied to >400,000 diverse compounds. Confirmed potent, selective inhibitors will be validated by determiningtheir ability to inhibit infectious viral assays and by ensuring that they are not cytotoxic. In vitro ADME assaysand preliminary SAR will prioritize analogs for further optimization. Strengths of this proposal include theproductive, collaborative research team; highly sensitive, homogeneous FPA screens; FPA counter-screens torapidly recognize and eliminate false positives; potential to identify broad inhibitors targeting hRSV and hMPV;and cellular assays to establish the target-specific function. In Phase II, priority validated inhibitors will bechemically optimized into lead compounds for efficacy and toxicity testing in animal models.