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

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 In
addition hRSV is a significant cause of disease in elderly populations and can often be fatal for patients with
compromised 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 The
development of safer more effective therapeutics is a major unmet medical need The goal of this project is to
address this need by discovering and developing inhibitors of hRSV and hMPV RNA synthesis for therapeutic
use by targeting the interaction between the viral nucleoprotein N and the viral P protein a cofactor for the
viral polymerase L This interaction is critical for viral RNA synthesis in cells infected with NNSVs an L N
complex is required for replication and P mediates interactions between L and the N RNA template The
strategy is to build and apply biochemical screens for inhibitors of the hRSV and hMPV N P interaction based
on fluorescence polarization This approach is based on a successful anti Ebola virus screening effort carried
out by this team to identify inhibitors of the interaction between the Ebola nucleoprotein eNP and the Ebola P
protein equivalent known as eVP Development and application of a primary fluorescence polarization
assay FPA followed by secondary assays including a counter screen FPA based on an unrelated interaction
resulted in the discovery of six specific eVP eNP interaction inhibitors with IC values ranging from M to
M Two of these compounds inhibited Ebola RNA synthesis in a cell based assay known as a
minigenome replication assay In Phase I these efforts will be extended to target this conserved viral
interaction by focusing on hRSV and hMPV which are of broad clinical importance Primary FPA screens for
inhibitors of the hRSV and hMPV N protein interactions with fluorophore labeled peptides from the
corresponding P proteins will be developed In addition biochemical e g biolayer interferometry BLI and
cellular e g split luciferase secondary assays with orthogonal read outs will be constructed to validate initial
hits and to assess cellular permeability and mechanism of action The primary and secondary assays will be
applied to andgt diverse compounds Confirmed potent selective inhibitors will be validated by determining
their ability to inhibit infectious viral assays and by ensuring that they are not cytotoxic In vitro ADME assays
and preliminary SAR will prioritize analogs for further optimization Strengths of this proposal include the
productive collaborative research team highly sensitive homogeneous FPA screens FPA counter screens to
rapidly 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 be
chemically optimized into lead compounds for efficacy and toxicity testing in animal models Narrative
Human respiratory syncytial virus hRSV and human metapneumovirus hMPV are the leading causes of
acute respiratory tract infections in infants worldwide Currently no vaccines are available and existing
therapeutics exhibit poor efficacy and present safety concerns The goal of this proposal is to discover and
develop novel safe inhibitors of hRSV and hMPV RNA synthesis for therapeutic use by targeting the interaction
between the viral nucleoprotein N and the viral P protein a cofactor for the viral polymerase L This
interaction is critical for viral RNA synthesis and viral replication