Targeting the essential interactions of the M2-1 protein of human respiratory syncytial virus (HRSV) for anti-viral drug development

Human respiratory syncytial virus (HRSV) is a non-segmented negative stranded RNA virus classified within the Mononegavirales order. HRSV is the leading cause of lower respiratory tract illness in young children and the immunocompromised, with over 250,000 annual fatalities worldwide. HRSV-mediated diseases are especially prevalent in developing countries, where no financially viable treatment exists. The M2-1 protein of HRSV represents a promising potential anti-viral target for the treatment of HRSV-mediated diseases. M2-1 is a transcription antiterminator with an essential role in viral gene expression. M2-1 binds both viral RNA and the polymerase co-factor phosphoprotein (P), and these interactions are essential for its anti-termination activity. Here, the crystal structure of M2-1 in complex with a P protein peptide was determined and used in addition to the unbound M2-1 crystal structure as a basis to identify potential anti-viral compounds. Structure based drug design (SBDD) of M2-1s interaction interface with RNA/ P produced hit compounds that were analysed through biophysical, structural and in cellulo methods including the use of a mini-genome and infectious HRSV assay. SBDD identified hit compounds that significantly inhibited the growth of HRSV. Subsequently, synthetic chemistry was used to generate libraries of molecules to establish structure-activity relationships (SAR) as well as assess their pharmacokinetic properties. Further, a novel facility that allowed assessment of fragment binders to HRSV M2-1 by X-ray crystallography was utilised to determine hit binders towards the entire M2-1 protein, including the previously targeted RNA/ P surface and allowed further rapid SAR. The work presented here represents an effective strategy to rationally design anti-viral compounds for the M2-1 protein of HRSV with potential applications for the related virus, bovine respiratory syncytial virus (BRSV).