Identification of Non-Nucleoside Inhibitors of the Respiratory Syncytial Virus Polymerase Complex

Abstract

Respiratory syncytial virus (RSV) represents a threat to infants, the elderly, and the immunocompromised. RSV entry blockers are in clinical trials, but escape mutations challenge their potential. In search of RSV inhibitors, we have integrated a signature resistance mutation into are combinant RSV virus and applied the strain to high-throughput screening. Counter screening of candidates returned 14 confirmed hits with activity in the nano- to low-micromolar range. All blocked RSV polymerase activity in minigenome assays. Compound 1a (GRP-74915) was selected for development based on activity (EC50=0.21 µM, selectivity index (SI) 40), and scaffold. Resynthesis confirmed potency of the compound, which suppressed viral RNA synthesis in infected cells. However, metabolic testing revealed a short half-life in the presence of mouse hepatocyte fractions. Metabolite tracking and chemical elaboration combined with 3D-quantitative structure-activity relationship modeling yielded analogs (i.e. 8n: EC50=0.06 µM, SI 500) that establish a platform for the development of a therapeutic candidate.

Development of an allosteric inhibitor class blocking RNA elongation by the respiratory syncytial virus polymerase complex

“…We also did not discover any entry inhibitors capable of blocking the pan-entry inhibitor-resistant (15) recombinant RSV-A2-L19F D489E -FireSMASh reporter virus used in this screen, which corroborates the conclusions of previous, independent screens that tested different chemical libraries against the same reporter strains (19,20). Combined, the results of these drug screens corroborate our earlier prediction that overcoming RSV pan-resistance with small-molecule entry inhibitors will be challenging (19,25,27).…”

“…To gain an initial understanding of how GRP-156784 responds to synthetic modification, we launched a ligand-driven SAR development strategy. All chemical analogs were tested for cytotoxicity and antiviral potency against RSV A2-L19F using an automated assay pipeline that we recently developed for standardized SAR profiling (19,25,27,28). The chemical scaffold was hypothetically divided into three distinct sections (Fig.…”

“…In our experience, ligand-driven QSARs show good predictive power for second-stage optimization, provided they are informed by meaningful training and test sets (27,(45)(46)(47). Particularly detrimental to model accuracy are data sets representing overlapping, but distinct, SARs, such as antiviral activity due to specific target binding and cytotoxic effects.…”

Orally Efficacious Broad-Spectrum Ribonucleoside Analog Inhibitor of Influenza and Respiratory Syncytial Viruses

“…Through a dual-pathogen (31,32) HTS campaign that affords the simultaneous identification of RSV and IAV inhibitors, we identified NHC, a pyrimidine ribonucleoside analog, as a hit candidate that integrates promising potency with a broadened indication spectrum. NHC was previously associated with antiviral activity against positivestrand RNA viruses (36-39), but PK/PD profiles have not been determined, and in vivo efficacy is untested.…”

“…Toward the ultimate goal of identifying developable broad-spectrum medications against influenza-like disease, we have established and validated a replicationcompetent dual RSV and influenza A virus (IAV) reporter virus-based high-throughput screening (HTS) assay that allows the simultaneous identification of IAV-specific, RSVspecific, and dually active, potentially broad-spectrum, hit candidates (30). The first implementation of this protocol against a large open discovery library of smallmolecule compounds has yielded promising target-specific inhibitors of RSV (31) and influenza virus (32), but broad-spectrum hits remained limited to undesirable scaffolds and clinically undevelopable compounds interfering with pyrimidine de novo synthesis (32)(33)(34). In this study, we applied the assay to a collection of ribonucleoside analogs.…”