Respiratory syncytial virus (RSV) is a major cause of respiratory illness in infants, immunocompromised patients, and the elderly. New antiviral agents would be important tools in the treatment of acute RSV disease. RSV encodes its own RNA-dependent RNA polymerase that is responsible for the synthesis of both genomic RNA and subgenomic mRNAs. The viral polymerase also cotranscriptionally caps and polyadenylates the RSV mRNAs at their 5 and 3 ends, respectively. We have previously reported the discovery of the first nonnucleoside transcriptase inhibitor of RSV polymerase through high-throughput screening. Here we report the design of inhibitors that have improved potency both in vitro and in antiviral assays and that also exhibit activity in a mouse model of RSV infection. We have isolated virus with reduced susceptibility to this class of inhibitors. The mutations conferring resistance mapped to a novel motif within the RSV L gene, which encodes the catalytic subunit of RSV polymerase. This motif is distinct from the catalytic region of the L protein and bears some similarity to the nucleotide binding domain within nucleoside diphosphate kinases. These findings lead to the hypothesis that this class of inhibitors may block synthesis of RSV mRNAs by inhibiting guanylylation of viral transcripts. We show that short transcripts produced in the presence of inhibitor in vitro do not contain a 5 cap but, instead, are triphosphorylated, confirming this hypothesis. These inhibitors constitute useful tools for elucidating the molecular mechanism of RSV capping and represent valid leads for the development of novel anti-RSV therapeutics.

RNA-dependent RNA polymerase from respiratory syncytial virus (RSV) is a multi-subunit ribonucleoprotein (RNP) complex that, in addition to synthesizing the full 15 222 nt viral genomic RNA, is able to synthesize all 10 viral mRNAs. We have prepared crude RNP from RSV-infected HEp-2 cells, based on a method previously used for Newcastle disease virus, and established a novel polyadenylation-dependent capture [poly(A) capture] assay to screen for potential inhibitors of RSV transcriptase activity. In this homogeneous assay, radiolabeled full-length polyadenylated mRNAs produced by the viral RNP are detected through capture on immobilized biotinylated oligo(dT) in a 96-well streptavidin-coated FlashPlate. Possible inhibitors identified with this assay could interfere at any step required for the production of complete RSV mRNAs, including transcription, polyadenylation and, potentially, co-transcriptional guanylylation. A specific inhibitor of RSV transcriptase with antiviral activity was identified through screening of this assay.

High-throughput screening is routinely employed as a method for the identification of novel hit structures. Large numbers of active compounds are typically procured in this way and must undergo a rigorous validation process. This process is described in detail for a collection of screening hits identified as inhibitors of IkappaB kinase-beta (IKKbeta), a key regulatory enzyme in the nuclear factor-kappaB (NF-kappaB) pathway. From these studies, a promising hit series was selected. Subsequent lead generation activities included the development of a pharmacophore hypothesis and structure-activity relationship (SAR) for the hit series. This led to the exploration of related scaffolds offering additional opportunities, and the various structural classes were comparatively evaluated for enzyme inhibition, selectivity, and drug-like properties. A novel lead series of thienopyridines was thereby established, and this series advanced into lead optimization for further development.

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