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The identification of novel antiretroviral agents is required to provide alternative treatment options for HIV-1-infected patients. The screening of a phenotypic cell-based viral replication assay led to the identification of a novel class of 4,5-dihydro-1H-pyrrolo[3,4-c]pyrazol-6-one (pyrrolopyrazolone) HIV-1 inhibitors, exemplified by two compounds: BI-1 and BI-2. These compounds inhibited early postentry stages of viral replication at a step(s) following reverse transcription but prior to 2 long terminal repeat (2-LTR) circle formation, suggesting that they may block nuclear targeting of the preintegration complex. Selection of viruses resistant to BI-2 revealed that substitutions at residues A105 and T107 within the capsid (CA) amino-terminal domain (CANTD) conferred high-level resistance to both compounds, implicating CA as the antiviral target. Direct binding of BI-1 and/or BI-2 to CANTD was demonstrated using isothermal titration calorimetry and nuclear magnetic resonance (NMR) chemical shift titration analyses. A high-resolution crystal structure of the BI-1:CANTD complex revealed that the inhibitor bound within a recently identified inhibitor binding pocket (CANTD site 2) between CA helices 4, 5, and 7, on the surface of the CANTD, that also corresponds to the binding site for the host factor CPSF-6. The functional consequences of BI-1 and BI-2 binding differ from previously characterized inhibitors that bind the same site since the BI compounds did not inhibit reverse transcription but stabilized preassembled CA complexes. Hence, this new class of antiviral compounds binds CA and may inhibit viral replication by stabilizing the viral capsid.

The emergence of resistance to existing classes of antiretroviral drugs necessitates finding new HIV-1 targets for drug discovery. The viral capsid (CA) protein represents one such potential new target. CA is sufficient to form mature HIV-1 capsids in vitro, and extensive structure-function and mutational analyses of CA have shown that the proper assembly, morphology, and stability of the mature capsid core are essential for the infectivity of HIV-1 virions. Here we describe the development of an in vitro capsid assembly assay based on the association of CA-NC subunits on immobilized oligonucleotides. This assay was used to screen a compound library, yielding several different families of compounds that inhibited capsid assembly. Optimization of two chemical series, termed the benzodiazepines (BD) and the benzimidazoles (BM), resulted in compounds with potent antiviral activity against wild-type and drug-resistant HIV-1. Nuclear magnetic resonance (NMR) spectroscopic and X-ray crystallographic analyses showed that both series of inhibitors bound to the N-terminal domain of CA. These inhibitors induce the formation of a pocket that overlaps with the binding site for the previously reported CAP inhibitors but is expanded significantly by these new, more potent CA inhibitors. Virus release and electron microscopic (EM) studies showed that the BD compounds prevented virion release, whereas the BM compounds inhibited the formation of the mature capsid. Passage of virus in the presence of the inhibitors selected for resistance mutations that mapped to highly conserved residues surrounding the inhibitor binding pocket, but also to the C-terminal domain of CA. The resistance mutations selected by the two series differed, consistent with differences in their interactions within the pocket, and most also impaired virus replicative capacity. Resistance mutations had two modes of action, either directly impacting inhibitor binding affinity or apparently increasing the overall s

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.

The emergence of resistance to existing classes of antiretroviral drugs necessitates finding new HIV-1 targets for drug discovery. The viral capsid (CA) protein represents one such potential new target. CA is sufficient to form mature HIV-1 capsids in vitro, and extensive structure-function and mutational analyses of CA have shown that the proper assembly, morphology, and stability of the mature capsid core are essential for the infectivity of HIV-1 virions. Here we describe the development of an in vitro capsid assembly assay based on the association of CA-NC subunits on immobilized oligonucleotides. This assay was used to screen a compound library, yielding several different families of compounds that inhibited capsid assembly. Optimization of two chemical series, termed the benzodiazepines (BD) and the benzimidazoles (BM), resulted in compounds with potent antiviral activity against wild-type and drug-resistant HIV-1. Nuclear magnetic resonance (NMR) spectroscopic and X-ray crystallographic analyses showed that both series of inhibitors bound to the N-terminal domain of CA. These inhibitors induce the formation of a pocket that overlaps with the binding site for the previously reported CAP inhibitors but is expanded significantly by these new, more potent CA inhibitors. Virus release and electron microscopic (EM) studies showed that the BD compounds prevented virion release, whereas the BM compounds inhibited the formation of the mature capsid. Passage of virus in the presence of the inhibitors selected for resistance mutations that mapped to highly conserved residues surrounding the inhibitor binding pocket, but also to the C-terminal domain of CA. The resistance mutations selected by the two series differed, consistent with differences in their interactions within the pocket, and most also impaired virus replicative capacity. Resistance mutations had two modes of action, either directly impacting inhibitor binding affinity or apparently increasing the overall s

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.