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AG7088 is a potent, irreversible inhibitor of human rhinovirus (HRV) 3C protease {inactivation rate constant (k obs /[I]} ‫؍‬ 1,470,000 ؎ 440,000 M ؊1 s ؊1 for HRV 14) that was discovered by protein structure-based drug design methodologies. In H1-HeLa and MRC-5 cell protection assays, AG7088 inhibited the replication of all HRV serotypes (48 of 48) tested with a mean 50% effective concentration (EC 50 ) of 0.023 M (range, 0.003 to 0.081 M) and a mean EC 90 of 0.082 M (range, 0.018 to 0.261 M) as well as that of related picornaviruses including coxsackieviruses A21 and B3, enterovirus 70, and echovirus 11. No significant reductions in the antiviral activity of AG7088 were observed when assays were performed in the presence of ␣ 1 -acid glycoprotein or mucin, proteins present in nasal secretions. The 50% cytotoxic concentration of AG7088 was >1,000 M, yielding a therapeutic index of >12,346 to >333,333. In a single-cycle, time-of-addition assay, AG7088 demonstrated antiviral activity when added up to 6 h after infection. In contrast, a compound targeting viral attachment and/or uncoating was effective only when added at the initiation of virus infection. Direct inhibition of 3C proteolytic activity in infected cells treated with AG7088 was demonstrated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis analysis of radiolabeled proteins, which showed a dose-dependent accumulation of viral precursor polyproteins and reduction of processed protein products. The broad spectrum of antiviral activity of AG7088, combined with its efficacy even when added late in the virus life cycle, highlights the advantages of 3C protease as a target and suggests that AG7088 will be a promising clinical candidate. MATERIALS AND METHODSCompounds. AG7088 and pleconaril (17) were synthesized at Agouron Pharmaceuticals, Inc. Pirodavir (1) was kindly provided by Janssen Research Foundation (Beerse, Belgium), and WIN 51711 (40) was kindly provided by Sterling Winthrop Research Institute (Collegeville, Pa.). Ganciclovir (Syntex Corp., Palo Alto, Calif.) was obtained from a local pharmacy, and acyclovir was purchased from Sigma (St. Louis, Mo.).Cells and virus strains. All numbered HRV serotypes, echovirus type 11 (EV 11), enterovirus type 70 (ETV 70), coxsackievirus types A21 (CAV 21) and B3 strain Nancy (CVB 3), human cytomegalovirus (HCMV) strain AD169, and herpes simplex virus type 1 (HSV-1) strain McIntyre were purchased from the American Type Culture Collection (ATCC; Manassas, Va.). HRV Hanks and a nasal lavage from a patient challenged with HRV Hanks were kindly provided by Ronald Turner from the Medical University of South Carolina, Charleston, S.C. HRV and coxsackievirus stocks were propagated, and antiviral assays were performed, in H1-HeLa cells (ATCC) incubated at 34 and 37°C, respectively. ETV 70, EV 11, and HCMV stocks were propagated, and antiviral assays were performed, in MRC-5 (ATCC) cells at 37°C. HSV-1 stocks were propagated, and antiviral assays were performed, in Vero (ATCC) cells incubated at 37°C. Vero cells ...

A new small-molecule inhibitor class that targets virion maturation was identified from a human immunodeficiency virus type 1 (HIV-1) antiviral screen. PF-46396, a representative molecule, exhibits antiviral activity against HIV-1 laboratory strains and clinical isolates in T-cell lines and peripheral blood mononuclear cells (PBMCs). PF-46396 specifically inhibits the processing of capsid (CA)/spacer peptide 1 (SP1) (p25), resulting in the accumulation of CA/SP1 (p25) precursor proteins and blocked maturation of the viral core particle. Viral variants resistant to PF-46396 contain a single amino acid substitution in HIV-1 CA sequences (CAI201V), distal to the CA/SP1 cleavage site in the primary structure, which we demonstrate is sufficient to confer significant resistance to PF-46396 and 3-O-(3,3-dimethylsuccinyl) betulinic acid (DSB), a previously described maturation inhibitor. Conversely, a single amino substitution in SP1 (SP1A1V), which was previously associated with DSB in vitro resistance, was sufficient to confer resistance to DSB and PF-46396. Further, the CAI201V substitution restored CA/SP1 processing in HIV-1-infected cells treated with PF-46396 or DSB. Our results demonstrate that PF-46396 acts through a mechanism that is similar to DSB to inhibit the maturation of HIV-1 virions. To our knowledge, PF-46396 represents the first small-molecule HIV-1 maturation inhibitor that is distinct in chemical class from betulinic acid-derived maturation inhibitors (e.g., DSB), demonstrating that molecules of diverse chemical classes can inhibit this mechanism.

Broadly cross-reactive neutralizing antibodies (bNabs) represent powerful tools to combat human immunodeficiency virus type 1 (HIV-1) infection. Here, we examined whether HIV-1-specific bNabs are capable of cross-neutralizing distantly related simian immunodeficiency viruses (SIVs) infecting central (Pan troglodytes troglodytes) (SIVcpzPtt) and eastern (Pan troglodytes schweinfurthii) (SIVcpzPts) chimpanzees (n = 11) as well as western gorillas (Gorilla gorilla gorilla) (SIVgor) (n = 1). We found that bNabs directed against the CD4 binding site (n = 10), peptidoglycans at the base of variable loop 3 (V3) (n = 5), and epitopes at the interface of surface (gp120) and membrane-bound (gp41) envelope glycoproteins (n = 5) failed to neutralize SIVcpz and SIVgor strains. In addition, apex V2-directed bNabs (n = 3) as well as llama-derived (heavy chain only) antibodies (n = 6) recognizing both the CD4 binding site and gp41 epitopes were either completely inactive or neutralized only a fraction of SIVcpzPtt strains. In contrast, one antibody targeting the membrane-proximal external region (MPER) of gp41 (10E8), functional CD4 and CCR5 receptor mimetics (eCD4-Ig, eCD4-Igmim2, CD4-218.3-E51, and CD4-218.3-E51-mim2), as well as mono- and bispecific anti-human CD4 (iMab and LM52) and CCR5 (PRO140, PRO140-10E8) receptor antibodies neutralized >90% of SIVcpz and SIVgor strains with low-nanomolar (0.13 to 8.4 nM) potency. Importantly, the latter antibodies blocked virus entry not only in TZM-bl cells but also in Cf2Th cells expressing chimpanzee CD4 and CCR5 and neutralized SIVcpz in chimpanzee CD4+ T cells, with 50% inhibitory concentrations (IC50s) ranging from 3.6 to 40.5 nM. These findings provide new insight into the protective capacity of anti-HIV-1 bNabs and identify candidates for further development to combat SIVcpz infection.

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