Elevated temperature triggers human respiratory syncytial virus F protein six-helix bundle formation.

Human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infection in infants, immunocompromised patients, and the elderly. The RSV fusion (F) protein mediates fusion of the viral envelope with the target cell membrane during virus entry and is a primary target for antiviral drug and vaccine development. The F protein contains two heptad repeat regions, HR1 and HR2. Peptides corresponding to these regions form a six-helix bundle structure that is thought to play a critical role in membrane fusion. However, characterization of six-helix bundle formation in native RSV F protein has been hindered by the fact that a trigger for F protein conformational change has yet to be identified. Here we demonstrate that RSV F protein on the surface of infected cells undergoes a conformational change following exposure to elevated temperature, resulting in the formation of the six-helix bundle structure. We first generated and characterized six-helix bundle-specific antibodies raised against recombinant peptides modeling the RSV F protein six-helix bundle structure. We then used these antibodies as probes to monitor RSV F protein six-helix bundle formation in response to a diverse array of potential triggers of conformational changes. We found that exposure of 'membrane-anchored' RSV F protein to elevated temperature (45-55 degrees C) was sufficient to trigger six-helix bundle formation. Antibody binding to the six-helix bundle conformation was detected by both flow cytometry and cell-surface immunoprecipitation of the RSV F protein. None of the other treatments, including interaction with a number of potential receptors, resulted in significant binding by six-helix bundle-specific antibodies. We conclude that native, untriggered RSV F protein exists in a metastable state that can be converted in vitro to the more stable, fusogenic six-helix bundle conformation by an increase in thermal energy. These findings help to better define the mechanism of RSV F-mediated membrane fusion and have important implications for the identification of therapeutic strategies and vaccines targeting RSV F protein conformational changes.

Anti-AIDS agents. 78. Design, synthesis, metabolic stability assessment, and antiviral evaluation of novel betulinic acid derivatives as potent anti-human immunodeficiency virus (HIV) agents.

In a continuing study of potent anti-HIV agents, seventeen 28,30-disubstituted betulinic acid (BA, 1) derivatives and seven novel 3,28-disubstituted BA analogues were designed, synthesized, and evaluated for in vitro antiviral activity. Among them, compound 21 showed an improved solubility and equal anti-HIV potency (EC(50) = 0.09 microM) when compared to HIV entry inhibitors 3b (IC9564, (3R,4S)-N'-[N-[3beta-hydroxy-lup-20(29)-en-28-oyl]-8-aminooctanoyl]-4-amino-3-hydroxy-6-methylheptanoic acid) and 4 (A43-D, [[N-[3beta-O-(3',3'-dimethylsuccinyl)-lup-20(29)-en-28-oyl]-7-aminoheptyl]carbamoyl]methane). Using a cyclic secondary amine to form the C-28 amide bond increased the metabolic stability of the derivatives significantly in pooled human liver microsomes. The most potent compounds 47 and 48 displayed potent anti-HIV activity with EC(50) values of 0.007 and 0.006 microM, respectively. These results are slightly better than that of bevirimat (2, 3',3'-dimethylsuccinylbetulinic acid), which is currently in phase IIb clinical trials. Compounds 47 and 48 should serve as attractive promising leads to develop next generation, metabolically stable, 3,28-disubstituted bifunctional HIV-1 inhibitors as clinical trials candidates.

Susceptibility of human immunodeficiency virus type 1 to the maturation inhibitor bevirimat is modulated by baseline polymorphisms in Gag spacer peptide 1.

In this study, we evaluated baseline susceptibility to bevirimat (BVM), the first in a new class of antiretroviral agents, maturation inhibitors. We evaluated susceptibility to BVM by complete gag genotypic and phenotypic testing of 20 patient-derived human immunodeficiency virus type 1 isolates and 20 site-directed mutants. We found that reduced BVM susceptibility was associated with naturally occurring polymorphisms at positions 6, 7, and 8 in Gag spacer peptide 1.

Bevirimat: a novel maturation inhibitor for the treatment of HIV-1 infection.

Existing antiretroviral treatments for HIV type-1 (HIV-1) disease are limited by problems of resistance and drug-drug interactions. Bevirimat is a novel HIV-1 maturation inhibitor with a mechanism of action that is distinct from other antiretroviral agents. Specific inhibition of the final rate-limiting step in Gag processing by bevirimat prevents release of mature capsid protein from its precursor (CA-SP1), resulting in the production of immature, non-infectious virus particles. Bevirimat inhibits replication of both wild-type and drug-resistant HIV-1 isolates in vitro, achieving similar 50% inhibitory concentration values with both categories. Serial drug passage studies have identified six single amino acid substitutions that independently confer bevirimat resistance. These resistance mutations occur at or near the CA-SP1 cleavage site, which is not a known target for resistance to other antiretroviral drugs. Bevirimat has demonstrated a consistent pharmacokinetic profile in healthy volunteers and HIV-infected patients, with peak plasma concentrations attained approximately 1-3 h after dosing. Plasma concentrations decrease in a log-linear manner with a mean plasma elimination halflife of 58-80 h, supporting once-daily dosing. Animal studies suggest that elimination of bevirimat is primarily by hepatic glucuronidation and hepatobiliary excretion. There is minimal renal elimination, with < 1% of the administered dose appearing in the urine. In responsive patients, bevirimat has demonstrated a robust dosedependent reduction in viral load (> 1.5 log10 copies/ml). Short-term administration (< or = 14 days) of bevirimat is well tolerated, even when used in combination with other antiretroviral agents. Further studies to evaluate the long-term efficacy and tolerability of bevirimat are currently underway.

Potent activity of the HIV-1 maturation inhibitor bevirimat in SCID-hu Thy/Liv mice.

BACKGROUND: The HIV-1 maturation inhibitor, 3-O-(3',3'-dimethylsuccinyl) betulinic acid (bevirimat, PA-457) is a promising drug candidate with 10 nM in vitro antiviral activity against multiple wild-type (WT) and drug-resistant HIV-1 isolates. Bevirimat has a novel mechanism of action, specifically inhibiting cleavage of spacer peptide 1 (SP1) from the C-terminus of capsid which results in defective core condensation. METHODS AND FINDINGS: Oral administration of bevirimat to HIV-1-infected SCID-hu Thy/Liv mice reduced viral RNA by >2 log(10) and protected immature and mature T cells from virus-mediated depletion. This activity was observed at plasma concentrations that are achievable in humans after oral dosing, and bevirimat was active up to 3 days after inoculation with both WT HIV-1 and an AZT-resistant HIV-1 clinical isolate. Consistent with its mechanism of action, bevirimat caused a dose-dependent inhibition of capsid-SP1 cleavage in HIV-1-infected human thymocytes obtained from these mice. HIV-1 NL4-3 with an alanine-to-valine substitution at the N-terminus of SP1 (SP1/A1V), which is resistant to bevirimat in vitro, was also resistant to bevirimat treatment in the mice, and SP1/AIV had replication and thymocyte kinetics similar to that of WT NL4-3 with no evidence of fitness impairment in in vivo competition assays. Interestingly, protease inhibitor-resistant HIV-1 with impaired capsid-SP1 cleavage was hypersensitive to bevirimat in vitro with a 50% inhibitory concentration 140 times lower than for WT HIV-1. CONCLUSIONS: These results support further clinical development of this first-in-class maturation inhibitor and confirm the usefulness of the SCID-hu Thy/Liv model for evaluation of in vivo antiretroviral efficacy, drug resistance, and viral fitness.

Anti-AIDS agents 73: structure-activity relationship study and asymmetric synthesis of 3-O-monomethylsuccinyl-betulinic acid derivatives.

3-O-3'(or 2')-Methylsuccinyl-betulinic acid (MSB) derivatives were separated by using recycle HPLC. The structures of four isomers were assigned by NMR and asymmetric synthesis. 3-O-3'S-Methylsuccinyl-betulinic acid (3'S-MSB, 4) exhibited potent anti-HIV activity with an EC(50) value of 0.0087microM and a TI value of 6.3x10(3), which is comparable to the data for bevirimat (DSB, PA-457), a current clinical trials drug that was also derived from betulinic acid. The anti-HIV potency of 4 was slightly better than that of AZT.

Phase I and II study of the safety, virologic effect, and pharmacokinetics/pharmacodynamics of single-dose 3-o-(3',3'-dimethylsuccinyl)betulinic acid (bevirimat) against human immunodeficiency virus infection.

Bevirimat [3-O-(3',3'-dimethylsuccinyl)betulinic acid] is the first in a new class of anti-human immunodeficiency virus (HIV) drugs that inhibit viral maturation by specifically blocking cleavage of the Gag capsid (CA) precursor, CA-SP1, to mature CA protein, resulting in defective core condensation and release of immature noninfectious virions. Four cohorts of six HIV-infected adults, with CD4 counts of >200 and plasma viral loads of 5,000 to 250,000 transcripts/ml and not currently receiving antiretroviral therapy, were randomized to receive a single oral dose of placebo, 75, 150, or 250 mg of bevirimat. Thirty blood samples for drug concentrations and 20 HIV RNA measures were collected from each subject over a 20-day period. Candidate pharmacokinetic/pharmacodynamic models were fit to individual subjects by maximum likelihood followed by Bayesian estimation; model discrimination was by corrected Akaike's Information Criterion. The bevirimat pharmacokinetics was well described by an oral two-compartment linear model (r(2), 0.98), with a mean (percent coefficient of variation) half-life of 60.3 (13.6) h and apparent oral clearance of bevirimat from the plasma compartment of 0.17 (18) liters/h. HIV RNA was modeled as being produced in infected CD4 cells, with bevirimat inhibiting infection of new CD4 cells thru a Hill-type function (r(2), 0.87). Single oral doses of bevirimat were well tolerated and demonstrated a dose-dependent reduction in viral load. The average maximum reduction from baseline following the 150- and 250-mg doses was greater than 0.45 log(10), with individual patients having reductions of greater than 0.7 log(10). No bevirimat resistance mutations were detected during the course of the study.

Determinants of activity of the HIV-1 maturation inhibitor PA-457.

3-O-(3',3'-dimethylsuccinyl) betulinic acid, also termed PA-457 or DSB, is a novel HIV-1 inhibitor that blocks virus maturation by disrupting cleavage of the capsid precursor, CA-SP1. To better define the molecular target for PA-457, we prepared a panel of mutant viruses with point deletions spanning the CA-SP1 cleavage domain and characterized each of these viruses for PA-457 sensitivity. Our results indicate that amino acid residues in the N-terminal half of SP1 serve as determinants of PA-457 activity, while residues in the C-terminal half of SP1 were not involved in compound activity. These findings support and extend previous observations that PA-457 is a specific inhibitor of CA-SP1 cleavage and identify the CA-SP1 domain as the primary viral determinant for this novel inhibitor of HIV-1 replication.

The discovery of a class of novel HIV-1 maturation inhibitors and their potential in the therapy of HIV.

Although HIV infection is now primarily treated with reverse transcriptase and protease inhibitors, HIV therapy must look toward new drugs with novel mechanism(s) of action to both improve efficacy and address the growing problem of drug resistance. Using natural products as a source of biologically active compounds, our drug discovery program has successfully optimised the natural product betulinic acid to the first-in-class maturation inhibitor 3-O-(3',3'-dimethylsuccinyl)-betulinic acid (DSB). DSB's unique viral target has been identified as a late step in Gag processing. Specifically, it inhibits the cleavage of the capsid precursor, CA-SP1, resulting in a block to the processing of mature capsid protein leading to a defect in viral core condensation. DSB represents a unique class of anti-HIV compounds that inhibit virus maturation and provide additional opportunities for anti-HIV therapy. In this review, the discovery of DSB and its mode of action are summarised. Anti-AIDS Agents part 64. For part 63 in the series, see YU D, LEE KH: Recent progress and prospects on plant-derived anti-HIV agents and analogs. In: Medicinal Chemistry of Bioactive Natural Products. XT Liang, WS Fang (Eds), Wiley, New York, USA (2005) (In Press).

3-O-Glutaryl-dihydrobetulin and related monoacyl derivatives as potent anti-HIV agents.

3-O-Acyl-betulin and -dihydrobetulin derivatives were prepared and evaluated for anti-HIV activity. 3-O-Glutaryl-dihydrobetulin (17) demonstrated extremely potent anti-HIV activity with an EC(50) value of 2 x 10(-5) microM and a TI value of 1.12 x 10(6). 3-O-(3',3'-Dimethylsuccinyl)- and 3-O-(3',3'-dimethylglutaryl)-dihydrobetulins (15, 16) were also potent anti-HIV compounds with EC(50) values of 0.0017 and 0.0013 microM, respectively, and TI values of 16,160 and 19,530, respectively.

Direct evidence that C-peptide inhibitors of human immunodeficiency virus type 1 entry bind to the gp41 N-helical domain in receptor-activated viral envelope.

While it has been established that peptides modeling the C-helical region of human immunodeficiency virus type 1 gp41 are potent in vivo inhibitors of virus replication, their mechanism of action has yet to be determined. It has been proposed, but never directly demonstrated, that these peptides block virus entry by interacting with gp41 to disrupt the formation or function of a six-helix bundle structure. Using a six-helix bundle-specific monoclonal antibody with isolate-restricted Env reactivity, we provide the first direct evidence that, in receptor-activated viral Env, C-peptide entry inhibitors bind to the gp41 N-helical coiled-coil to form a peptide/protein hybrid structure and, in doing so, disrupt native six-helix bundle formation.