A betulinic acid-based compound, bevirimat (BVM), inhibits HIV-1 maturation by blocking a late step in protease-mediated Gag processing: the cleavage of the capsid-spacer peptide 1 (CA-SP1) intermediate to mature CA. Previous studies showed that mutations conferring resistance to BVM cluster around the CA-SP1 cleavage site. Single amino acid polymorphisms in the SP1 region of Gag and the C terminus of CA reduced HIV-1 susceptibility to BVM, leading to the discontinuation of BVM's clinical development. We recently reported a series of "second-generation" BVM analogs that display markedly improved potency and breadth of activity relative to the parent molecule. Here, we demonstrate that viral clones bearing BVM resistance mutations near the C terminus of CA are potently inhibited by second-generation BVM analogs. We performed de novo selection experiments to identify mutations that confer resistance to these novel compounds. Selection experiments with subtype B HIV-1 identified an Ala-to-Val mutation at SP1 residue 1 and a Pro-to-Ala mutation at CA residue 157 within the major homology region (MHR). In selection experiments with subtype C HIV-1, we identified mutations at CA residue 230 (CA-V230M) and SP1 residue 1 (SP1-A1V), residue 5 (SP1-S5N), and residue 10 (SP1-G10R). The positions at which resistance mutations arose are highly conserved across multiple subtypes of HIV-1. We demonstrate that the mutations confer modest to high-level maturation inhibitor resistance. In most cases, resistance was not associated with a detectable increase in the kinetics of CA-SP1 processing. These results identify mutations that confer resistance to second-generation maturation inhibitors and provide novel in-sights into the mechanism of resistance. IMPORTANCE HIV-1 maturation inhibitors are a class of small-molecule compounds that block a late step in the viral protease-mediated processing of the Gag polyprotein precursor, the viral protein responsible for the formation of virus particles. The first-in-class HIV-1 maturation inhibitor bevirimat was highly effective in blocking HIV-1 replication, but its activity was compromised by naturally occurring sequence polymorphisms within Gag. Recently developed bevirimat analogs, referred to as "second-generation" maturation inhibitors, overcome this issue. To understand more about how these second-generation compounds block HIV-1 maturation, here we selected for HIV-1 mutants that are resistant to these compounds. Selections were performed in the context of two different subtypes of HIV-1. We identified a small set of mutations at highly conserved positions within the capsid and spacer peptide 1 domains of Gag that confer resistance. Identification and analysis of these maturation inhibitor-resistant mutants provide insights into the mechanisms of resistance to these compounds.

Maturation of HIV-1 particles encompasses a complex morphological transformation of Gag via an orchestrated series of proteolytic cleavage events. A longstanding question concerns the structure of the C-terminal region of CA and the peptide SP1 (CA–SP1), which represents an intermediate during maturation of the HIV-1 virus. By integrating NMR, cryo-EM, and molecular dynamics simulations, we show that in CA–SP1 tubes assembled in vitro, which represent the features of an intermediate assembly state during maturation, the SP1 peptide exists in a dynamic helix–coil equilibrium, and that the addition of the maturation inhibitors Bevirimat and DFH-055 causes stabilization of a helical form of SP1. Moreover, the maturation-arresting SP1 mutation T8I also induces helical structure in SP1 and further global dynamical and conformational changes in CA. Overall, our results show that dynamics of CA and SP1 are critical for orderly HIV-1 maturation and that small molecules can inhibit maturation by perturbing molecular motions.

Antiretroviral therapy has led to a profound improvement in the clinical care of HIV-infected patients. However, drug tolerability and the evolution of drug resistance have limited treatment options for many patients. Maturation inhibitors are a new class of antiretroviral agents for treatment of HIV-1. They act by interfering with the maturation of the virus by blocking the last step in Gag processing: the cleavage of the capsid-spacer peptide 1 (CA-SP1) intermediate to mature CA by the viral protease (PR). The first-in-class maturation inhibitor bevirimat (BVM) failed against a subset of HIV-1 isolates in clinical trials due to polymorphisms present in the CA-SP1 region of the Gag protein. Sequence analysis indicated that these polymorphisms are more common in non-clade B strains of HIV-1 such as HIV-1 clade C. Indeed, BVM was found to be ineffective against HIV-1 clade C molecular clones tested in this study. A number of BVM analogs were synthesized by chemical modifications at the C-28 position to improve its activity. The new BVM analogs displayed potent activity against HIV-1 clade B and C and also reduced infectivity of the virus. This study identifies novel and broadly active BVM analogs that may ultimately demonstrate efficacy in the clinic.

Concomitant with the release of human immunodeficiency virus type 1 (HIV-1) particles from the infected cell, the viral protease cleaves the Gag polyprotein precursor at a number of sites to trigger virus maturation. We previously reported that a betulinic acid-derived compound, bevirimat (BVM), blocks HIV-1 maturation by disrupting a late step in protease-mediated Gag processing: the cleavage of the capsid-spacer peptide 1 (CA-SP1) intermediate to mature CA. BVM was shown in multiple clinical trials to be safe and effective in reducing viral loads in HIV-1-infected patients. However, naturally occurring polymorphisms in the SP1 region of Gag (e.g., SP1-V7A) led to a variable response in some BVM-treated patients. The reduced susceptibility of SP1-polymorphic HIV-1 to BVM resulted in the discontinuation of its clinical development. To overcome the loss of BVM activity induced by polymorphisms in SP1, we carried out an extensive medicinal chemistry campaign to develop novel maturation inhibitors. In this study, we focused on alkyl amine derivatives modified at the C-28 position of the BVM scaffold. We identified a set of derivatives that are markedly more potent than BVM against an HIV-1 clade B clone (NL4-3) and show robust antiviral activity against a variant of NL4-3 containing the V7A polymorphism in SP1. One of the most potent of these compounds also strongly inhibited a multiclade panel of primary HIV-1 isolates. These data demonstrate that C-28 alkyl amine derivatives of BVM can, to a large extent, overcome the loss of susceptibility imposed by polymorphisms in SP1.

BACKGROUND:Bevirimat, the prototype Human Immunodeficiency Virus type 1 (HIV-1) maturation inhibitor, is highly potent in cell culture and efficacious in HIV-1 infected patients. In contrast to inhibitors that target the active site of the viral protease, bevirimat specifically inhibits a single cleavage event, the final processing step for the Gag precursor where p25 (CA-SP1) is cleaved to p24 (CA) and SP1.RESULTS:In this study, photoaffinity analogs of bevirimat and mass spectrometry were employed to map the binding site of bevirimat to Gag within immature virus-like particles. Bevirimat analogs were found to crosslink to sequences overlapping, or proximal to, the CA-SP1 cleavage site, consistent with previous biochemical data on the effect of bevirimat on Gag processing and with genetic data from resistance mutations, in a region predicted by NMR and mutational studies to have α-helical character. Unexpectedly, a second region of interaction was found within the Major Homology Region (MHR). Extensive prior genetic evidence suggests that the MHR is critical for virus assembly.CONCLUSIONS:This is the first demonstration of a direct interaction between the maturation inhibitor, bevirimat, and its target, Gag. Information gained from this study sheds light on the mechanisms by which the virus develops resistance to this class of drug and may aid in the design of next-generation maturation inhibitors.

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.

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.