A betulinic acid-based compound, bevirimat (BVM), the first-in-class HIV-1 maturation inhibitor, acts by blocking 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 to be safe and effective in reducing viral loads in HIV-1-infected patients. However, single-amino-acid polymorphisms in the SP1 region of Gag reduced HIV-1 susceptibility to BVM in patients.
We carried out an extensive medicinal chemistry campaign to identify BVM derivatives that demonstrate increased potency against consensus clade B strains of HIV-1 and are active against primary isolates with polymorphisms in SP1. Compound activity was tested in assays that measure CA-SP1 processing and virus replication kinetics. Selection experiments were performed to identify mutations that confer resistance to these novel compounds and a variety of virological, structural, and molecular approaches were applied to elucidate the mechanism of resistance for each mutant. To evaluate the effect of Gag polymorphisms and resistance mutations on the kinetics of Gag processing, pulse-chase radiolabeling assays were performed.
We identified a set of BVM derivatives that are more potent than BVM against WT HIV-1 and show robust antiviral activity against SP1 polymorphic strains and clinical isolates. The best of these analogs retain significant activity against BVM-resistant mutants. Selection experiments identified an Ala-to-Val mutation at SP1 residue 1 (SP1-A1V). In addition, we also selected for the mutation CA-P157A, located in the major homology region (MHR) of CA. Remarkably, the P157A mutant was resistant to not only BVM and the second-generation BVM analogs but also to the structurally distinct maturation inhibitor PF-46396. Pulse-chase data demonstrate that CA-SP1 processing kinetics for P157A are similar to those of the WT. Analysis of the HIV-1 database reveals that Ala1 of SP1 and Pro157 of CA are conserved in ~99.95% of available sequences.
This study identifies a panel of BVM derivatives that display marked improvements relative to BVM in antiviral potency and breadth of activity. The characterization of resistant mutants provides novel insights into the structure of the maturation inhibitor-binding site and the role of SP1 and the CA MHR in virus assembly and maturation. This study will support ongoing clinical development of this class of inhibitors.