Maturation Inhibitors (MI), a new class of anti-HIV-1 compounds, act by blocking the maturation of virions into infectious particles. Bevirimat (BVM), a first-in-class betulinic acid-based compound, acts by blocking a late step in protease-mediated Gag processing: the cleavage of the capsid-spacer peptide 1 (CA-SP1) intermediate into mature CA. BVM was shown to be safe and effective in reducing viral loads in patients. However, polymorphisms in the SP1 region of Gag reduced HIV-1 susceptibility to BVM in patients, effectively halting BVM’s clinical development.
We carried out extensive screening to identify BVM derivatives with both increased potency against multiple HIV-1 clades and activity against primary isolates containing polymorphisms in SP1. Compound activity was tested in biochemical and biological assays measuring CA-SP1 processing and virus replication kinetics. Selection experiments with both clade B and clade C isolates were performed to identify mutations that confer resistance. Virological, structural, and molecular approaches were applied to elucidate the mechanism of resistance for each mutant. Further, combination assays with MIs alongside protease or integrase inhibitors were performed to determine inhibitor synergy or antagonism.
We identified a set of BVM derivatives that are markedly more potent than BVM against clade B HIV-1 and show robust activity against SP1 polymorphic strains, clinical isolates, and some BVM-resistant mutants. Selection experiments with a clade B isolate identified an SP1-A1V mutation, and a CA-P157A mutation located in the major homology region (MHR) of CA. Selections with a clade C isolate identified several mutations in SP1. 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. Analysis of the HIV-1 database reveals that Ala1 of SP1 and Pro157 of CA are conserved in ~99.95% of available sequences. To date, combination assays with potent MI candidates have shown no antagonism with protease or integrase inhibitors.
This study identifies a panel of BVM derivatives that display improvements upon BVM in antiviral potency and breadth of activity. The characterization of resistant mutants provides 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 supports ongoing clinical development of this class of inhibitors.