Abstract
ABSTRACTHIV-1 maturation inhibitors are a novel class of antiretroviral compounds that consist of two structurally distinct chemical classes: betulinic acid derivatives and the pyridone-based compound PF-46396. It is currently believed that both classes act by similar modes of action to generate aberrant noninfectious particles via inhibition of CA-SP1 cleavage during Gag proteolytic processing. In this study, we utilized a series of novel analogues with decreasing similarity to PF-46396 to determine the chemical groups within PF-46396 that contribute to antiviral activity, Gag binding, and the relationship between these essential properties. A spectrum of antiviral activity (active, intermediate, and inactive) was observed across the analogue series with respect to CA-SP1 cleavage and HIV-1 (NL4-3) replication kinetics in Jurkat T cells. We demonstrate that selected inactive analogues are incorporated into wild-type (WT) immature particles and that one inactive analogue is capable of interfering with PF-46396 inhibition of CA-SP1 cleavage. Mutations that confer PF-46396 resistance can impose a defective phenotype on HIV-1 that can be rescued in a compound-dependent manner. Some inactive analogues retained the capacity to rescue PF-46396-dependent mutants (SP1-A3V, SP1-A3T, and CA-P157S), implying that they can also interact with mutant Gag. The structure-activity relationships observed in this study demonstrate that (i) the tert-butyl group is essential for antiviral activity but is not an absolute requirement for Gag binding, (ii) the trifluoromethyl group is optimal but not essential for antiviral activity, and (iii) the 2-aminoindan group is important for antiviral activity and Gag binding but is not essential, as its replacement is tolerated.IMPORTANCE Combinations of antiretroviral drugs successfully treat HIV/AIDS patients; however, drug resistance problems make the development of new mechanistic drug classes an ongoing priority. HIV-1 maturation inhibitors are novel as they target the Gag protein, specifically by inhibiting CA-SP1 proteolytic cleavage. The lack of high-resolution structural information of the CA-SP1 target in Gag has hindered our understanding of the inhibitor-binding pocket and maturation inhibitor mode of action. Therefore, we utilized analogues of the maturation inhibitor PF-46396 as chemical tools to determine the chemical components of PF-46396 that contribute to antiviral activity and Gag binding and the relationship between these essential properties. This is the first study to report structure-activity relationships of the maturation inhibitor PF-46396. PF-46396 is chemically distinct from betulinic acid-derived maturation inhibitors; therefore, our data provide a foundation of knowledge that will aid our understanding of how structurally distinct maturation inhibitors act by similar modes of action.
Highlights
HIV-1 maturation inhibitors are a novel class of antiretroviral compounds that consist of two structurally distinct chemical classes: betulinic acid derivatives and the pyridone-based compound PF-46396
We demonstrate that the tert-butyl group in the PF-46396 phenyl ring is essential for antiviral activity and cannot be replaced with a hydrogen, methyl, methoxy, trifluoromethyl, or chloro group
The contacts made by the tert-butyl group almost certainly improve the Gag binding efficiency, because levels of incorporated StA-MAT-23 are ϳ30% lower than those of PF-46396 in WT immature virus particles
Summary
HIV-1 maturation inhibitors are a novel class of antiretroviral compounds that consist of two structurally distinct chemical classes: betulinic acid derivatives and the pyridone-based compound PF-46396. Correct proteolytic processing of Gag is essential for the formation of infectious HIV-1 particles, as mutations that disrupt the cleavage of individual sites or alter the order in which sites are cleaved result in aberrant particles that have significantly reduced infectivity [13, 15, 21,22,23,24,25,26]. Protease inhibitors (PIs) have been developed, which target HIV-1 PR enzyme activity, preventing all cleavage events in Gag and Gag-Pol and resulting in noninfectious immature virus particles. The structural organization of maturation inhibitor-treated particles suggests that, in addition to preventing CA-SP1 cleavage, maturation inhibitors stabilize the immature Gag lattice and that both of these modes of action may contribute to the generation of noninfectious particles and, the inhibitory activity of maturation inhibitors [36, 37]
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