Mechanism by Which PF-3758309, a Pan Isoform Inhibitor of p21-Activated Kinases, Blocks Reactivation of HIV-1 Latency
The “block and lock” strategy represents a potential method for achieving a sterilizing cure for HIV-1 infection. Here, “block” refers to a compound’s ability to suppress latent HIV-1 proviral transcription, while “lock” refers to its ability to induce permanent silencing of the provirus. In previous work, we identified PF-3758309, a pan-isoform inhibitor of p21-activated kinases (PAKs), as a strong inhibitor of HIV-1 latency reversal, and the aim of this study was to investigate the mechanisms involved. We observed that both 24ST1NLESG cells (a cell model of HIV-1 latency) and purified CD4+ naïve and central memory T cells show high levels of PAK2 expression and lower levels of PAK1 and PAK4. Reducing PAK1 or PAK2 expression in 24ST1NLESG cells resulted in a modest yet statistically significant decrease in HIV-1 latency reversal, while reducing PAK4 had no effect. Conversely, overexpression of PAK1 markedly increased latency reversal. A phospho-protein array analysis indicated that PF-3758309 downregulates the NF-κB signaling pathway, suggesting this pathway as the likely mechanism by which PF-3758309 inhibits latency reversal. Additionally, cellular thermal shift assays combined with liquid chromatography and mass spectrometry were used to determine if PF-3758309’s activity was due to off-target effects. In both 24ST1NLESG cells and peripheral blood mononuclear cells, PF-3758309 bound to mitogen-activated protein kinase 1 and protein kinase A; however, silencing either kinase had no effect on HIV-1 latency reversal. Overall, our findings suggest that PAK1 and PAK2 are critical in maintaining HIV-1 latency.