Chemoproteomics Reveals FAM114A2 as a Functional Target of Wikstroelide E for Reversal of HIV-1 Latency

The latent reservoir of HIV-1 is a hidden fortress for escaping from the immune system and preventing antiretroviral therapy. The reversal of latent reservoirs is one of the key components of the HIV eradication strategy. Many natural diterpenoids exhibit a high activity of HIV latency reactivation. However, their functional targets are largely unknown. In this study, a daphnane diterpene named Wikstroelide E is identified with very high activity for reversing the latent HIV-1 with an EC₅₀ of 2.14 ± 0.11 nM in 2D10 cells and 2.06 ± 0.20 nM in J-Lat A2 cells by using flow cytometry. We confirmed that Wikstroelide E could activate latent HIV-1 via the PKC β-NF-κB signal pathway using large-scale quantitative proteomics analysis as well as biological validations. Furthermore, photoreactive probe #2-PP was synthesized through modification of the C₂₀-OH of Wikstroelide E with a diazirine alkyne tag for covalent bonding to the target proteins in cells. A total of 15 potential targets were identified for Wikstroelide E by using quantitative chemoproteomics. Among them, FAM114A2 was verified as the functional target for HIV-1 latency reversal through the combination of RNAi experiments, pull-down assays, and colocalization analysis in cells. Knockdown of FAM114A2 suppressed the HIV-1 latency reversal effect of PKC agonist-type latency reversal agents (LRAs) but not those of non-PKC agonist-type LRAs. FAM114A2 serves as a functional target of Wikstroelide E for reversing HIV-1 latency and is also implicated in other PKC agonist-driven HIV-1 latency reversal. Our results provide valuable information for understanding the molecular mechanisms of Wikstroelide E for HIV latency reversal, which highlights diterpenoids as a promising source for developing therapeutic LRAs.