Identification of novel diimide and hydrazone scaffolds for synergistic HIV-1 shock and kill strategies

Combination antiretroviral therapy (ART) is currently the standard-of-care to control HIV replication in infected individuals. However, since this treatment mainly targets viral enzymes, the presence of latently-infected reservoirs and the emergence of drug resistance remain major barriers to viral eradication. To overcome this, a "Shock and Kill" approach has been developed, using "latency-reversing agents" (LRAs) to activate latent proviruses ("shock"), leading to viral elimination by ART and immune-mediated clearance ("kill"). We previously screened a small-molecule library on monocytic and CD4⁺ T cell lines latently infected with HIV, and identified two novel LRA candidates (compounds #C1 and #C3). Based on their structural features, we now identified two related derivatives, compounds #C2 and #C4. Compounds #C1 and #C2 possess diimide structures, whereas #C3 and #C4 have hydrazone structures. All four compounds synergistically enhanced LRA activity when combined with JQ1 or PKC activators in J-Lat 10.6 cells. Notably, combining #C4 with JQ1 further enhanced HIV reactivation showing limited responsiveness to JQ1 alone in J-Lat 10.6 model cells. In ex vivo purified CD4⁺ T cells from ART-suppressed individuals, #C4 elicited a statistically significant, donor-matched increase in HIV-1 transcription (ratio paired t-test, P = 0.0137, n = 8), whereas JQ1 did not reach significance under the same paired analysis despite a comparable mean magnitude. Notably, #C4 additionally induced measurable reactivation in donors with limited responsiveness to JQ1 alone. RNA-sequencing analyses indicated that compound #C4, JQ1 and PKC activators mediated LRA activity through distinct molecular mechanisms. Collectively, these findings delineate a structurally novel class of LRA candidates that engage a latency-reversal mechanism distinct from that of JQ1 and canonical PKC signaling, and thereby support further investigation of their utility within combinatorial strategies aimed at HIV-1 reservoirs.