Structure-Activity Relationship Analysis of Benzotriazine Analogues as HIV-1 Latency-Reversing Agents

Abstract

“Shock and kill” therapeutic strategies toward HIV eradication are based on the transcriptional activation of latent HIV with a latency-reversing agent (LRA) and the consequent killing of the reactivated cell by either the cytopathic effect of HIV or an arm of the immune system. We have recently found several benzotriazole and benzotriazine analogues that have the ability to reactivate latent HIV by inhibiting signal transducer and activator of transcription 5 (STAT5) SUMOylation and promoting STAT5 binding to the HIV long terminal repeat and increasing its transcriptional activity. To understand the essential structural groups required for biological activity of these molecules, we performed a systematic analysis of >40 analogues. First, we characterized the essential motifs within these molecules that are required for their biological activity. Second, we identified three benzotriazine analogues with similar activity. We demonstrated that these three compounds are able to increase STAT5 phosphorylation and transcriptional activity. All active analogues reactivate latent HIV in a primary cell model of latency and enhance the ability of interleukin-15 to reactivate latent HIV in cells isolated from aviremic participants. Third, this family of compounds also promote immune effector functions in vitro in the absence of toxicity or global immune activation. Finally, initial studies in mice suggest lack of acute toxicity in vivo. A better understanding of the biological activity of these compounds will help in the design of improved LRAs that work via inhibition of STAT5 SUMOylation.

An ultrasensitive planar array p24 Gag ELISA to detect individual HIV-1 viral particles and infected cells

“…This assay can also be performed directly using tissue culture media. For example, it could be used to evaluate the LRA activity of compounds in preclinical development or to evaluate the size of the replication competent reservoir calculating infectious units per million (IUPM), as recently done with the dELISA (DEVO assay) [20][21][22]25 ; or to evaluate increase in HIV protein translation ex vivo using cell lysates. Our results did show a better compatibility using FBS than HS, as the latter has a higher background and reduces the sensitivity of the assay.…”

“…We and others had previously evaluated the digital ELISA to measure p24 release upon stimulation of cells isolated from aviremic PLWH with different LRAs 20,22,25 . As such, being able to measure p24 using a lower volume with a similar sensitivity can potentially allow the performance of viral release kinetics upon stimulation 20 .…”

The Current Status of Latency Reversing Agents for HIV-1 Remission

“…However, this activating effect is observed only in combination with IL-2 because IL-2 binding to its cellular receptor triggers the recruitment of Janus family kinases 1 and 3 ( JAK1/JAK3), which then transduce the signal by phosphorylating specific tyrosine residues in STAT5 CTD, thereby maintaining STAT5 phosphorylated and activated status (131,134). More recently, a new generation of benzotriazine analogs was evaluated for HIV-1 latency reversal, and three of them induce HIV-1 gene expression similarly to the original HOAt and HODHBt compounds (135). As observed with the first generation of benzotriazole and benzotriazine analogs, this new generation of drugs has no effect on cellular viability or on global immune activation (135).…”

Potential Utility of Natural Killer Cells for Eliminating Cells Harboring Reactivated Latent HIV-1 Following the Removal of CD8+ T Cell-Mediated Pro-Latency Effect(s)

“…Another group of LRAs that promote HIV-1 reactivation are benzotriazole derivatives, such as 3-hydroxy-1,2,3-benzotriazin-4(3H)-one (HODHBt), 1-hydroxybenzotriazol (HOBt), and 1-hydroxy-7-amino benzotriazole (HOAt). HODHBt has been shown to reactivate latent HIV-1 in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15) [ 55 , 56 ]. This class of LRAs impairs signal transducer and activator of transcription 5 (STAT5) SUMOylation and increases STAT5 activity and occupancy of the HIV-1 5′LTR [ 55 ].…”