UC Irvine

Suppression of human immunodeficiency virus (HIV) infection is achieved with antiretroviral therapy (ART), halting viral spread but not eliminating cells already infected with HIV. Eradication and cure approaches are currently hindered by long-lived memory CD4+ T cells harboring non-expressing yet functional integrated provirus, collectively called the latent HIV reservoir. This dissertation presents the characterization and development of novel protein kinase C (PKC) modulators, a promising experimental class of latency reversing agents (LRAs). In collaboration with the Wender lab from Stanford University, who are bioorganic chemists specializing in drug design and synthesis, we have utilized different approaches to improve existing PKC modulators and develop new synthetic PKC modulators inspired by naturally-occurring scaffolds. We investigated their utility in HIV cure approaches through in vitro and ex vivo assays and in vivo using both wild-type mice and HIV-infected and ART-suppressed humanized mouse models. LRAs induce HIV protein expression, allowing host immune cells to recognize and kill the previously latently-infected cells through viral cytopathic effects or immunological targeting. Designed synthetic PKC modulators have proven efficacious in latency reversal in vitro using HIV latency cell lines, ex vivo using patient-derived reservoirs, and in vivo using humanized mice. However, no LRA has yet been identified that can safely and effectively deplete all latent virus, indicating further improvements are needed for currently available approaches. Here, I studied a family of prodrugs and analogs derived from bryostatin-1, a lead PKC modulator in HIV studies, and newly designed and synthesized prodrug and analog formulations of tigilanol tiglate (EBC-46), which has largely been studied as a cancer treatment but is relatively unexplored as an HIV LRA. In this dissertation I first present an exploration into the mechanisms by which PKC modulators affect HIV latency. Bryostatin-1 and SUW133 (a better-performing bryostatin-1 analog) induce the production of cytokines and other factors by peripheral blood mononuclear cells (PBMCs) and isolated NK cells, including some that at high concentrations can independently induce latent HIV expression. However, we found that these secreted factors do not indirectly cause HIV latency reversal, indicating that the capacity to induce expression of latent HIV is a direct effect of compound on target cells, not indirectly mediated through cytokines or other secreted factors. Next, we explored which PKC isoforms are required for latency reversal in vitro and demonstrated that bryostatin-1 and analog SUW133 utilize different PKC isoforms than EBC-46 and the better-performing EBC-46 analog SUW400, providing mechanistic insights into the different intracellular pathways that can induce latency reversal. In work directed at improving the in vivo tolerability of PKC modulators for HIV cure efforts, we identified and characterized a lead SUW133 prodrug formulation (SUW322) in vitro using HIV latency cell line models and uninfected PBMC assays. The slow-release prodrug formulation also had delayed activity as designed. In further work, we evaluated a new series of designed and synthesized PKC modulators based on various naturally occurring scaffolds, including EBC-46 and SUW400, silicon (Si)-based analogs, and prodrug formulations of these novel compounds. We found that several of the new designed synthetic PKC modulators reversed latency in cell line models and induced early T cell activation markers in primary uninfected T cells at lower concentrations than natural PKC modulators, suggesting these novel compounds may be useful in future work. Lastly, we advanced lead compound SUW400 into in vivo models. We determined tolerability in vivo using C57/bl6 mice and tested our lead compound for its HIV latency reversal ability in LRA-treated HIV-infected but ART-suppressed humanized mice. SUW400 was tolerated at higher concentrations and was able to reverse latency in some SUW400-treated humanized mice. Thus, the newly synthesized “next generation” PKC modulator compounds are strong candidates for further preclinical assessment in HIV cure approaches. Finally, I summarize the findings described in this dissertation and present them in the broader context of the overall HIV field, along with potential future directions to further improve upon HIV cure approaches. Together, these studies provide new insights into HIV latency and advance a multi-pronged approach to improving protein kinase C modulators with the goal of future development of a safe, effective, scalable HIV cure.