Highly potent, synthetically accessible prostratin analogs induce latent HIV expression in vitro and ex vivo

Abstract

Highly active antiretroviral therapy (HAART) decreases plasma viremia below the limits of detection in the majority of HIV-infected individuals, thus serving to slow disease progression. However, HAART targets only actively replicating virus and is unable to eliminate latently infected, resting CD4(+) T cells. Such infected cells are potentially capable of reinitiating virus replication upon cessation of HAART, thus leading to viral rebound. Agents that would eliminate these reservoirs, when used in combination with HAART, could thus provide a strategy for the eradication of HIV. Prostratin is a preclinical candidate that induces HIV expression from latently infected CD4(+) T cells, potentially leading to their elimination through a virus-induced cytopathic effect or host anti-HIV immunity. Here, we report the synthesis of a series of designed prostratin analogs and report in vitro and ex vivo studies of their activity relevant to induction of HIV expression. Members of this series are up to 100-fold more potent than the preclinical lead (prostratin) in binding to cell-free PKC, and in inducing HIV expression in a latently infected cell line and prostratin-like modulation of cell surface receptor expression in primary cells from HIV-negative donors. Significantly, selected members were also tested for HIV induction in resting CD4(+) T cells isolated from infected individuals receiving HAART and were found to exhibit potent induction activity. These more potent agents and by extension related tunable analogs now accessible through the studies described herein should facilitate research and preclinical advancement of this strategy for HIV/AIDS eradication.

Keywords: HIV latency; NF-κB; PKC-δ; bryostatin.

Fig. 1.

Pharmacophore model highlighting triad of oxygenation proposed for binding to the PKC-C1 regulatory domain in diacylglycerol (1), phorbol-12-myristate-13-acetate (2), prostratin (3; R = CH₃), and DPP (4; R = CH₂Ph).

Fig. 2.

Synthesis of C20-O-trityl-prostratin-ol (10). Reagents and conditions: (a) H₂SO₄, H₂O, 90 °C, 40% yield; (b) trityl chloride, pyridine, 50 °C, 94–96% yield; (c) N₂H₄•H₂O, AcOH, MeOH; (d) iPr₂NEt, toluene, 140 °C, then Pb(OAc)₄, CH₂Cl₂, −78 °C → room temperature, 50–59% yield over two steps; (e) hν (350 nM), EtOAc, 64% yield; and (f) Ba(OH)₂•8H₂O, MeOH, 89% yield.

Fig. 3.

Summary of analogs synthesized.

Fig. 4.

Treatment of resting CD4⁺ T cells with costimulation (via bead-based antibody ligation of CD3 and CD28), natural products, or analogs. Results are from a single experiment and are representative of two to four similar experiments.

Fig. 5.

Summary of viral induction from huPBMC from fully suppressed HIV-positive donors. (A) Amount of viral induction after 2 d of treatment with compounds 11c and 11f at four different concentrations. (B) Amount of viral induction after 4 d of treatment with compounds 11c and 11f at four different concentrations. (C) Amount of viral induction after 3 d of exposure to compound 11c (50 nM) in samples from six different patients. (D and E) Amount of viral induction after 3 d of exposure to compound 11c (50 nM), 11f (50 nM), and prostratin (multiple doses).