Aphios Corporation Presents “Combination Therapy for Treating HIV Latency” at HIV DART 2008 in Puerto Rico
December 10, 2008
Background
HIV infects several cell types during the course of infection and progression to acquired immune deficiency syndrome (AIDS). The persistence of latent HIV-infected cellular reservoirs represents the major hurdle to virus eradication with highly active anti-retroviral therapy (HAART). Reactivation of the latent reservoirs could allow effective targeting and possible eradication of the virus. Experiments with relatively specific PKCs inhibitors suggest that Bryostatin 1, a potent PKC modulator, reactivates HIV-1 latency thorough the PKC pathway. We thus investigated biochemical targets downstream of PKC.

Methods
Bryostatin 1 was extracted and purified from Bugula neritina utilizing a supercritical fluid with a polar co-solvent followed by downstream chromatographic purification and crystallization. Jurkat-LAT-GFP cells were stimulated with increasing concentrations of bryostatin-1 and the phosphorylation and degradation of the NF-?B inhibitor I?Ba. The phosphorylation (activation) of the MAPKs, ERK and JNK, were investigated by Western blot using specific mAbs. The percentage of GFP+ cells was analyzed by flow cytometry in an EPIC XL flow cytometer (Beckman-Coulter Inc. CA, USA). Jurkat LAT-GFP cells were pretreated with inhibitors for 30 min at the indicated dose and then stimulated with Bryostatin 1 (10 nM) for 6h.

Results
Bryostatin 1 induced phosphorylation and degradation of I?Ba, and also the activation of the MAPKs, ERK1+2 and JNK1+2 in a concentration dependent manner. Bryostatin 1 at the concentration of 10 nM does not induce I?Ba phosphorylation and degradation and JNK activation but fully reactivates HIV-1 latency. Therefore, therapeutic activity of Bryostatin 1 for HIV-1 latency can be achieved at concentrations that do not activate signal transduction pathways (i.e. NF-?B and AP-1) that may result in negative side effects.

In addition to its HIV-1-latency antagonizing activity, Bryostatin 1 also downregulates, at 10 nM concentration, the expression of the human HIV-1 receptors CD4 and CXCR4 and prevents de novo HIV-1 infection as measured by virus-induced cytotoxicity assays (EC50 of 26 nM).

Bryostatin 1 also synergizes with Histone Deacetylase (HDAC) inhibitors (valproic acid and TSA) to antagonize HIV-1 latency. HDAC inhibitors alone do not significantly reactivate HIV-1 latency but reduces the concentration of Bryostatin 1 (at least one order of magnitude). Bryostatin 1 at 1 nM concentration can induce HIV-1 reactivation in the presence of therapeutically relevant concentrations of valproic acid. Thus, the therapeutic activity of Bryostatin 1 can be drastically improved in humans by utilizing a HDAC inhibitor in combination therapy.