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, re-activates 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 down-regulates, 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 Deacetylases (HDAC)
inhibitors (valproic acid and TSA) to antagonise 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.
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