The results of this study demonstrate that the daphnane diterpene gnidimacrin activated HIV-1 production from chronically infected cells, inhibited HIV-1 R5 virus infection of PBMCs, and killed HIV-1 chronically infected cells at picomolar concentrations. Several other protein kinase C agonists were shown to activate HIV-1 replication and down regulate HIV-1 receptors at nanomolar concentrations. These protein kinase C agonists include prostratin, P-13S, DPP, I3A, SJ23B, and bryostatin 
. Most of these compounds are natural products isolated from plants. Among these compounds, prostratin was the best studied for its potential as an adjuvant therapeutic with HAART to eradicate HIV-1 from its latent reservoirs. Successful semi-synthesis of prostratin has further raised the interest of developing this compound for potential HIV-1 eradication 
. Thus, prostratin was chosen as a control in this study for its regulatory effect on HIV-1 replication.
The results of this study clearly indicate that gnidimacrin is at least 1,000 fold more effective than prostratin in activation of HIV-1 replication in the latently infected ACH-2 and U1 cells. The effective doses for HIV-1 activation were equivalent to the cytotoxic doses of both compounds. In contrast, U937, the parental cell line of U1, was at least 1,000 fold less sensitive to the cytotoxic effect of gnidimacrin. The differential sensitivity between U1 and U937 to gnidimacrin suggests that activation of HIV-1 replication could potentially eliminate latently infected cells. Gnidimacrin was also at least 1,000 fold more potent than prostratin in inhibiting HIV-1 infection. Gnidimacrin was particularly effective against R5 virus infection of PBMCs. The average EC50 for gnidimacrin against the 12 tested R5 viruses was approximately 9 pM. Such a potent inhibitory activity is likely sufficient to inhibit the R5 viruses purged from latent viral reservoirs.
Inhibition of R5 HIV-1 strains by gnidimacrin correlated with down regulation of CCR5 on PBMCs (). CCR5 was down regulated to background level on PBMCs, whereas CD4 and CXCR4 were only partially down regulated by gnidimacrin. Although the ×4 virus NL4-3 replication in MT4 cells is very sensitive to gnidimacrin, ×4 virus replication in PBMCs was only partially inhibited by gnidimacrin and prostratin. Down regulation of HIV-1 receptors does not explain this discrepancy because similar partial down regulation of CD4 and CXCR4 was observed on both PBMCs and MT4 cells (, ). One possible explanation is that a small fraction of CD4 and CXCR4 on PBMCs, but not on MT4 cells, are sufficient for HIV-1 infection; as a result, partial down regulation of CD4 and CXCR4 potently inhibits HIV-1 infection of MT4 cells but is not effective against ×4 virus infection of PBMCs.
Like gnidimacrin, prostratin was able to completely inhibit R5 virus, but only partially inhibited ×4 virus infection of PBMCs in this study (). The fact that gnidimacrin was ineffective against all tested five viruses that use CXCR4 for entering PBMCs further support the notion that CCR5 down regulation is a key mechanism responsible for the potent anti-R5 virus activity. Although the anti-HIV-1 activity of prostratin in cell lines is well documented, the effect of prostratin on HIV-1 infection of PBMCs is not conclusive. Prostratin was shown to inhibit both R5 and ×4 virus infection of activated PBMCs 
. In contrast, it has also been shown that prostratin and the ingenol I3A were inactive against NL4-3 (an ×4 virus) infection of activated PBMCs 
. It is possible that inhibition of ×4 virus infection by gnidimacrin is dependent on cell types and the activation states of the cells.
Protein kinase C is a large family with four conventional isozymes PKCα, PKCβI, PKCβII and PKCγ; four novel isozymes, PKCδ, -ε, -η, and -θ; the atypical isozymes PKCζ and PKCι 
. Enzastaurin is a selective inhibitor for PKCβI and PKCβII 
. The strong antagonistic effect of enzastaurin on the anti-HIV-1 activity of gnidimacrin suggests that protein kinase C beta is the major protein kinase C isozyme responsible for the potent anti-HIV-1 activity. We propose that gnidimacrin inhibits HIV-1 by activating protein kinase C beta, which in turn strongly down regulates CCR5 and partially internalizes CD4 and CXCR4.
In conclusion, the results of this study suggest that protein kinase C beta is the target of gnidimacrin. Activation of protein kinase C beta by gnidimacrin causes internalization of HIV-1 receptors CD4, CXCR4, and CCR5, which prevents HIV-1 from entering the cells. Gnidimacrin is an extremely potent HIV-1 regulator that activates HIV-1 replication in chronically infected cells and inhibits R5 HIV-1 strains at low picomolar concentrations. The potent dichotomous activities make gnidimacrin an attractive adjuvant therapeutic candidate for the possibility of HIV-1 eradication. Future challenges in studying this potent anti-HIV-1 agent include identification of the specific cellular pathways that impart this extremely potent dichotomous activity of gnidimacrin.