Novel PI3K/Akt Inhibitors Screened by the Cytoprotective Function of Human Immunodeficiency Virus Type 1 Tat

The PI3K/Akt pathway regulates various stress-related cellular responses such as cell survival, cell proliferation, metabolism and protein synthesis. Many cancer cell types display the activation of this pathway, and compounds inhibiting this cell survival pathway have been extensively evaluated as anti-cancer agents. In addition to cancers, several human viruses, such as HTLV, HPV, HCV and HIV-1, also modulate this pathway, presumably in order to extend the life span of the infected target cells for productive viral replication. The expression of HIV-1 Tat protein exhibited the cytoprotective effect in macrophages and a human microglial cell line by inhibiting the negative regulator of this pathway, PTEN. This cytoprotective effect of HIV-1 appears to contribute to the long-term survival and persistent HIV-1 production in human macrophage reservoirs. In this study we exploited the PI3K/Akt dependent cytoprotective effect of Tat-expressing CHME5 cells. We screened a collection of compounds known to modulate inflammation, and identified three novel compounds: Lancemaside A, Compound K and Arctigenin that abolished the cytoprotective phenotype of Tat-expressing CHME5 cells. All three compounds antagonized the kinase activity of Akt. Further detailed signaling studies revealed that each of these three compounds targeted different steps of the PI3K/Akt pathway. Arctigenin regulates the upstream PI3K enzyme from converting PIP2 to PIP3. Lancemaside A1 inhibited the movement of Akt to the plasma membrane, a critical step for Akt activation. Compound K inhibited Akt phosphorylation. This study supports that Tat-expressing CHME5 cells are an effective model system for screening novel PI3K/Akt inhibitors

SAMHD1 restricts HIV-1 infection in dendritic cells (DCs) by dNTP depletion, but its expression in DCs and primary CD4+ T-lymphocytes cannot be upregulated by interferons

BACKGROUND: SAMHD1 is an HIV-1 restriction factor in non-dividing monocytes, dendritic cells (DCs), macrophages, and resting CD4(+) T-cells. Acting as a deoxynucleoside triphosphate (dNTP) triphosphohydrolase, SAMHD1 hydrolyzes dNTPs and restricts HIV-1 infection in macrophages and resting CD4(+) T-cells by decreasing the intracellular dNTP pool. However, the intracellular dNTP pool in DCs and its regulation by SAMHD1 remain unclear. SAMHD1 has been reported as a type I interferon (IFN)-inducible protein, but whether type I IFNs upregulate SAMHD1 expression in primary DCs and CD4(+) T-lymphocytes is unknown. RESULTS: Here, we report that SAMHD1 significantly blocked single-cycle and replication-competent HIV-1 infection of DCs by decreasing the intracellular dNTP pool and thereby limiting the accumulation of HIV-1 late reverse transcription products. Type I IFN treatment did not upregulate endogenous SAMHD1 expression in primary DCs or CD4(+) T-lymphocytes, but did in HEK 293T and HeLa cell lines. When SAMHD1 was over-expressed in these two cell lines to achieve higher levels than that in DCs, no HIV-1 restriction was observed despite partially reducing the intracellular dNTP pool. CONCLUSIONS: Our results suggest that SAMHD1-mediated reduction of the intracellular dNTP pool in DCs is a common mechanism of HIV-1 restriction in myeloid cells. Endogenous expression of SAMHD1 in primary DCs or CD4(+) T-lymphocytes is not upregulated by type I IFNs

CD8\u3csup\u3e+\u3c/sup\u3e T Cells Responding to Influenza Infection Reach and Persist at Higher Numbers Than CD4\u3csup\u3e+\u3c/sup\u3e T Cells Independently of Precursor Frequency

The activation, localization, phenotypic changes, and function of CFSE-labeled naive influenza-specific CD8+ and CD4+ T cells following influenza infection were examined. Response of adoptively transferred CD8+ T cells was seen earliest in draining lymph node. Highly activated cells were found later in the lung, airways, and spleen, were cytolytic, and expressed IFN-γ upon restimulation. Similar amounts of division at early time points, but higher numbers of CD8+ T cells, were detected at 9 and 30 days postinfection after cotransfer of CD4+ and CD8+ T cells followed by infection. Transfer of much smaller numbers of CD4+ and CD8+ T cells led to more extensive expansion but the same difference in final number between the two cell types. These studies demonstrate how CD8+ and CD4+ T cells respond to influenza at early time points postinfection and the differential kinetics of antigenspecific CD4+ and CD8+ T cells

Pleckstrin Homology Domain of Akt Kinase: A Proof of Principle for Highly Specific and Effective Non-Enzymatic Anti-Cancer Target

<div><p>While pharmacological inhibition of Akt kinase has been regarded as a promising anti-cancer strategy, most of the Akt inhibitors that have been developed are enzymatic inhibitors that target the kinase active site of Akt. Another key cellular regulatory event for Akt activation is the translocation of Akt kinase to the cell membrane from the cytoplasm, which is accomplished through the pleckstrin homology (PH) domain of Akt. However, compounds specifically interacting with the PH domain of Akt to inhibit Akt activation are currently limited. Here we identified a compound, lancemaside A (LAN-A), which specifically binds to the PH domain of Akt kinase. First, our mass spectra analysis of cellular Akt kinase isolated from cells treated with LAN-A revealed that LAN-A specifically binds to the PH domain of cellular Akt kinase. Second, we observed that LAN-A inhibits the translocation of Akt kinase to the membrane and thus Akt activation, as examined by the phosphorylation of various downstream targets of Akt such as GSK3β, mTOR and BAD. Third, in a co-cultured cell model containing human lung epithelial cancer cells (A549) and normal human primary lung fibroblasts, LAN-A specifically restricts the growth of the A549 cells. LAN-A also displayed anti-proliferative effects on various human cancer cell lines. Finally, in the A549-luciferase mouse transplant model, LAN-A effectively inhibited A549 cell growth with little evident cytotoxicity. Indeed, the therapeutic index of LAN-A in this mouse model was >250, supporting that LAN-A is a potential lead compound for PH domain targeting as a safe anti-cancer Akt inhibitor.</p> </div

Western blot analysis of LAN-A treated A549 cell lysates.

<p>(<b>A</b>) A549 cells were left untreated or treated with 5, 10 or 20 µM LAN-A for 24 h. Western blot analysis was conducted on cell lysates (<b>Supplementary </b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050424#pone-0050424-g002" target="_blank"><b>Figure 2</b></a>) and indicated a reduction in phosphorylated Akt (Ser⁴⁷³). (<b>B–E</b>) Analysis of Akt downstream targets is shown. The phosphorylation levels for GSK3β, IKK-α, mTOR and BAD were examined and are reduced in LAN-A treated groups. (<b>F</b>) Cellular PTEN remained constant with LAN-A treatment. (<b>G</b>) Phospho-PDK1 level remained unchanged with LAN-A treatment. (<b>H</b>) PI3K level remained unchanged with LAN-A treatment. Data sets were done in duplicate. ANOVA analysis was performed on the data sets with * = <i>p</i><0.05, ** = <i>p</i><0.01 and *** = <i>p</i><0.001.</p

Effects of LAN-A on the growth of A549-luc-c8 cells <i>in vivo</i>.

<p>(<b>A</b>) Tumor volumes were measured from days 10–45 in nude mice treated with control (con) and 10 or 20 mg/kg/day LAN-A (9 mice/group). (<b>B</b>) At day 45, mice were injected with luciferin reagent for luminescence live imaging. Relative intensities for 5 mice/group are shown, and the relative intensity was indicated compared to that of the control (control = 1). (<b>C</b>) Mice were sacrificed and tumors removed. Tumors were measured by Maestro <i>In Vivo</i> Multispectral Imaging System and relative volume plotted. ANOVA analysis was performed on data sets, and statistically significant differences from the control group are indicated with asterisks (* = <i>p</i><0.05).</p