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

Abstract

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.

Ginsenoside Rh1 Eliminates the Cytoprotective Phenotype of Human Immunodeficiency Virus Type 1-Transduced Human Macrophages by Inhibiting the Phosphorylation of Pyruvate Dehydrogenase Lipoamide Kinase Isozyme 1

“…The primary human monocyte-derived macrophages were isolated from the blood of volunteers and differentiated as previously described. 17) Cell Survival Assay Tat-expressing CHME5 cells and human primary macrophages that had been transduced with the HIV-1 vector expressing eGFP were treated with 50 µg/mL LPS and 10 µg/mL CHX stress in the presence or absence of Rh1 (5, 10, 20 µM) for 48 h. Subsequently, the cells were trypsinized and mixed with Trypan blue solution and the ratio of dead cells to total cells (150-250 cells) was calculated. Alternatively, the cells were trypsinized, stained with PI (1 µg/mL), and measured by using a fluorescence activated cell sorter (FACS; C6 Flow Cytometer System).…”

Clionosterol and Ethyl Cholestan-22-enol Isolated from the Rhizome of <i>Polygala tenuifolia</i> Inhibit Phosphatidylinositol 3-Kinase/Akt Pathway

“…5) Tat-expressing CHME5 cells were treated with 50 µg/mL LPS and 10 µg/mL CHX stress in the presence or absence of test agents. 17) Then the cells were trypsinized, mixed with trypan blue solution and the dead cells were calculated. Or the cells were trypsinized, stained with PI (1 µg/ mL) for 15 min at room temperature and measured by flow cytometry (C6 Flow Cytometer ® System, Ann Arbor, MI, U.S.A.).…”

“…Others were measured within 90 min of LPS/CHX treatment. The cell culture condition for each western analysis is performed according to the previous method of Kim et al 17) RESULTS AND DISCUSSION When PT was extracted with EtOAc and BuOH successively, EtOAc fraction of PT most potently abolished the cytoprotective effect of Tat-transduced CHME5 cells. Therefore, we applied EtOAc fraction of PT to silica gel column chromatography and obtained 12 fractions, and their abolishing effect against the cytoprotective CHME5 cells (Fig.…”

Metabolism of Ginsenoside Rb1 by Human Intestinal Microflora and Cloning of Its Metabolizing β-<small>D</small>-Glucosidase from <i>Bifidobacterium longum</i> H-1

“…However, the molecular weight of H-1 rBglX was different from that of recombinant β-glucosidases from B. breve, Bacillus subtilis and Streptomyces coelicolor (average molecular weight of 50 kDa), [34][35][36] and from β-glucosidases purified from Bifidobacterium sp. strain SEN. 37,38) Addition of divalent cations, like Cu 2+ strongly inhibited H-1 rBglX activity, whereas it hardly inhibited the activity of other recombinant enzyme (remaining activity was 73%). Some of the β-D-glucosidases possess broad spectrum activities, such as β-D-galacosidase and β-D-fucosidase activities including β-D-glucosidase activity.…”

“…1,26) The metabolites arctigenin and ginsenoside Rd exhibit more potent biological effects in vitro than their parental compounds. 37,38) However, the biological activities of loganetin, a metabolite of loganin, have not been studied. By employing rBglX, we could easily isolate loganetin and arctiin from their glycosides (yield, >85%), respectively.…”