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Abstract
In the previous study, we found that flavonoids and ginsenosides exhibited high eliminate rates of human immunodeficiency virus type 1 (HIV-1) D3-transfected macrophages. Based on these findings, here we synthesized the derivatives of gallic acid, including methyl gallate, methyl 4-O-methyl gallate, methyl 3,4-O-dimethyl gallate, and methyl 3,4,5-O-trimethyl gallate and measured their cellular toxic effects against HIV-1-infected macrophages. Of these, treatment with methyl 4-O-methyl gallate in the presence of lipopolysaccharide (LPS) and cycloheximide (CHX) most effectively eliminated HIV-1-transfected cytoprotective human microglial CHME5 cells and HIV-1-D3-infected human primary macrophages. Furthermore, these strongly inhibited LPS/CHX-induced phosphorylation of phosphoinositide 3-kinase (PI3K), pyruvate dehydrogenase lipoamide kinase isozyme 1 (PDK1), Akt, and glycogen synthase kinase-3β (GSK-3β) in the Tat-transfected cells and HIV-1-D3-infected human primary macrophages. These findings suggest that methyl 4-O-methyl gallate may be a promising candidate for eliminating HIV-1 infected macrophages by blocking PI3K/Akt signaling pathway.
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Figure 1.
Synthesis of gallic acid and its derivatives. (Compound 1), gallic acid; (compound 2), methyl gallate; (compound 3), methyl 4-O-methyl gallate; (compound 4), methyl 3,4-O-dimethyl gallate; (compound 5), methyl 3,4,5-O-trimethyl gallate. cat., catalytic; DMF, dimethylformamide; rt, room temperature.
Figure 2.
The cytotoxic effects of gallic acid derivatives against cytoprotective HIV-1 Tat-transfected CHME5 cells. HIV-1 Tat-transfected CHME5 cells were treated with test compounds (0, 5, 10, and 20 μM) or miltefosine (MF, 20 μM) for 48 h. (Compound 1), gallic acid; (compound 2), methyl gallate; (compound 3), methyl 4-O-methyl gallate; (compound 4), methyl 3,4-O-dimethyl gallate; (compound 5), methyl 3,4,5-O-trimethyl gallate in the absence or presence of LPS/CHX. Cell death was determined by the trypan blue staining assay. All values are mean ± S.D. (n = 4). ∗, p <0.05 compared with 0 μM treatment group.
Figure 3.
The cytotoxic effects of gallic acid and methyl 4-O-methyl gallate against cytoprotective HIV-1 Tat-transfected CHME5 cells. (A) Cytotoxic effects of gallic acid (compound 1) and 4-methoxy methyl gallate (compound 3) were determined using trypan blue staining assay. (B) Cytotoxic effects of gallic acid (compound 1) and 4-methoxy methyl gallate (compound 3) by the calcein AM/PI using a flow cytometer (C6 Flow Cytometer® System) and a fluorescence microscope. HIV-1 Tat-transfected CHME5 cells were treated with LPS/CHX in the absence or presence of test compounds (0, 10, and 20 μM) or miltefosine (MF, 20 μM) for 48 h. Trypsinized cells stained with calcein AM/PI were measured by a flow cytometer. All values are the mean ± S.D. (n = 4). (C) Cytotoxicity of test compounds was measured using a fluorescence microscope. Cells were stained with calcein AM/PI to distinguish between dead (red) and live (green) cells. Images (merged red and green fields) are representatives of 3 experiments conducted in duplicate. ∗, p < 0.05 compared with LPS/CHX treatment group.
Figure 4.
Cytotoxic effects of gallic acid and methyl 4-O-methyl gallate against HIV-1-D3-infected human primary macrophages. (A) Cytotoxic effects of gallic acid (compound 1) and 4-methoxy methyl gallate (compound 3) were determined using trypan blue staining assay. (B) HIV-1-D3-infected primary macrophages were treated with LPS/CHX in the absence or presence of gallic acid (compound 1) or methyl 4-O-methyl gallate (compound 3) (0, 5, 10, and 20 μM). Cytotoxic effects of gallic acid (compound 1) and methyl 4-O-methyl gallate (compound 3) by PI/FACS assay. Cells stained with PI were examined by a flow cytometer. All values are mean ± S.D. (n = 4). (C) Cytotoxicity of compounds was determined using a fluorescence microscope. Cells were stained with PI to distinguish between dead (red) and live (green) cells. Images (merged red and green fields) are representatives of 3 experiments conducted in duplicate. ∗, p < 0.05 compared with LPS/CHX treatment group.
Figure 5.
Effects of gallic acid and 4-methoxy methyl gallate on the phosphorylation of PI3K, PDK1, Akt, GSK3β, mTOR and β-actin in LPS/CHX-stimulated HIV-1-D3-infected human primary macrophages. (A) Effects of gallic acid (compound 1). (B) Effect of methyl 4-O-methyl gallate (compound 3). HIV-1-D3-transfected primary macrophages were treated with LPS/CHX in the absence or presence of gallic acid (compound 1), methyl 4-O-methyl gallate (compound 3) (0, 5, 10, and 20 μM) for 120 min. Proteins were measured by immunoblotting. #, p < 0.05 compared with normal control; ∗, p < 0.05 compared with LPS/CHX treatment group.
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