Fig 1.
Characterization of TIQ-15 as a Gαi-based CXCR4 antagonist.
(A) Structures of TIQ-15, AMD3100, and maraviroc used in this study. (B) SDF-1α inhibits while TIQ-15 or AMD3100 promotes SDF-1α/Forskolin-induced cAMP production. CXCR4-Glo cells were stimulated with 1 μM forskolin to initiate cAMP production. Cells were also co-treated with 28 nM of SDF-1α or 28 nM SDF-1α plus various dosages of AMD3100 or TIQ-15. Luminescence was measured, and the IC50 values were calculated. (C) TIQ-15 inhibits SDF-1α-mediated chemotaxis. Resting CD4+ T cells were pretreated with TIQ-15 from 640 pM to 50 μM (5-fold dilution) or DMSO (1%, control) for 1 hour at 37°C, and then assayed for migration towards SDF-1α (50 nM) in trans-well assays. Results are expressed as the relative percentage of migrating cells. (D) TIQ-15 inhibits SDF-1α-mediated cofilin activation. Resting CD4+ T cells were treated with TIQ-15 (10 μM) or DMSO (1%, control) for 1 hour at 37°C prior to stimulation with SDF-1α (50 ng/ml). Cells were fixed, stained with an anti-p-cofilin antibody, and then analyzed with flow cytometer.
Fig 2.
TIQ-15 inhibits NefM1-CXCR4-mediated cell depolarization in Jurkat T cells.
Jurkat cells were treated with 10 ng/ml of the NefM1 peptide (TNAACAWLEAQ) and different doses of TIQ-15 or AMD3100 for 24 hours. (A) Untreated; (B) NefM1 treated; (C) NefM1 + AMD3100 (1000 nM); (D) NefM1 + TIQ-15 (50 nM). Cells were stained with JC-1 and imaged with epi-fluorescent microscopy. Image processing was conducted with Image-Pro 2.0. (E) IC50 determinations for TIQ-15 (1 nM) and AMD3100 (474 nM) inhibition of NefM1 depolarization (Numerical and graphical data analyses were conducted using SigmaPlot 10).
Fig 3.
TIQ-15 inhibits HIV infection of primary and transformed CD4 T cells.
(A) TIQ-15 inhibits HIV infection of Rev-CEM-GFP-Luc reporter cell. Cells were pretreated with TIQ-15 at 3 different dosages for 1 hour at 37°C, and then treated with HIV-1(NL4-3) for 2 hours. Cells were washed and cultured in the absence of TIQ-15 for 48–72 hours, analyzed with flow cytometry. Propidium iodide (PI) was used to ensure GFP quantification only in the viable cell population. (B) Quantification of anti-HIV potency. Rev-CEM-GFP-Luc cells were pretreated with TIQ-15 at different dosages (from 10 μM to 25.6 pM, 5-fold dilution) or DMSO for 1 hour at 37°C, and then treated with HIV-1(NL4-3) for 2 hours. Cells were washed and cultured in the absence of TIQ-15 for 72 hours, and quantified for luciferase activity. (C) Quantification of TIQ-15 cytotoxicity. Rev-CEM-GFP-Luc cells were similarly treated with TIQ-15 from 50 μM to 16 nM (5-fold dilution) or DMSO for 1 hour at 37°C, washed, cultured for 72 hours, analyzed with flow cytometry for drug cytotoxicity. Results are expressed as the relative percentage of viable cells. (D) TIQ-15 inhibits HIV infection of blood resting CD4 T cells. Cells from 3 donors were treated with TIQ-15 at different dosages or with DMSO for 1 hour at 37°C, and then infected with HIV-1(NL4-3) for 2 hours. Cells were washed and cultured (with IL-7, 1 ng/mL) in the absence of TIQ-15 for 5 days, and then activated with anti-CD3/CD28 magnetic beads (4 beads per cell). Viral replication was measured by HIV-1 p24 release.
Fig 4.
TIQ-15 blocks HIV entry and downregulates CXCR4.
(A) TIQ-15 dosage-dependent downregulation of CXCR4. Resting CD4 T cells were treated with TIQ-15 (10 μM) or DMSO control for 1 hour at 37°C. Cells were stained with a PE/Cy5-labeling anti-CXCR4 antibody and analyzed with flow cytometer (top panel). A3R5 CD4 T cells were treated with different doses of TIQ-15 (10 μM to 1 nM) and similarly analyzed (lower panel). (B) TIQ-15 does not affect CD4 surface density. TIQ-15-treated resting CD4 T cells were stained with a FITC-labeling anti-CD4 antibody. (C) TIQ-15 does not inhibit VSV-G pseudotyped HIV-1. Rev-CEM-GFP-Luc cells were treated with TIQ-15 (50 μM) or DMSO for 1 hour at 37°C, and then infected with HIV-1(VSV-G) or HIV-1(NL4-3) for 2 hours. Cells were washed and cultured in the absence of TIQ-15 for 48 to 72 hours, and analyzed with flow cytometry. (D) TIQ-15 blocks HIV entry. CEM-SS cells were treated with TIQ-15 (10 μM), AMD3100 (10 μM) or DMSO for 1 hour at 37°C, and then infected with BlaM-Vpr containing HIV-1(NL4-3) for 4 hours. Cells were analyzed with a BlaM-Vpr-based entry assay. (E) Lack of HIV DNA in TIQ-15-treated and HIV-infected T cells. CEM-SS cells were treated with TIQ-15 (10 μM) or DMSO for 1 hour at 37°C, and then infected with a single cycle HIV-1(NL4-3)(Env) that was pseudotyped with HIV-1 gp160. Following infection, viral DNA synthesis was followed in a time course and quantified using real-time PCR. (F) TIQ-15 moderately inhibits low-dose HIV-1(AD8) infection of Rev-A3R5-GFP reporter cells. Cells were treated with 10 μM of TIQ-15 for 1 hour, infected with HIV-1(AD8) for two hours. Cells were washed and cultured in the absence of TIQ-15 for 48 hours, and analyzed with flow cytometry. (G) TIQ-15 does not affect CCR5 surface density. A3R5 CD4 T cells were treated with TIQ-15 (10 μM and 50 μM) or control for 1 hour at 37°C. Cells were stained with a PE/Cy5-labeling anti-CCR5 antibody and analyzed with flow cytometer.
Fig 5.
TIQ-15 inhibits HIV clinical isolates of X4, R5, and mixed tropisms.
Human PBMCs were cultured, pre-treated with TIQ-15 at various dosages, and then infected with HIV for 7 days. Cell-free supernatants were collected and analyzed for reverse transcriptase activity. TIQ-15 potently inhibits infection of PBMCs by two X4- and one dual-tropic viruses (A). TIQ-15 is moderately active against three R5-tropic and one dual-tropic viruses (B). Compound cytotoxicity was measured by MTS assays.
Table 1.
Inhibition of clinical isolates of HIV-1 in PBMCs by TIQ-15.
Table 2.
Determination of the anti-HIV-1 synergy for combination doses of TIQ-15 and Maraviroc.
Table 3.
Summary of results on TIQ-15.