Aromatic disulfides as potential inhibitors against interaction between deaminase APOBEC3G and HIV infectivity factor

Abstract

APOBEC3G (A3G) is a member of cytosine deaminase family with a variety of innate immune functions. It displays activities against retrovirus and retrotransposon by inhibition of virus infectivity factor (Vif)-deficient HIV-1 replication. The interaction between A3G N-terminal domain and Vif directs the cellular Cullin 5 E3-ubiquitin ligase complex to ubiquitinate A3G, and leads to A3G proteasomal degradation, which is a potential target for anti-HIV drug. Currently, there are very few reports about stable small molecules targeting the interaction between A3G and Vif. In this study, we screened two series of small molecules containing carbamyl sulfamide bond or disulfide bond as bridges of two different aromatic rings. Five asymmetrical disulfides were successfully identified against interaction between A3G and Vif with the IC ₅₀ values close to or smaller than 1 μM, especially, not through covalently binding with A3G or Vif. They restore the A3G expression in the presence of Vif by inhibiting Vif-induced A3G ubiquitination and degradation. This study opens a way to the discovery of new anti-HIV drugs.

Keywords: APOBEC3G; HIV-1; Vif; aromatic disulfide; interaction.

Figure1

Chemicals were found to bind to A3G-sNTD or rVif (A) Chemical library design of WL and ZXM series of compounds (highlighted in dashed box) based on the reported aromatic inhibitors. (B) The binding affinity of MM-2 to A3G-sNTD measured by SPR assay. (C) Kinetics fitting of A3G-sNTD binding with rVif. (D) Response levels of interactions between rVif and different small molecules. Vertical axis indicates the response unit of rVif chips with small molecules flow through; horizontal axis shows the cycle numbers during SPR assay. In (D), different cycles stand for different samples; blue dots represent samples with small molecules; green dots represent starting-up samples at the beginning of experiment; red dots represent samples for correction every 32 cycles; red dashed lines indicate RU=150 and RU=100 respectively. Small molecules with high response are labeled.

Figure2

Measurements of fluorescent intensity (FI) in 293T cells under the treatment with small molecules FI of cells transfected with A3G-HA and A3G-Vif plasmids were treated with ZXM2-6 (A), WL1-3 (B) and other 20 small molecules (C,D). Vertical axes indicate the normalized FI. The averaged FI of A3G-HA cells is set as 100%; the number of asterisks stands for the statistical difference between the FI of A3G-Vif cells with and without treatment with small molecules. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 analyzed by One-way ANOVA, Holm-Sidak’s multiple comparisons method.

Figure3

EGFP-A3G fluorescence is recovered dose-dependently by small molecules Measurement of FI (A) indicates the dose-dependent recovery of EGFP-A3G fluorescence (down) by five small molecules ZXM1-1 (B), ZXM1-2 (C), ZXM1-12 (D), ZXM2-3 (E) and ZXM2-10 (F), respectively. Vertical axes indicate normalized FI. The averaged FI of A3G-HA cells is set as 100%. Concentrations of small molecules in A3G-Vif cells are 0, 5, 10, 20, 50 μM, respectively. The number of asterisks stands for the statistical difference between the FI of A3G-Vif cells with and without treatment with small molecules. **P<0.01, ***P<0.001, ****P<0.0001 analyzed by One-way ANOVA, Holm-Sidak’s multiple comparisons method.

Figure4

EGFP-A3G expression level is increased with increasing concentrations of small molecules Recovery of EGFP-A3G expression levels of under the treatment with the chemicals (A) ZXM1-1, (B) ZXM1-2, (C) ZXM1-12, (D) ZXM2-3 and (E) ZXM2-10 at different concentrations, respectively.

Figure5

Compounds block the interactions between A3G and Vif-CBFβ-ELOB-ELOC (VCBC) complex (A) Kinetics fitting of the binding of A3G-FL with VCBC complex measured by SPR assay. (B–F) The inhibition constants of the chemicals ZXM1-1 (B), ZXM1-2 (C), ZXM1-12 (D), ZXM2-3 (E) and ZXM2-10 (F) against the interaction between A3G-FL and VCBC complex, measured by SPR assay.

Figure6

Small molecules inhibit Vif-mediated polyubiquitination and degradation of A3G (A,B) Effects of different small molecules on the fluorescence intensity of intracellular EGFP-A3G at a concentration of 20 μM. Vertical axes indicate normalized FI. The average FI of A3G-HA cells without small molecules is set as 100%. The concentration of small molecules in 293T cells is 20 μM. Number of asterisks stands for the statistical difference between the FI of A3G-Ub-Vif cells with and without treatment with small molecules. ****P<0.0001. (C) The small molecules inhibit polyubiquitination of APOBEC3G. 293T cells were transfected with expression vectors for EGFP-fused A3G and HA-tagged ubiquitin, in the presence or absence of co-transfection of the Vif expression vector, and treated with small molecules for 36 h. Cell lysates were immunoprecipitated with anti-GFP antibody and analyzed by western blotting using anti-ubiquitin antibody, anti-EGFP antibody and anti-Vif antibody.