Identification of a novel HIV-1 inhibitor targeting Vif-dependent degradation of human APOBEC3G protein

Abstract

APOBEC3G (A3G) is a cellular cytidine deaminase that restricts HIV-1 replication by inducing G-to-A hypermutation in viral DNA and by deamination-independent mechanisms. HIV-1 Vif binds to A3G, resulting in its degradation via the 26 S proteasome. Therefore, this interaction represents a potential therapeutic target. To identify compounds that inhibit interaction between A3G and HIV-1 Vif in a high throughput format, we developed a homogeneous time-resolved fluorescence resonance energy transfer assay. A 307,520 compound library from the NIH Molecular Libraries Small Molecule Repository was screened. Secondary screens to evaluate dose-response performance and off-target effects, cell-based assays to identify compounds that attenuate Vif-dependent degradation of A3G, and assays testing antiviral activity in peripheral blood mononuclear cells and T cells were employed. One compound, N.41, showed potent antiviral activity in A3G(+) but not in A3G(-) T cells and had an IC50 as low as 8.4 μM and a TC50 of >100 μM when tested against HIV-1Ba-L replication in peripheral blood mononuclear cells. N.41 inhibited the Vif-A3G interaction and increased cellular A3G levels and incorporation of A3G into virions, thereby attenuating virus infectivity in a Vif-dependent manner. N.41 activity was also species- and Vif-dependent. Preliminary structure-activity relationship studies suggest that a hydroxyl moiety located at a phenylamino group is critical for N.41 anti-HIV activity and identified N.41 analogs with better potency (IC50 as low as 4.2 μM). These findings identify a new lead compound that attenuates HIV replication by liberating A3G from Vif regulation and increasing its innate antiviral activity.

Keywords: Antiviral Agent; Cytidine Deaminase; High Throughput Screening (HTS); Human Immunodeficiency Virus (HIV); Viral Protein; Virology.

FIGURE 1.

High throughput screening for inhibitors of HIV-1 Vif-APOBEC3G interaction. A, TR-FRET-based assay used to screen for inhibitors of HIV-1 Vif-APOBEC3G interaction. The interaction between purified 1–94 GST-Vif and biotinylated APOBEC3G peptide (amino acids 110–148, a surrogate for the Vif-binding site) is detected by europium (donor fluorophore)-labeled anti-GST antibody and streptavidin-UL (Ulight (UL)) (acceptor fluorophore). B, screening pipeline to identify inhibitors of HIV-1 Vif-APOBEC3G interaction. Criteria and cutoffs for selecting active hits and positive controls associated with each step of the screen are shown. The median z-values calculated for each of the sets in the primary screen (B) were 0.84, 0.70, and 0.77. C, dose-response TR-FRET assay and counter screen testing of compound N.41 for specificity and activity validation. The z-values calculated for the counter screens and cytoxicity assays ranged from 0.70 to 0.91.

FIGURE 2.

N.41 attenuates APOBEC3G degradation by Vif. A, fluorescent cell-based screen to identify molecules that attenuate Vif-mediated degradation of YFP-A3G. 293T cells expressing YFP-A3G with or without Vif were treated with DMSO or 40 μm concentrations of tested compounds for 20 h, then FI of treated cell lysates was measured. The change in FI (percentage change relative to untreated (DMSO) controls) in compound-treated cells expressing YFP-A3G and Vif is plotted on the x axis. Compounds for which the % increase in FI is above a specified cutoff (% increase in FI_Comp > % increase in FI_DMSO + 2 S.D., red dashed line) are identified as active hits. To identify compounds that potentially target Vif, -fold increase in YFP-A3G levels was calculated as the change in FI in compound-treated cells expressing YFP-A3G relative to untreated (DMSO) control cells (y axis). Shown is a representative experiment and cutoff (-fold increase FI(YFP-A3G)_DMSO + 1 S.D., blue dashed line) used to classify hit compounds into two groups corresponding to those predicted to target Vif (below the cutoff) or A3G (above the cutoff). Compound C.18, identified in the cell-based screen as a false positive that significantly increases YFP levels, was included as a positive control for molecules that enhance YFP-A3G levels. B, N.41 likely stabilizes endogenous A3G protein levels in CEM cells. CEM T cells were treated with 0, 10, and 20 μm N.41 for 48 h. Endogenous A3G and β-tubulin protein levels in CEM cell lysates were detected by Western blotting. Results are representative of two independent experiments.

FIGURE 3.

N.41 inhibits HIV-1 replication in PBMCs and T cells in an A3G-dependent manner. A, antiviral activity against HIV-1 replication in PBMCs. N.41 was evaluated in dose-response assays using a high test concentration of 100 μm and half-log dilutions. Treated PBMCs were infected with the indicated HIV-1 isolates (HIV-1_Ba-L, 92RW016, 92UG021, or JV1083), and 7 days post-infection virus replication and cell viability were measured by RT and MTS assays. Shown is the percentage of virus replication and cell viability in N.41-treated cells relative to untreated controls in 1 of 3 independent experiments. B, N.41 has A3G-dependent antiviral activity against HIV-1 replication in T cells. H9 and SupT1 T cells were infected with wild-type or ΔVif HIV-1NL4-3_. At 3 h post infection, cells were washed 3 times and incubated with medium containing 0, 5, 10, 20 and 40 μm N.41. Ritonavir (protease inhibitor with antiviral activity, included as a positive control) was tested at 0.5 and 5 μm. At 6 days post infection, virus replication and cell viability were measured by p24 ELISA and MTS assays. Results are representative of two independent experiments. C, HIV-1 spreading infection in T cells. CEM and CEM-SS T cells were infected with HIV-1NL4-3, and 3 h post-infection cells were treated with 0, 2.5, 5, and 10 μm N.41. Virus production from treated cells was tested 3, 6, and 9 days post-infection by RT assay.

FIGURE 4.

N.41 increases HIV-1 virion incorporation of A3G and decreases virus infectivity. A, 293T cells were co-transfected with 0, 200, or 400 ng of A3G-3xHA (left panel) or 250 ng of A3F-V5 or 250 ng A3C-V5 (right panels) and either pNLX HIV-1 ΔEnv or pNLX HIV-1 ΔEnv ΔVif plasmids. VSV-G was used for pseudotyping in single round infections. At 5 h post transfection, the media was replaced with fresh media supplemented with DMSO or 40 μm N.41. At 40 h post transfection, supernatants containing virus were collected, and producer cells were lysed. A3G, A3F, A3C, Vif, p24 Gag, and β-tubulin protein levels in producer cell lysates were analyzed by Western blotting. WCL, whole cell lysates (upper panels). Compound N.41 increases A3G incorporation into HIV-1 virions. Virions normalized for equivalent RT units were purified through 20% sucrose. A3G, A3F, A3C, Vif, and p24 Gag protein levels in virion lysates were detected by Western blotting (lower panels). Results are representative of two independent experiments. B, N.41 reduces infectivity of viruses produced from cells expressing A3G but not A3C or A3F proteins. TZM-bl reporter cells were infected with viruses corresponding to 4000 RT units. Luminescence values from infected cells were measured 48 h post infection. Shown is the percentage of infection relative to infection of untreated producer cells. *, p value is based on Student's t test. Results are representative of two independent experiments each done in duplicate.

FIGURE 5.

N.41 targets Vif-A3G interaction. A, N.41 attenuates Vif-mediated degradation of huA3G but not agmA3G. 293T cells were co transfected with 75 ng of pCDNA3 (empty vector), huA3G-3xHA, or agmA3G-HA and pNLX, HIV-1 ΔEnv, or pNLX HIV-1 ΔEnv ΔVif plasmids. VSV-G was used for pseudotyping in single-round infections. At 5 h post transfection, media was replaced with fresh media supplemented with DMSO (untreated controls) or 25 μm N.41. At 40 h post transfection, supernatants containing virus were collected, and producer cells were lysed. A3G, Vif, and β-tubulin protein levels in producer cell lysates were analyzed by Western blotting (upper panel; WCL, whole cell lysates). To assess A3G incorporation into virions, virions normalized for equivalent RT units were purified through 20% sucrose. A3G, Vif, and p24 Gag protein levels in virion lysates were detected by Western blotting (bottom panel). Results are representative of two independent experiments. B, N.41 treatment reduces infectivity of viruses produced from cells expressing huA3G but not agmA3G proteins. TZM-bl reporter cells were infected with the indicated virus (4000 RT units). Luminescence from infected cells was measured 48 h post infection. Shown is the percentage of infection relative to levels in untreated producer cells. *, p value based on Student's t test. Results are representative of two independent experiments each performed in duplicate. C, N.41 attenuates Vif-A3G interaction in in vitro binding assays. Recombinant GST-Vif proteins were incubated with 293-apo lysate (293 cells stably expressing A3G-HA). GST-Vif pulldown was performed in the presence of DMSO or 40 μm N.41. GST-Vif-A3G complexes were detected by Western using anti-GST and anti-HA antibodies. D, N.41 attenuates HIV-1 Vif-A3G interaction in 293T cells in co-immunoprecipitation assays. 293T cells transfected with huA3G-3xHA and empty vector (−) or pNLX HIV-1 ΔEnv or pNLX HIV-1 ΔEnvΔVif plasmids were treated with 40 μm N.41 or DMSO for 40 h. A Western blot of cell lysates or anti-HA co-immunoprecipitated proteins was probed using anti-Vif and anti-HA antibodies.

FIGURE 6.

N.41 molecule optimization. A, 16 commercially available molecules were identified as potential N.41 analogs for followup studies. B, effect of N.41 analogs on endogenous A3G protein levels in CEM cells. CEM T cells were treated with 0, 10, and 20 μm N.41 and its analogs for 48 h. Endogenous A3G and β-tubulin protein levels in CEM cell lysates were detected by Western blotting. β-Tubulin protein levels also served to evaluate viability of treated cells. Values shown below A3G blots represent relative A3G protein levels in treated CEM cells determined by densitometry using ImageJ software and normalization to A3G protein levels in untreated cells (DMSO control). β-Tubulin was used as an internal loading control. Results are representative of two independent experiments.

FIGURE 7.

Effect of compound N.41 and its most potent analogs on A3G virion incorporation, viral infectivity, and viral replication in PBMCs. A, compound N.41 and its analogs 3 and 12 increase A3G virion incorporation. 293T cells were co-transfected with or without 100 ng of huA3G-3xHA and pNLX HIV-1 ΔEnv plasmids. VSV-G was used for pseudotyping HIV-1 envelope for single-round infections. At 5 h post transfection, media were replaced with fresh media supplemented with DMSO (untreated controls) or 40 μm N.41 or its analogs 3 and 4 or 20 μm concentration of analog 12. At 40 h post-transfection, supernatants containing virus were collected, and producer cells were lysed. A3G, Vif, and β-tubulin protein levels in producer cell lysates were analyzed by Western blotting (upper panel; WCL, whole cell lysates).To assess A3G incorporation into virions, virions normalized for equivalent RT units were purified through 20% sucrose. A3G, Vif, and p24 Gag protein levels in virus lysates were detected by Western blotting (bottom panel). Results are representative of two independent experiments. B, effect of N.41 and analogs 3, 4, and 12 on infectivity of viruses produced from cells expressing A3G. TZM-bl reporter cells were infected with viruses corresponding to 4000 RT units. Luminescence from infected cells was measured 48 h post infection. Percentage of infection is relative to virus infection of untreated producer cells. *, p value based on Student's t test. Results are representative of two independent infection experiments each carried out in duplicate. C, antiviral activity of the most potent N.41 analogs against HIV-1 replication in PBMCs. N.41 and its analogs were evaluated in dose-response assays using a 100 μm high test concentration and half-log dilutions. Compound-treated PBMCs were infected by the indicated HIV1 isolates (HIV-1_Ba-L or NL4−3), and 7 days post infection virus replication and cell viability were measured by RT and MTS assays. Shown is the percentage virus replication and cell viability in compound-treated cells relative to levels measured in untreated cells. The table shows the virus isolates used for infections, and IC₉₀, IC₅₀, TC₅₀, and Therapeutic Index of the tested compounds.