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. Author manuscript; available in PMC: 2014 Jan 24.
Published in final edited form as: Chem Biol. 2013 Jan 24;20(1):82–91. doi: 10.1016/j.chembiol.2012.11.005

Effector Kinase Coupling Enables High-Throughput Screens for Direct HIV-1 Nef Antagonists with Anti-retroviral Activity

Lori A Emert-Sedlak 1, Purushottam Narute 1, Sherry T Shu 1, Jerrod A Poe 1, Haibin Shi 2, Naveena Yanamala 2, John Jeff Alvarado 2, John S Lazo 3,4, Joanne I Yeh 2, Paul A Johnston 3,5, Thomas E Smithgall 1,3
PMCID: PMC3559019  NIHMSID: NIHMS425165  PMID: 23352142

Abstract

HIV-1 Nef, a critical AIDS progression factor, represents an important target protein for antiretroviral drug discovery. Because Nef lacks intrinsic enzymatic activity, we developed an assay that couples Nef to the activation of Hck, a Src-family member and Nef effector protein. Using this assay, we screened a large, diverse chemical library and identified small molecules that block Nef-dependent Hck activity with low micromolar potency. Of these, a diphenylpyrazolo compound demonstrated sub-micromolar potency in HIV-1 replication assays against a broad range of primary Nef variants. This compound binds directly to Nef via a pocket formed by the Nef dimerization interface and disrupts Nef dimerization in cells. Coupling of non-enzymatic viral accessory factors to host cell effector proteins amenable to high-throughput screening may represent a general strategy for the discovery of new antimicrobial agents.

INTRODUCTION

The year 2011 marked the 30th anniversary of the HIV/AIDS pandemic with 25 million AIDS-related deaths world-wide and 33 million people currently infected with the virus. The course of the disease changed dramatically with the advent of antiretroviral drugs, which target HIV-1 enzymes critical to the viral life cycle as well as fusion of the virus with the host cell (Temesgen et al., 2006). While cocktails of these drugs have extended the life expectancy of infected individuals, they do not clear the virus and require life-long administration. Chronic drug therapy, coupled with the remarkable mutational capacity of HIV-1, continues to drive drug resistance (Gupta et al., 2009). The emergence of multi-drug resistant strains of HIV-1, together with uncertain prospects for an effective vaccine, underscores the urgent need for new antiretrovirals with mechanisms of action complementary to existing agents.

In addition to viral enzymes and structural proteins, the HIV-1 genome encodes a unique set of accessory factors (Vpr, Vpu, Vif, and Nef) that are essential for viral pathogenesis and represent underexplored targets for new anti-retroviral drug discovery (Malim and Emerman, 2008). HIV-1 Nef is particularly attractive in this regard, as it enhances HIV infectivity, promotes viral replication, and enables immune escape of HIV-infected cells (O’Neill et al., 2006; Joseph et al., 2005). Nef lacks known biochemical activity, functioning instead through interactions with a myriad of host cell proteins. These interactions provide a molecular basis for many Nef functions, including downregulation of viral (CD4/CXCR4/CCR5) and immune (MHC-I) receptors from the host cell surface. Nef-mediated receptor internalization is believed to prevent superinfection and enhance viral release, while MHC-I downregulation promotes evasion of immune surveillance by the host.

A critical role for Nef in HIV disease has also been established in animal models as well as AIDS patients. Nef is required for the high-titer replication of both HIV and SIV in vivo, and is essential for the development of AIDS-like disease in non-human primates (Herna and Saksela, 2000; Geyer et al., 2001; Arold and Baur, 2001; Kestler et al., 1991). Furthermore, targeted expression of Nef in the T-cells and macrophages of transgenic mice induces a severe AIDS-like syndrome, strongly supporting an essential role for this single viral protein in HIV-1 pathogenesis (Hanna et al., 1998; Jolicoeur, 2011). The phenotype of these Nef-transgenic mice recapitulates many aspects of human AIDS, including profound immunodeficiency, loss of CD4+ T cells, thymic atrophy, persistent T-cell activation, as well as kidney, spleen, and lung pathology. In contrast, HIV strains with defective nef alleles have been isolated from patients with long-term, non-progressive HIV infections (Kirchhoff et al., 1995; Deacon et al., 1995). Similarly, CD4+ T-cell depletion and immunosuppression was greatly delayed in a cohort of individuals infected with a Nef-deficient HIV-1 quasispecies, providing strong clinical evidence that Nef is essential for disease progression in humans (Dyer et al., 1997; Deacon et al., 1995). Taken together, these findings provide a strong rationale for the discovery and development of Nef-directed antiretroviral drugs.

Identification of small molecule Nef antagonists as drug leads has been hampered by the lack of an assay for Nef function compatible with high-throughput screening (HTS). Previously, we reported the development of an in vitro kinase assay that couples Nef to the activation of the Src-family kinase, Hck (Emert-Sedlak et al., 2009). Hck is strongly expressed in macrophages, a major HIV-1 host cell type, and serves a key effector role in Nef-dependent HIV-1 replication and downregulation of MHC-I (Narute and Smithgall, 2012; Dikeakos et al., 2010; Emert-Sedlak et al., 2009; Atkins et al., 2008). Using this assay to screen a small kinase-biased library, we identified a unique diphenylfuranopyrimidine kinase inhibitor that also blocks Nef-dependent HIV-1 replication (Narute and Smithgall, 2012; Emert-Sedlak et al., 2009). In the present study, we automated this kinase-coupled Nef assay, enabling HTS of a much larger and more diverse chemical library of more than 220,000 compounds. Subsequent concentration-response experiments identified a subset of 62 compounds with low micromolar potency for Nef-dependent kinase activation. Of these, four compounds emerged that potently inhibit both HIV-1 infectivity and replication in multiple cell lines. In particular, we discovered a unique diphenylpyrazolo compound that inhibits Nef-dependent HIV-1 replication with sub-micromolar potency. This compound is broadly active against HIV-1 replication supported by Nef alleles representative of all major subtypes of HIV-1. Furthermore, surface plasmon resonance studies show that this compound binds to Nef at a novel site formed at the Nef dimerization interface, supporting a mechanism of action involving direct interaction with the viral protein. These studies demonstrate that coupling Nef to Hck, a natural host cell effector kinase amenable to HTS, enables discovery of direct small molecule antagonists for this critical HIV virulence factor. This kinase coupling strategy may be more generally useful for other drug discovery targets where direct assays are not readily available.

RESULTS AND DISCUSSION

High throughput screening for direct Nef antagonists

Previously we described a screening assay for inhibitors of Nef-dependent Src-family kinase (SFK) activation (Emert-Sedlak et al., 2009). Implementation of this assay on a pilot scale with a kinase-biased library identified 4-amino substituted diphenylfuranopyrimidines (DFPs) as potent inhibitors of Nef-dependent SFK activation and HIV replication (Narute and Smithgall, 2012; Emert-Sedlak et al., 2009). One critical feature of this screening assay is that Hck kinase activation is completely dependent upon Nef, thereby providing a functional readout of Nef activity in vitro. We reasoned that screening a larger and more diverse compound library with this assay might identify compounds that bind directly to Nef rather than the active site of Hck, which is the likely mechanism of action of the 4-amino DFP compounds (Emert-Sedlak et al., 2009).

Using the Nef-coupled kinase assay, we screened the NIH Molecular Library Screening Center Network (MLSCN) collection of more than 220,000 diverse chemical structures. This fully automated HTS campaign yielded 364 confirmed ‘hit’ compounds with IC50 values for Nef-induced Hck activation of less than 20 μM (overall hit rate of ~ 0.1%; Table S1). Assay results from a representative plate are shown in Figure 1A and illustrate that kinase activity is entirely dependent upon Nef. Overall, nearly 700 × 384-well plates were screened, with a composite Z′-factor (Zhang et al., 1999) of 0.83 ± 0.12, indicative of a remarkably robust HTS assay (Figure 1B). Interestingly, the 4-amino DFP inhibitors of Nef-dependent Hck activation first identified in the pilot screen described above, were also flagged as active in the large-scale screen. All 364 hit compounds were then re-assayed in 10-point concentration-response assays against Nef-induced Hck vs. Hck alone. These experiments identified 66 compounds with at least a 3-fold preference for inhibition of Nef-activated Hck (Tables S1 and S2). These compounds were then assayed for anti-HIV activity as well as cytotoxicity in U87MG astroglioma cells (Figure S1) and CEM-T4 lymphoblasts (Figure S2). Our previous work has established that HIV replication is dependent upon Nef in both of these cell lines (Narute and Smithgall, 2012; Emert-Sedlak et al., 2009). In U87MG cells, 24 of the 62 compounds (39%) showed more than 50% inhibition of HIV-1 replication at a concentration of 1 μM without toxicity. The response rate in CEM-T4 cells was somewhat lower, with 11 of the 62 hit compounds (18%) blocking replication by more than 50% under the same conditions. Five compounds inhibited HIV-1 replication in both cell lines with low to sub-micromolar potency and without appreciable cytotoxicity. The structures of these compounds, their IC50 values for inhibition of the Hck:Nef complex vs. Hck alone, as well as their effects on HIV-1 replication are shown in Figure 2.

Figure 1. High-throughput screening for inhibitors of Nef-dependent Hck activity.

Figure 1

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The NIH Molecular Libraries Screening Centers Network (MLSCN) library (~220,000 compounds) was screened using the FRET-based Nef:Hck in vitro kinase assay as described in the text. (a) Scatterplot of results from a representative 384-well plate. Under these conditions, Hck is inactive when added by itself (blue circles), while addition of a 10-fold molar excess of Nef induces Hck kinase activation, demonstrating the Nef dependence of the assay (red circles). Compounds were screened at 20 μM under conditions where Hck activity is completely dependent on Nef (grey circles), with > 50% inhibition defined as a ‘hit’ (dashed line). (b) Z′-factors for each 384-well plate for the entire high-throughput screening campaign. Of 694 plates screened, 684 passed with Z′-factors ≥ 0.5 (98.5% pass rate); plates that failed due to robotic error were rescreened and subsequently passed (see also Table S1).

Figure 2. Inhibitors of Nef-dependent Hck activity also block Nef-dependent HIV replication and infectivity.

Figure 2

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(a) Chemical structures of the compounds identified by the Nef:Hck HTS campaign and subsequently shown to block Nef-dependent enhancement of HIV-1 replication in two different cell lines are shown, along with the IC50 values for each compound against Hck alone vs. the Nef:Hck complex. Two compounds from the screen share a diphenyldiazenylpyrazole substructure (highlighted in red), differing only in the placement of a single chlorine atom. (b) HIV replication. Compounds (1 μM) shown in (a) were added to cultures of U87MG and CEM-T4 cells in 96-well plates, followed by infection with wild-type HIV-1 NL4-3 (50 pg p24 equivalents/ml) 1 h later. Viral output for the DMSO-treated control cultures was consistently greater than 100-fold over the HIV input, typically ranging from 9,000–12,000 p24 equivalents/ml. Virus replication was assessed by p24 ELISA after 4 days (U87MG) or 9 days (CEM-T4). Data are expressed as the mean percent inhibition as compared to control cultures incubated with the carrier solvent (DMSO) ± S.E.M. (n=4). Results of replication and cytotoxicity experiments with all 66 hit compounds from the primary screen are shown in Figures S1 and S2. (c) Infectivity assays. Compounds (3 μM) were added to cultures of the reporter cell line TZM-bl followed by infection with either wild-type or Nef-defective (ΔNef) HIV NL4-3 in 96-well plates. After 48 hours, relative virus infectivity was assessed as luciferase production in infected cells. Results are plotted as the mean percent of HIV-1 infectivity observed in control cells incubated with the carrier solvent DMSO ± S.E.M. (n=3). In the absence of Nef, infectivity is reduced by about 50% (ΔNef; dashed line shown for reference). See also Figures S1 and S2.

Because HIV-1 Nef is also known to enhance HIV-1 infectivity (Vermeire et al., 2011; Spina et al., 1994; Chowers et al., 1994; Aiken and Trono, 1995), we also evaluated the impact of these five compounds on infectivity using the TZM-bl reporter cell line (Platt et al., 1998; Derdeyn et al., 2000). In this system, infectivity is measured as stimulation of luciferase reporter gene expression driven by the HIV-1 LTR in response to infection with HIV-1, and this effect is enhanced by HIV-1 Nef. As shown in Figure 2C, four of the five compounds suppressed Nef-dependent enhancement of HIV-1 infectivity, providing further support for a Nef-directed antiretroviral mechanism of action.

Diphenylpyrazoles block Nef-dependent HIV-1 replication and SFK activation

Of the five compounds with Nef-dependent anti-HIV activity, two share a diphenyldiazenylpyrazole substructure, differing only in the placement of a single chlorine atom (Figure 2A). Of these, (E)-4-((3-chlorophenyl)diazenyl)-5-hydroxy-3-(4-nitrophenyl)-1H-pyrazole-1-carbothioamide, referred to hereafter as ‘B9,’ exhibited remarkable selectivity for Hck inhibition in the presence of Nef (Figure 3A). B9 blocked the kinase activity of the Nef:Hck complex in vitro with an IC50 value of 2.8 μM, while its activity against Hck alone was > 20 μM. B9 also showed weak activity against other Src-family members in vitro, with IC50 values > 20 μM for c-Src, Lck and Lyn. While these findings support a Nef-dependent mechanism of action, the possibility of direct action on cellular kinases or other proteins cannot be formally ruled out.

Figure 3. Inhibition of Nef-dependent Hck activity and HIV-1 replication by the diphenylpyrazolo compound, B9.

Figure 3

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(a) Concentration-response curves for B9 were generated with the Nef:Hck complex (circles) vs. Hck alone (squares) using the Z′-lyte kinase assay. For Hck alone, approximately 5-fold more kinase protein was added to achieve a similar level of activity as the Nef:Hck complex. Under these conditions, B9 inhibits Nef-dependent Hck activation with an IC50 value in the low micromolar range (2.8 μM), while the IC50 value for Hck alone is > 20 μM. Kinase assays were performed three times in quadruplicate, and the data represent percent inhibition as compared to the DMSO vehicle control ± S.E.M. (b) CEM-T4 cells were infected with wild-type HIV-1 NL4-3 (grey bars) or the corresponding Nef-defective mutant (ΔNef; black bars) in the presence of the B9 concentrations shown. Viral replication was assessed 9 days later by p24 ELISA. Input virus for HIV-1 ΔNef was increased by ten-fold relative to wild-type to compensate for the reduced infectivity and replication of Nef-defective virus in CEM-T4 cells (Narute and Smithgall, 2012). This experiment was done in triplicate and data are represented as percent of HIV-1 replication relative to the DMSO vehicle control ± S.E.M. (c) TZM-bl cells were infected with wild-type (gray bars) and ΔNef (black bar) HIV NL4-3 in the presence of the B9 concentrations shown, and infectivity was assessed as luciferase activity 48 h later. This experiment was repeated three times in triplicate and the data are represented as percent infectivity relative to the DMSO control ± S.E.M. In the absence of Nef, infectivity is reduced by about 50% (dashed line shown for reference).

To investigate whether the antiretroviral activity of B9 was dependent upon the expression of Nef, we compared the impact of B9 on replication of wild-type and Nef-defective HIV-1 in CEM-T4 cells. As shown in Figure 3B, B9 blocked wild-type HIV-1 replication with an IC50 value in the 100–300 nM range, while replication of Nef-defective HIV-1 was unaffected at the highest concentration tested (3.0 μM). B9 also inhibited Nef-mediated enhancement of HIV-1 infectivity in a concentration-dependent manner in the reporter cell line, TZM-bl (Figure 3C). Together, these results strongly support a Nef-dependent antiretroviral mechanism of action for B9.

We next evaluated whether B9 is broadly active against the diverse Nef alleles that comprise the majority of HIV-1 M-group clades. For these experiments, we used a set of recombinant HIV-1 NL4-3 chimeras in which the NL4-3 Nef sequence is replaced with representative Nef sequences derived from the M-group HIV-1 subtypes A1, A2, B, C, F1, F2, G, H, J, K, as well as the laboratory strain, SF2 (Narute and Smithgall, 2012). As shown in Figure 4A, B9 inhibited the replication of all eleven HIV-1 Nef chimeras with an IC50 value of ~ 300 nM in CEM-T4 cells, demonstrating that the compound is broadly active against HIV replication supported by a wide range of HIV-1 Nef proteins. As observed previously, the compound had no effect on the replication of Nef-defective HIV replication in this experiment.

Figure 4. Inhibition of HIV-1 Nef chimera replication and endogenous SFK activation in CEM-T4 cells by the diphenylpyrazolo compound, B9.

Figure 4

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(a) CEM-T4 cells (1 × 104 per well of a 96-well plate) were infected with wild-type HIV-1 NL4-3, a Nef-defective mutant (ΔNef), or the indicated Nef chimeras in a final culture volume of 200 μl. Input virus for HIV-1 ΔNef was increased by ten-fold relative to wild-type to compensate for the reduced infectivity and replication of Nef-defective virus in CEM-T4 cells (Narute and Smithgall, 2012). B9 was added to the cultures to final concentrations of 0.3 and 1.0 μM, and viral replication was determined by p24 ELISA 10 days later. Data are expressed as the mean percent of HIV-1 replication observed in control cultures incubated with the carrier solvent (0.1 % DMSO) ± S.D. (n=6). (b) CEM-T4 cells were infected with wild-type HIV-1 NL4-3, a Nef-defective mutant (ΔNef), or the indicated Nef chimeras in a final culture volume of 10 ml in the presence of B9 (1 μM) or the DMSO carrier solvent as a control (Con). The infected cells were lysed and Src-family kinase proteins were immunoprecipitated with a pan-specific antibody and protein G-sepharose beads. The SFK activation state was assessed by immunoblotting with a phosphospecific antibody against the activation loop phosphotyrosine residue common to all Src-family members (pY418). Control blots were performed on cell lysates for HIV-1 Gag proteins (p55, p40, and p24), Nef, as well as actin as a loading control. Results from uninfected cells are shown in the far right lane (No virus). This experiment was repeated twice with comparable results.

Recently, we showed that infection of CEM-T4 cells with HIV-1 causes a sustained increase in endogenous SFK activation that is dependent upon Nef (Narute and Smithgall, 2012). We therefore explored whether the inhibition of Nef-dependent kinase activation observed with B9 in vitro could also be observed in inhibitor-treated cells under conditions of viral replication block. For these studies, CEM-T4 cells were infected with wild-type HIV-1 NL4-3, the eleven Nef chimeras, and the Nef-defective mutant in the presence or absence of B9. Endogenous SFK activity was assessed in infected cell lysates by immunoblotting with an antibody that recognizes the phosphotyrosine residue in the activation loop of active SFKs (pY418). As shown in Figure 4B, wild-type but not Nef-defective HIV infection stimulated endogenous SFK activation, consistent with previous results(Narute and Smithgall, 2012). B9 treatment completely inhibited Nef-dependent SFK activation at a concentration of 1.0 μM. These results provide important evidence that B9 blocks Nef-mediated SFK activation in HIV-infected cells, which may represent one part of its antiretroviral mechanism of action.

Diphenylpyrazoles interact directly with HIV-1 Nef

The data presented above show that the diphenylpyrazole compound B9 potently inhibits Nef-dependent enhancement of HIV-1 replication as well as endogenous SFK activation across all M-group HIV-1 Nef alleles tested, suggesting that Nef is the target for this compound in HIV-infected cells. To explore possible binding sites for B9 on the Nef structure, docking studies were performed using AutoDock Vina(Trott and Olson, 2010) and the crystal structure of HIV-1 Nef in complex with a SFK SH3 domain [PDB: 1EFN (Lee et al., 1996)]. All docking studies used an unbiased approach, with the grid covering the entire structure of the Nef dimer. This analysis predicted two energetically favorable binding sites for PPD-B9, one of which localizes to the Nef dimer interface (site 1) while the other maps to the surface of each monomer (site 2; Figure 5A). The predicted binding energy for the Nef dimer interface (site 1) was more favorable than that for the other site (−8.5 vs. −7.2 kcal/mol). Docking of the compound to site 1 is predicted to involve a network of polar contacts with residues Gln104, Gln107, and Asn126 from both Nef monomers via a pocket that is formed at the dimer interface (Figure 5B). In contrast, binding site 2 is shallower and involves a single polar contact with Asn126. All predicted binding site residues within 4 Å of B9 along with the number of B9 conformations accommodated by each site are summarized in Table S3.

Figure 5. Docking studies predict direct interaction of B9 with the HIV-1 Nef dimer interface.

Figure 5

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(a) The two “halves” of the Nef dimer (PDB: 1EFN) are modeled in green and blue, respectively (Nef-A and Nef-B). B9 is docked at the two most energetically favored binding sites (Sites 1 and 2). The structure of B9 is shown for reference (right). (b) Close-up view of the predicted B9 binding sites. Site 1 is more energetically favored and nestles between the α-helices that form the dimer interface. Here B9 is predicted to form an extensive network of polar contacts with Nef residues Gln104, Gln107, and Asn126. Site 2 is positioned on the surface of each Nef monomer, away from the dimerization interface, and also makes a polar contact with Asn126. A single site 2 interaction of B9 with the Nef dimer is shown for simplicity. B9 also docks to the SIV Nef dimerization interface; see Figure S3. For additional details of docking results, see Table S3.

To determine whether B9 binds directly to Nef, we next performed SPR studies using immobilized Nef and a range of compound concentrations. As shown in Figure 6A, B9 interaction with Nef was readily detected by this approach, demonstrating saturable binding at each B9 concentration tested. The resulting SPR sensorgrams fit a heterogenous ligand-parallel reaction model (Kuroki and Maenaka, 2011) (where Nef is ‘ligand’), which yielded Kd values of 860 ± 58 nM and 1.72 ± 0.23 nM. These binding data are consistent with the docking results, which predicted two binding sites (Figure 5). The SPR data were also consistent with a two-state model, in which B9 binding to a lower affinity site is predicted to induce a conformational change to a higher affinity binding site (Figure 6B). The overall Kd value resulting from this two-state analysis was 1.79 ± 0.11 nM, which is in close agreement with the heterogeneous ligand model.

Figure 6. B9 binding to Nef requires the predicted binding pocket residue Asn126.

Figure 6

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(a) Surface Plasmon Resonance. Recombinant purified HIV-1 Nef-SF2 was immobilized on the surface of a Biacore CM5 chip and B9 was flowed past Nef at the concentrations shown. The flow path was switched back to buffer after 180 s to induce B9 dissociation (arrow). The resulting sensorgrams (black lines) were best-fit by a heterogeneous ligand (Nef in this case) model (red lines) supporting the presence of two distinct binding sites with Kd values of 860 ± 58 nM and 1.72 ± 0.23 nM. (b) SPR data were also fit by a two-state model, which yielded a Kd value 1.79 ± 0.11 nM for the final Nef:B9 complex. (c) SPR analysis was repeated with wild-type (WT) Nef and three Nef mutants in which Asn126 is replaced with Leu, Gln, or Ala as shown. B9 was held constant at 10 μM and bound readily to wild-type Nef but not to any of the N126 mutants. (d) Nef Asn126 mutants retain their ability to activate Hck. Downregulated Hck was assayed in vitro using the Z′-Lyte kinase assay and Tyr2 peptide substrate either alone or in the presence of a 10-fold molar excess of wild-type Nef or the three Asn126 mutants shown. All four Nef proteins produced an equivalent shift of the Hck activation curve to the left, indicating that mutagenesis of Asn126 does not affect its ability to bind and activate Hck (see also Table S3).

To validate the B9 binding site predicted by AutoDock Vina (Figure 5), we created a series of three Nef mutants with Ala, Leu, and Gln substitutions for Asn126 which is predicted to contact the ligand in both binding sites. Each of these mutants was expressed and purified in recombinant form and compared to wild-type Nef in terms of B9 binding by SPR. As shown in Figure 6C, none of these mutants demonstrated detectable binding to B9, supporting a critical role for Asn126 in B9 binding. To verify that the Nef mutants were properly folded, we also performed SPR analysis with the Hck SH3 domain, which binds to a site that is dependent upon the three-dimensional fold of Nef (Lee et al., 1996) but is distinct from the B9 binding site. All three Nef mutants bound to the Hck SH3 domain with similar kinetics and affinities as wild-type Nef, demonstrating that the Asn126 mutations did not cause a global disturbance in the Nef structure (data not shown). These Nef mutants also activated recombinant Hck to the same extent as wild-type Nef in the in vitro kinase assay (Figure 6D). The dramatic impact of Nef Asn126 mutagenesis on B9 binding may relate to its predicted role in forming polar contacts with B9 in both predicted binding sites. This residue is conserved in all of the Nef alleles present in the HIV-1 Nef chimeras used to demonstrate B9-sensitive viral replication in Figure 4, consistent with the broad-spectrum anti-HIV activity observed with this compound.

In addition to HIV-1 Nef, we also performed docking studies of B9 with a recent X-ray crystal structure of SIV Nef (Kim et al., 2010). In this structure, SIV Nef also packs as a dimer, although the nature of the dimer interface is distinct from that of HIV-1 Nef (Figure S3A). Remarkably, B9 was also predicted to dock to an energetically favorable site formed by the SIV Nef dimer interface (Table S3). This result led us to test whether B9 impacts SIV replication and infectivity. For the replication assay, CEM-174 cells were infected with the pathogenic SIV quasispecies ΔB670 (Seman et al., 2000) over a range of B9 concentrations, and assayed for SIV replication as p27 Gag release. As shown in Figure S3B, B9 blocked SIV replication with an IC50 value of about 1.0 μM. We also evaluated the impact of B9 on SIV ΔB670 infectivity using the TZM-bl reporter cell line described above for HIV. As shown in Figure S3C, B9 blocked SIV infectivity with an IC50 value of about 3 μM. These results provide further support for the idea that compounds targeting the Nef dimer may be broadly effective as Nef antagonists. Previous studies have shown that the Nef dimerization interface is very sensitive to mutagenesis, with single amino acid substitutions in this region compromising Nef functions related to receptor downregulation and HIV-1 replication (Poe and Smithgall, 2009; Liu et al., 2000). These studies suggest that small molecules such as B9, which bind to the dimer interface and influence the conformation or stability of the dimer, may have a major impact on Nef function.

The diphenylpyrazole B9 blocks Nef dimerization in cells

Data presented above demonstrate that B9 binds directly to Nef and suggest that it may influence Nef dimerization as a mechanism of action. To test this idea, we used a bimolecular fluorescence complementation (BiFC) assay previously developed for Nef in our laboratory (Poe and Smithgall, 2009). In this assay, Nef is expressed as a pair of fusion proteins with non-fluorescent fragments of YFP in 293T cells. Nef dimerization juxtaposes the YFP fragments, which then refold to form the fluorescent YFP structure. Nef dimerization requires four conserved hydrophobic side chains that lock together to form a helical interface (Ile109, Leu112, Tyr115, Phe121; Figure 7A). Replacement of these residues with aspartic acid (Nef-4D mutant) results in a dramatic loss of the Nef-BiFC signal, providing a negative control for the assay. To test the effect of B9 on Nef dimerization by BiFC, 293T cells were transfected with wild-type Nef-BiFC fusion proteins and incubated with B9 over a range of concentrations (1–6 μM). As shown in Figure 7B, B9 treatment resulted in a concentration-dependent loss in the Nef BiFC signal, with suppression more dramatic that that observed with the Nef-4D mutant at the highest concentration tested (6 μM). All cultures were immunostained with a Nef antibody to establish that B9 interferes with dimerization rather than Nef protein expression. These data support the idea that B9 inhibits Nef dimerization as part of its mechanism of action.

Figure 7. B9 inhibits Nef dimerization in cells.

Figure 7

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A) Molecular model of the Nef dimerization interface, based on the crystal structure of the Nef:SH3 complex (Lee et al., 1996). Hydrophobic side chains that contribute to dimerization are indicated; substitution of these residues with aspartate (Nef-4D mutant) dramatically reduces Nef dimerization as determined by fluorescence complementation assay (Poe and Smithgall, 2009). B) Human 293T cells were transfected with Nef-BiFC constructs and incubated with B9 over a range of concentrations. The Nef-4D mutant was included as a negative control. Following incubation for 48 hours, the cells were fixed, stained with a Nef antibody and Texas-red, and analyzed by two-color fluorescence microscopy. The top panel shows representative images from cells expressing the wild-type Nef (Nef-WT) in the absence or presence of B9 (6 μM) as well as the dimerization defective mutant, Nef-4D. The bottom panel shows the result of image analysis, in which BiFC (Nef dimerization) to immunofluorescence (Nef expression) intensity ratios were calculated for at least 150 cells. Data were normalized to the untreated Nef-WT control, and represent the mean ± S.D. C) Nef-4D fails to activate Hck. Downregulated Hck was assayed in vitro using the Z′-Lyte kinase assay and Tyr2 peptide substrate either alone or in the presence of a 10-fold molar excess of either wild-type Nef (Nef-WT) or the Nef-4D mutant.

The observation that B9 prevents Nef dimerization suggests an allosteric mechanism for B9-mediated inhibition of Hck activation. This raises the question of whether the dimerization interface contributes to Hck activation in the Z’Lyte assay, which was used to discover B9. To test this possibility, the dimerization-defective Nef-4D mutant was expressed and purified in recombinant form, and tested for its ability to activate Hck in the Z’Lyte assay. Figure 7C shows that Nef-4D is completely defective for Hck activation in vitro. Previous studies have shown that recombinant Nef-4D retains its capacity for SH3 domain binding (Poe and Smithgall, 2009), indicating that this mutation does not globally influence Nef protein folding. These results support the idea that B9 inhibits Hck activation by binding to the Nef dimerization interface, thereby preventing Nef dimerization and juxtaposition of associated Hck kinase domains. This observation complements a previous observation from our group showing that enforced dimerization of Nef leads to hyperactivation of Hck in a cell-based system (Ye et al., 2004).

SIGNIFICANCE

Work presented here describes a new strategy for the discovery of small molecule antagonists that interact directly with Nef, a major virulence factor for HIV-1. Nef lacks intrinsic biochemical activity and therefore is not directly amenable to HTS. To circumvent this issue, we employed an assay in which activation of a natural Nef effector protein (the Src-family kinase Hck) is completely dependent upon the presence of the viral protein. Using this approach, we were able to perform a robust, fully automated HTS campaign. This effort uncovered a unique diphenylpyrazolo compound which binds directly to Nef and displays remarkably potent Nef-dependent antiretroviral activity. While a few other small molecules have been reported that interact with Nef (Dikeakos et al., 2010; Betzi et al., 2007; Chutiwitoonchai et al., 2011), these compounds are of lower potency than B9 in cell-based assays for Nef function, raising the possibility of off-target effects. Furthermore, compounds directed to the Nef-SH3 interface failed to function in cell-based anti-viral assays (Betzi et al., 2007). In contrast, B9 inhibits Nef-dependent HIV replication in the submicromolar range and is broadly active against all M-group HIV-1 variants. This potent anti-Nef activity appears to result from disruption of dimerization, a Nef function implicated in many Nef functions. B9 is an attractive chemical probe for future studies of Nef biology as well as an exciting new lead for antiretroviral drug development.

EXPERIMENTAL PROCEDURES

Recombinant protein expression and purification

Recombinant Hck-YEEI was expressed in Sf9 insect cells as an N-terminal His-tagged fusion protein and purified as described elsewhere (Trible et al., 2006). Full-length HIV-1 Nef proteins (SF2 allele; wild-type and Asn126 mutants) were expressed in E. coli with an N-terminal His-tag and purified as described (Trible et al., 2007; Narute and Smithgall, 2012).

In vitro kinase assay and chemical library screening

Screening assays were conducted in 384-well plates in a final volume of 10 μl per well using the Z′-lyte kinase assay system and Tyr2 peptide substrate (Life Technologies) as described elsewhere (Emert-Sedlak et al., 2009). Compounds were added to each well (20 μM final concentration) and incubated at room temperature with a preformed complex of Hck-YEEI (15 ng/well) and Nef (1:10 molar ratio, 75 ng/well) for 30 minutes. Reactions were initiated by the addition of ATP (100 μM) and peptide substrate (1 μM), and incubated at room temperature for 45 min. Reactions were terminated with 5 μl stop reagent as per the manufacturer’s protocol and fluorescence ratios were calculated as described elsewhere (Emert-Sedlak et al., 2009; Trible et al., 2006). The chemical library for this screen was provided to the University of Pittsburgh Drug Discovery Institute through the NIH Molecular Libraries Screening Center Network initiative, and consisted of about 220,000 compounds at the time the primary screen was initiated. The 1495 hit compounds from the primary screen were counter-screened for autofluorescence by repeating the assay in the absence of Hck and Nef proteins. A second counter-screen for development reagent (protease) inhibitors was conducted against assay reagents and a tyrosine-phosphorylated Tyr2 control peptide in the absence of Hck and Nef. As per NIH requirements, the complete set of assay results from the primary screen has been deposited in PubChem (UID: 463187).

HIV assays

Viral stocks were prepared by transfection of 293T cells (ATCC) with wild-type and Nef-defective (ΔNef) proviral genomes (NL4-3 strain) and amplified in the T-cell line, MT2 (NIH AIDS Research and Reference Reagent Program) as previously described (Narute and Smithgall, 2012; Poe and Smithgall, 2009; Emert-Sedlak et al., 2009). Viral replication was assessed in the U87MG astroglioma cell line engineered to express the HIV-1 co-receptors CD4 and CXCR4 (Narute and Smithgall, 2012; Poe and Smithgall, 2009; Emert-Sedlak et al., 2009) or in the T-lymphoblast cell line, CEM-T4 (Narute and Smithgall, 2012). Both the U87MG and CEM-T4 cell lines support HIV-1 replication in a Nef-dependent manner, and were obtained from the NIH AIDS Research and Reference Reagent Program. Compounds were solubilized in DMSO, and added to the cell culture medium 1 h prior to infection with HIV. Viral replication was monitored for either 4 days (U87MG) or 9 days (CEM-T4) by measuring p24 Gag protein levels in the culture supernatant using standard ELISA-based techniques (Narute and Smithgall, 2012; Poe and Smithgall, 2009; Emert-Sedlak et al., 2009). HIV-1 infectivity was measured using the reporter cell line, TZM-bl (Platt et al., 1998; Derdeyn et al., 2000) (NIH AIDS Research and Reference Reagent Program). Cells were grown in 96-well plates (2.5 × 104) 8 h prior to virus infection to permit adherence. Compounds were pre-incubated with wild-type HIV-1 for 4 h prior to addition to the cells in a final volume of 200 μl. After 48 h at 37 °C, the cells were washed with PBS and lysed in luciferase lysis buffer (Promega) by rocking for 15 min. Lysates (40 μl) were transferred to white 96-well plates and 50 μl luciferase reagent (Promega) was injected into each well. Readings were recorded with a delay time of 2 sec and an integration period of 10 sec.

The effect of B9 on Nef-mediated activation of endogenous SFK activity was evaluated in CEM-T4 cells. Cells (1 × 105) were infected with 50 pg p24 equivalents/ml of wild-type HIV-1 NL4-3, a Nef-defective mutant (ΔNef), or the indicated Nef chimeras in a final culture volume of 10 ml in the presence of 1 μM PPD-B9 or the DMSO carrier solvent alone as a control. The infected cells were lysed eight days later and SFK proteins were immunoprecipitated with a pan-specific antibody as described elsewhere (Narute and Smithgall, 2012). SFK activity was assessed by immunoblotting each immunoprecipitate with a phosphospecific antibody against the activation loop phosphotyrosine residue common to all Src family members (pY418; Life Technologies). Control blots were performed on cell lysates for HIV-1 Gag proteins (p55, p40, and p24), Nef, as well as actin as a loading control.

SIV Assays

Stocks of the pathogenic SIV quasispecies ΔB670 (Seman et al., 2000) were the generous gift of Dr. Michael Murphey-Corb, University of Pittsburgh. SIV replication assays were conducted in CEM-174 cells (Salter et al., 1985). B9 was solubilized in DMSO and added to the cell culture medium 1 h prior to infection with SIV. Viral replication was assayed 5 d later as p27 Gag protein levels by ELISA (ZeptoMetrix). SIV infectivity was measured using the reporter cell line, TZM-bl, as described above for HIV. B9 was pre-incubated with SIV for 4 h prior to addition to the cells.

Cytotoxicity assays

U87MG or CEM-T4 cells were plated with compounds in DMSO carrier solvent in 96-well plates and incubated at 37 °C. After 72 hours, cytotoxicity was assessed using the Cell Titer Blue reagent (Promega) and the manufacturer’s protocol.

Surface plasmon resonance (SPR)

Recombinant full-length Nef (SF2 strain) with an N-terminal His-tag was expressed in bacteria and purified as described elsewhere (Narute and Smithgall, 2012). Nef was then exchanged into HBS-EP buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05 % v/v P20 surfactant) and concentrated with an Amicon Ultra 10 kDa molecular weight cutoff spin concentrator. SPR analysis was performed on a BIAcore 3000 instrument (GE Healthcare) using a four-channel CM5 biosensor chip at 25 °C. The Nef protein was covalently attached to the CM5 chip via standard amine coupling chemistry (Murphy et al., 2006; Jason-Moller et al., 2006). B9 (as analyte) was prepared in PBS buffer (10 mM Na2HPO4, 1.8 mM KH2PO4, 2.7 mM KCl, 137 mM NaCl) with 1% DMSO and flowed past the immobilized Nef protein channel and a reference channel on the biosensor CM5 chip at a flow rate of 50 μl/min for 3 min over a range of concentrations (see Figure 6). The initial binding reaction was followed by dissociation for 5 min, and the chip surface was regenerated using HBS-EPD buffer HBS-EPD buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05 % v/v P20, 1 mM DTT) at a flow rate of 50 μl/min for 10 min. The sensorgram curves recorded at each B9 concentration were assessed in triplicate, corrected for buffer effects, and fitted with the heterogeneous ligand-parallel reaction model using the BIAevaluation 4.1 software suite (GE Healthcare). In this model, one analyte (B9 in this case) interacts with two independent binding sites on the ligand (two predicted binding sites on Nef in this case), i.e., A + B1 + B2 ↔ AB1 + AB2, where A is the analyte while B1 and B2 represent the independent binding sites. In addition, the data were also fit to a two-state binding model, in which binding of B9 induces a conformational change in Nef to stabilize the binding. The two-state model assumes two states of Nef-B9 complexes, AB and AB*, which correspond to simple binding of B9 to Nef (AB) and a conformational change to a stable complex (AB*), i.e., A + B ↔ AB ↔ AB*. See Kuroki and Maenaka for further details of these SPR data fitting models (Kuroki and Maenaka, 2011).

Molecular docking

The 3D structure of B9 was docked to the crystal structures of both HIV-1 Nef (Lee et al., 1996) (PDB: 1EFN; without the SH3 domain) and SIV mac239 Nef (Kim et al., 2010) (PDB: 3IK5) in their dimeric conformations using AutoDock Vina (Trott and Olson, 2010) available at http://vina.scripps.edu. The three-dimensional structures of compound, B9 and the Nef dimers were first converted from pdb into pdbqt format using MGL Tools (Sanner, 1999). The structure of each Nef dimer was treated as the receptor and was kept rigid during the docking routine. In contrast, rotatable bonds in the structure of B9 imparted flexibility on the ligand. A grid box was centered at the 43.76, 18.61, 37.94 (HIV Nef) and 26.43, −7.16, −23.57 (SIV Nef) coordinates with 60Å units in the x, y and z directions to cover the entire structure in each case. Docking of B9 to both Nef dimer structures returned 9 lowest-energy conformations of the ligand. Of these, the Nef:B9 complexes showing the lowest binding energies and the greatest number of conformations in a cluster were chosen for further study (see Table S3).

BiFC Assay for Nef dimerization

The effect of B9 on Nef dimerization was assessed using a cell-based BiFC assay and our published method (Poe and Smithgall, 2009). Briefly, 293T cells were plated on glass coverslips and allowed to attach overnight. Cells were then treated with B9 or the DMSO carrier solvent alone for 30 min prior to transfection with the Nef-BiFC plasmid pair using XtremeGene9 and the manufacturer’s protocol (Roche). Forty-eight h later, cells were fixed and stained with anti-Nef antibodies, and immunostained cells were visualized with secondary antibodies conjugated to Texas red. Two-color immunofluorescent images were recorded at fixed exposure times for each channel using a Nikon TE300 inverted microscope with epifluorescence capability and a SPOT CCD high-resolution digital camera and software (Diagnostic Instruments). Image analysis was performed to determine mean pixel intensities in the BiFC (dimerization) and immunofluorescence (expression) channels of individual cells using Image J. BiFC to immunofluorescence ratios were calculated for at least 150 cells from each condition and are presented as percent of ratios obtained with DMSO-treated cells expressing wild-type Nef.

Supplementary Material

01

Highlights.

  • Nef is a critical HIV virulence factor lacking biochemical activity amenable to HTS

  • HTS for inhibitors of Nef-mediated Hck kinase activation identified Nef antagonists

  • Hit compound B9 blocks Nef-dependent HIV replication with submicromolar potency

  • B9 binds directly to purified HIV Nef in vitro and blocks Nef dimerization in cells

Acknowledgments

This work was supported by National Institutes of Health grants R01 AI057083, R21 AI077444, and X01 MH083223 (to T.E.S.). The authors would like to thank Dr. Michael Corb, University of Pittsburgh School of Medicine, for supplying the primary SIV isolate, ΔB670, and the NIH AIDS Reference Reagent Program for cell lines and antibodies.

Footnotes

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