- Open Access
Discovery of a diaminoquinoxaline benzenesulfonamide antagonist of HIV-1 Nef function using a yeast-based phenotypic screen
© Trible et al.; licensee BioMed Central Ltd. 2013
- Received: 13 April 2013
- Accepted: 31 October 2013
- Published: 14 November 2013
HIV-1 Nef is a viral accessory protein critical for AIDS progression. Nef lacks intrinsic catalytic activity and binds multiple host cell signaling proteins, including Hck and other Src-family tyrosine kinases. Nef binding induces constitutive Hck activation that may contribute to HIV pathogenesis by promoting viral infectivity, replication and downregulation of cell-surface MHC-I molecules. In this study, we developed a yeast-based phenotypic screen to identify small molecules that inhibit the Nef-Hck complex.
Nef-Hck interaction was faithfully reconstituted in yeast cells, resulting in kinase activation and growth arrest. Yeast cells expressing the Nef-Hck complex were used to screen a library of small heterocyclic compounds for their ability to rescue growth inhibition. The screen identified a dihydrobenzo-1,4-dioxin-substituted analog of 2-quinoxalinyl-3-aminobenzene-sulfonamide (DQBS) as a potent inhibitor of Nef-dependent HIV-1 replication and MHC-I downregulation in T-cells. Docking studies predicted direct binding of DQBS to Nef which was confirmed in differential scanning fluorimetry assays with recombinant purified Nef protein. DQBS also potently inhibited the replication of HIV-1 NL4-3 chimeras expressing Nef alleles representative of all M-group HIV-1 clades.
Our findings demonstrate the utility of a yeast-based growth reversion assay for the identification of small molecule Nef antagonists. Inhibitors of Nef function discovered with this assay, such as DQBS, may complement the activity of current antiretroviral therapies by enabling immune recognition of HIV-infected cells through the rescue of cell surface MHC-I.
- Src-family kinases
- MHC-I downregulation
- Small molecule Nef antagonists
HIV-1 nef encodes a small myristoylated protein required for optimal viral replication and AIDS pathogenesis [1, 2]. Deletion of nef from the HIV-related simian immunodeficiency virus prevents AIDS-like disease progression in rhesus macaques . In addition, expression of the nef gene alone is sufficient to induce an AIDS-like syndrome in transgenic mice very similar to that observed upon expression of the complete HIV-1 provirus [4, 5]. In humans, nef sequence variability and function correlate with HIV disease progression over the course of infection [6, 7]. Indeed, long-term non-progressive HIV infection has been associated with nef-defective strains of HIV in some cases [8–10]. These and other studies identify the HIV-1 Nef accessory protein as a key molecular determinant of AIDS.
Nef lacks any known intrinsic enzymatic or biochemical function and instead exploits multiple host cell signaling pathways to optimize conditions for viral replication and AIDS progression [11, 12]. Growing evidence identifies the Src family kinases (SFKs) as key molecular targets for Nef . One important example is Hck, a Src family member expressed in macrophages that binds strongly to Nef via an SH3-mediated interaction [14, 15]. Nef binding leads to constitutive Hck activation [16, 17], which may be important for macrophage survival  and productive infection by M-tropic HIV . In addition, activation of Hck, Lyn or c-Src is a critical first step in the downregulation of cell-surface MHC-I by Nef, which enables immune escape of HIV-infected cells [20–22]. Transgenic mice expressing a Nef mutant lacking a highly conserved PxxPxR motif essential for activation of Hck and other SFKs showed no evidence of AIDS-like disease . When the Nef-transgenic mice were crossed into a hck-null background, appearance of the AIDS-like phenotype was delayed with reduced mortality . These observations support an essential role for Nef interactions with Hck and other SFKs in multiple aspects of AIDS pathogenesis.
In this report, we describe the development of a yeast-based screen to identify inhibitors of Nef signaling through SFKs. First, we established that co-expression with Nef leads to constitutive activation of Hck in yeast by the same biochemical mechanism observed in mammalian cells. The active Nef:Hck complex induced growth arrest in yeast that was reversed with a known SFK inhibitor, providing a basis for a simple yet powerful screen for novel compounds. Using this system, we screened a small chemical library of drug-like heterocycles and identified a diaminoquinoxaline benzenesulfonamide analog that potently blocks Nef-dependent HIV replication and MHC-I downregulation. Docking studies and differential scanning fluorimetry assays support direct interaction of this compound with Nef as its mechanism of action. Small molecules that interfere with Nef-mediated downregulation of MHC-I molecules may represent powerful adjuvants to existing antiretroviral drugs by thwarting the viral strategy of immune evasion.
Hck-YEEI models Csk-downregulated Hck in yeast
Previous work has shown that ectopic expression of active c-Src induces growth arrest in yeast [24–27]. Co-expression of C-terminal Src kinase (Csk), a negative regulator of SFKs , rescues Src-mediated growth suppression by phosphorylating the c-Src negative regulatory tail and repressing kinase activity [26, 29–31]. Using a similar yeast-based system, we have previously shown that other members of the Src kinase family also induce yeast growth arrest in a Csk-reversible manner . Co-expression of HIV-1 Nef selectively overcomes Csk-mediated negative regulation for Hck, Lyn, and c-Src, resulting in kinase re-activation and growth arrest . These observations suggest that the yeast system may provide the basis for an inhibitor screen, as compounds which block Nef-induced SFK signaling are predicted to rescue cell growth.
Nef activates Hck-YEEI in yeast by the same molecular mechanism observed in mammalian cells
A second structural determinant of Nef interaction with SH3 involves a hydrophobic pocket formed by several conserved non-polar side chains in the Nef core (Phe90, Trp113, Tyr120; Figure 3A). These residues interact with SH3 Ile96, a residue unique to the RT loops of the Hck and Lyn SH3 domains  (Figure 3A). Substitution of Tyr120 within this Nef hydrophobic pocket with isoleucine (Nef-Y120I) disrupts Nef-mediated Hck activation in a rodent fibroblast model system . Similarly, Nef-Y120I was unable to activate Hck-YEEI in yeast and failed to produce growth suppression (Figure 3B). These data show that Nef recognizes and activates Hck-YEEI in yeast through the same mechanism observed in mammalian cells.
Chemical inhibition of Nef:Hck-YEEI activity restores yeast growth
Yeast cultures expressing the Nef:Hck-YEEI complex were then used to screen a chemical library of 2496 discrete heterocyclic compounds. In the first pass, each compound was tested in duplicate at 10 μM for its ability to increase the growth of Nef:Hck-YEEI cultures relative to controls incubated with the carrier solvent alone. From this primary screen, 170 compounds were observed to restore growth of Nef:Hck-YEEI cultures by at least 10% over untreated controls. These compounds were then re-screened at 10 μM in comparison to 5 μM A-419259, the control SFK inhibitor described above. Of these, fifteen compounds were observed to rescue growth to at least 25% of the values observed with A-419259-treated positive controls. Each of these compounds was then re-purchased and tested a third time over a range of concentrations to verify growth recovery of Nef:Hck-YEEI cultures compared with A-419259. Figure 4C shows the resulting rank order of these compounds relative to the A-419259 control response. Though the activities of these compounds were lower than those observed with the original library, the rank order of their activities remained the same.
Hit compounds from the Nef:Hck-YEEI yeast screen block Nef-dependent HIV replication
Because DQBS was identified as an inhibitor of Nef-dependent SFK activation, we next explored whether it affected Nef-dependent activation of endogenous SFK activity in the context of HIV-1 infection. For these experiments, CEM-T4 cells were infected with wild-type or Nef-defective HIV-1 over a range of DQBS concentrations. Endogenous SFK proteins were then immunoprecipitated from the infected cell lysates, and immunoblotted with a phosphospecific antibody against the activation loop phosphotyrosine (pY418). As shown in Figure 6B, HIV-1 infection resulted in Nef-dependent SFK activation loop tyrosine phosphorylation, and this effect was inhibited by about 50% in the presence of DQBS. This result shows that DQBS interferes with this Nef-dependent signaling function as part of its mechanism of action.
DQBS inhibits Nef-mediated MHC-I downregulation
We next explored the mechanism of the DQBS-dependent block in Nef-induced downregulation of MHC-I. An essential first step in this pathway involves Nef-mediated assembly of a multi-kinase complex including an SFK, Syk/Zap-70, and a class I PI3K [20, 21]. To determine whether DQBS affected assembly of this complex, H9 cells were co-infected with recombinant Hck and Nef vaccinia viruses in the presence or absence of DQBS. Nef immunoprecipitates were then prepared and probed for associated Hck and the p85 regulatory subunit of PI3K. Figure 7B shows that DQBS treatment reduced the amount of both Hck and p85 associated with Nef. DQBS treatment also completely blocked Nef-dependent activation of Zap-70 (Figure 7C). Using an in vitro kinase assay, we were unable to detect direct inhibition of Zap-70 or Hck by DQBS (Figure 7D), suggesting that its effects on kinase activity are mediated through Nef. Taken together, these findings suggest that DQBS prevents Nef-dependent downregulation of MHC-I by interfering with assembly of the multi-kinase complex and preventing the activation of Zap-70 downstream. Inhibition of Zap-70 may also contribute to the anti-retroviral efficacy of this compound (see Discussion).
Docking studies predict direct binding of DQBS to Nef
Docking of the small molecule Nef antagonist DQBS to HIV-1 Nef
Binding energy (kcal/mol)
Nef residues within 4 Å of DQBS
Nef subunit 1 (blue in Figure 8): Gln104, Asp108, Pro122, Asp123
Nef subunit 2 (green in Figure 8): Gln104, Asp108, Gln107, Asp111, Leu112, Pro122, Gln125, Asn126, Tyr127
Pro78, Met79, Thr80, Tyr81, Asp123, Trp124, Asn126, Leu137, Thr138, Phe129, Tyr202
Gln104, Gln107, Gln125, Asn126, Tyr127, Thr128, Pro129, Arg134, Leu137, Tyr202
Met79, Tyr82, Asn126, Leu137, Thr138, Phe139, His193, Tyr202, Phe203
Leu91, Lys94, Gly95, Gly96, Leu97, Leu100, Arg106, Ile109, Leu110, Trp113
Docking routines for DQBS based on an individual Nef subunit from the same crystal structure returned two sites with binding energies of −7.9 kcal/mol (Table 1). Both of these involve Asn126, which was also implicated in docking poses based on the dimer. A third putative DQBS binding site on Nef (−7.7 kcal/mol) involves Trp113, which is involved in Nef interaction with the SH3 domains of Src-family kinases (see Figure 3). In addition, Trp113 is essential for Nef binding to PACS-2, a trafficking protein critical to the assembly of the multi-kinase complex that initiates the Nef-dependent MHC-I downregulation pathway . This aspect of the docking model is consistent with our observations that DQBS destabilizes the multi-kinase complex and prevents activation of Zap-70 in the context of Nef-induced MHC-I downregulation (Figure 7). Overall, the docking studies raise the possibility that DQBS may interact with multiple sites on Nef, providing a mechanistic basis for its potent activity against several Nef functions (see Discussion).
Direct interaction of DQBS with Nef by differential scanning fluorimetry
Comparison of the anti-HIV activities of DQBS with other Nef antagonists
In this report we describe the discovery of a unique antagonist of the HIV-1 accessory protein, Nef, using a yeast-based screening assay. This assay exploits the growth-suppressive actions of Src-family kinases on yeast cell growth [26, 31]. In our case, we engineered yeast strains to co-express Nef and the Src-family kinase Hck, one of the best-characterized Nef target proteins. Nef interacts with Hck and switches on its kinase activity by binding to its SH3 domain, resulting in growth arrest. Hit compounds were selected based on their ability to rescue growth suppression by the Nef:Hck complex. One advantage of this approach is that non-selective cytotoxic compounds cannot rescue growth and therefore do not score as false positives. Remarkably, two of the top five compounds identified in the yeast screen were subsequently found to block Nef-dependent HIV-1 replication in vitro. One of these, the 2,3-diaminoquinoxaline analog DQBS, not only blocked Nef-dependent HIV-1 replication with submicromolar potency across a wide spectrum of Nef subtypes, but was also shown to reverse MHC-I downregulation by Nef.
DQBS was isolated from a chemical library biased towards heterocyclic structures that resemble protein kinase inhibitors, raising the possibility that it may target the ATP-binding site of Nef-activated SFKs or Zap-70 rather than Nef directly. However, using an in vitro kinase assay and recombinant purified Hck and Zap-70, we were unable to detect direct inhibition of kinase activity by DQBS. A more likely mechanism of action for DQBS involves direct interaction with Nef, thereby interfering with recruitment and activation of SFKs and other Nef effector proteins. This possibility is supported by docking studies, which predicted several energetically favorable binding sites for DQBS on the Nef structure. Remarkably, several of the Nef residues predicted to interact with DQBS have been previously identified in a similar docking study of the Nef antagonist, B9, including Gln104, Gln107, and Asn 126 . The observation that two independent screens yielded Nef antagonists with overlapping predicted binding sites suggests that this region may represent a hot spot for Nef inhibitor development. Direct interaction of DQBS with Nef is supported by differential scanning fluorimetry assays presented here, which showed that this compound causes thermal destabilization of the Nef protein in a concentration-dependent manner.
One exciting feature of DQBS is its potent activity against Nef-dependent downregulation of MHC-I, which is believed to allow HIV-infected cells to escape immune surveillance. The extent of MHC-I downregulation in simian immunodeficiency virus (SIV)-infected macaques correlates directly with the severity of disease progression, supporting a critical role for this immune evasive mechanism in vivo . A possible mechanism by which DQBS blocks this critical Nef function is suggested by the way in which Nef initiates MHC-I downregulation in HIV-infected cells. During the first two days following infection, Nef triggers MHC-I downregulation by an endocytic program termed the signaling mode. By three days post-infection, Nef switches to a stoichiometric mode of downregulation to prevent newly synthesized MHC-I molecules from reaching the cell surface . One early step in the signaling mode involves Nef-dependent assembly of a multi-kinase complex involving a Src-family member (Hck, c-Src or Lyn, depending upon the cell lineage), Zap-70 or Syk, and a class I PI3K [20, 22]. Data presented in Figure 7 support a model in which DQBS interferes with the initial assembly of this kinase complex, resulting in a complete block in the activation of Zap-70. Control kinase assays show that DQBS does not impact Zap-70 or Hck activity directly, supporting a mechanism of action that is mediated through Nef. In addition, computational docking studies show that DQBS binding may influence the accessibility of Nef Trp113, which is required for interaction with SFK SH3 domains as well as the PACS-2 trafficking protein that triggers SFK/Zap-70/PI3K complex assembly [22, 44].
Interestingly, Zap-70 has also been implicated in HIV-1 replication and viral spread [58, 59], suggesting that DQBS may interfere with HIV-1 replication by blocking Nef-dependent Zap-70 activation in CEM-T4 cells and other T-cell hosts. Future work will address whether DQBS can similarly inhibit HIV-1 replication in macrophages, which express the Zap-70 homolog, Syk. The finding that Syk interchanges with Zap-70 in the Nef-assembled multi-kinase complex in cells of the monocytic lineage supports this possibility . The potent inhibition of Nef-dependent HIV-1 replication in U87MG/CD4/CXCR4 cells reported here may also involve Syk, which exhibits a more widespread expression pattern than Zap70 . Taken together, these findings support an inhibitory mechanism in which DQBS binds directly to Nef and interferes with its activation of a common intermediate (Zap-70 or Syk) in both the MHC-I downregulation pathway and in HIV-1 replication.
Another Nef-binding compound, the Streptomyces natural product derivative known as ′2c’, has also been reported to affect Nef-dependent MHC-I downregulation [20, 61] and viral infectivity. NMR studies showed that 2c interacts primarily with Nef through a cleft formed by the central β-sheet and the C-terminal α-helices. While the 2c binding site is distinct from those for DQBS on Nef presented here, neither binding site overlaps with Nef structural features involved in SH3 binding and SFK recruitment. These observations suggest an allosteric mechanism of action for both compounds. Compared to DQBS, however, the potency of 2c is lower in terms of both MHC-I downregulation and antiviral activity. This difference may relate to a weaker binding affinity of 2c for Nef as well as the possibility that DQBS may occupy multiple sites on the Nef structure that are important for MHC-I downregulation as well as viral growth.
Antiretroviral agents currently used for the treatment of AIDS target the viral reverse transcriptase, integrase and protease or block virus-host cell fusion . Data presented here with the compound DQBS support the idea that the HIV-1 accessory protein Nef represents an alternative target for antiretroviral drug action. This compound not only inhibits enhancement of HIV replication by Nef, but also reverses Nef-mediated downregulation of MHC-I, raising the exciting possibility that it may enhance recognition of HIV-infected cells by cytotoxic T-cells. The growing number of HIV strains resistant to conventional antiretroviral therapy [63, 64] combined with the lack of an HIV vaccine underscore the need for new anti-HIV drugs. Work presented here shows that compounds targeting HIV-1 Nef may provide a new avenue for anti-HIV therapy, and demonstrates the potential of a yeast-based, phenotypic screen based on the complex of an HIV-1 accessory protein with a host cell kinase as a route to their discovery.
Yeast expression vectors
Coding sequences for human Csk and Hck as well as HIV-1 Nef (SF2 strain) were modified by PCR to introduce a yeast translation initiation sequence (AATA) immediately 5′ to the ATG start codon. The coding sequence for Hck was subcloned downstream of the Gal10 promoter in the pYC2/CT vector (Invitrogen), which carries the CEN6/ARSH4 sequence for low-copy replication. The Csk and Nef coding sequences were subcloned downstream of the Gal1 and Gal10 promoters, respectively, in the yeast expression vector pESC-Trp (Stratagene). The coding sequence of the wild-type Hck tail (YQQQP) was modified by PCR to encode the high-affinity SH2-binding sequence, YEEIP, as described elsewhere [32, 65]. The Nef-PA mutant, in which prolines 72 and 75 are replaced with alanines, has also been described elsewhere .
Yeast growth suppression assay
S. cerevisiae strain YPH 499 (Stratagene) was co-transformed with pESC-Ura (or pYC2/CT) and pESC-Trp plasmids containing the genes of interest via electroporation (BioRad Gene Pulser II). Yeast were selected for three days at 30°C on standard synthetic drop-out plates lacking uracil and tryptophan (SD/-U-T) with glucose as the sole carbon source to repress protein expression. Positive transformants were grown in liquid SD/-U-T medium plus glucose, normalized to OD600nm = 0.2 in water, and then spotted in four-fold dilutions onto SD/-U-T agar plates containing galactose as the sole carbon source to induce protein expression. Duplicate plates containing glucose were also prepared to control for yeast loading (data not shown). Plates were incubated for three days at 30°C and imaged on a flatbed scanner. Yeast patches appear as dark spots against the translucent agar background. All growth suppression assays were repeated at least three times starting with randomly selected independent transformed clones and produced comparable results; representative examples are shown. For the liquid growth assay, yeast strain W303a (gift of Dr. Frank Boschelli, Wyeth Pharmaceuticals) was co-transformed with the required plasmids, seeded at an initial density of OD600nm = 0.05 units in SD/-U-T medium, and incubated for 21 h at 30°C. The control inhibitor A-419259 was added with DMSO as carrier solvent to a final concentration of 0.1%.
Immunoblotting from yeast cultures
Aliquots of the yeast cultures used for the spot assay were grown in SD/-U-T medium plus galactose for 18 h. Cells were pelleted, treated with 0.1 N NaOH for 5 min at room temperature , and normalized with SDS-PAGE sample buffer to 0.02 OD600nm units per μl. Aliquots of each lysate (0.2 OD600nm units) were separated via SDS-PAGE, transferred to PVDF membranes, and probed for protein phosphotyrosine content with a combination of the anti-phosphotyrosine antibodies PY99 (Santa Cruz Biotechnology) and PY20 (Transduction Laboratories). Immunoblots were also performed with antibodies to Csk (C-20; Santa Cruz), Hck (N-30; Santa Cruz), actin (MAB1501; Chemicon International) and Nef (monoclonal Hyb 6.2; NIH AIDS Research and Reference Reagent Program).
Yeast inhibitor screen
Yeast strain W303a was co-transformed with Hck-YEEI and Nef expression plasmids and grown to an OD600nm of 0.05. Cells (100 μl) were plated in duplicate wells of a 96-well plate in the presence of each compound from the ChemDiv kinase-biased inhibitor library (ChemDiv, Inc., San Diego, CA). All compounds were initially screened at 10 μM with 0.5% DMSO as carrier solvent. Control wells contained 0.5% DMSO to define the extent of growth arrest as well as cells transformed with Hck-YEEI plus the Nef-2PA mutant to define maximum outgrowth. Each plate also contained wells with 5 μM A-419259 as a positive control for drug-mediated growth reversion. Cultures were incubated at 30°C, and the OD600nm was measured at 0 and 22 h. Those compounds which induced a 10% or greater increase in yeast growth relative to the DMSO control were further assayed in triplicate and compared against A-419259-mediated growth reversion. Compounds from this secondary screen which recovered yeast growth to at least 25% of that observed with A-419259 were obtained in powder form from the provider of the original library (ChemDiv) and assayed a third time in triplicate at 1, 3, 10, and 30 μM in comparison with 5 μM A-419259.
HIV-1 replication assays were conducted using the HIV-1 strain NL4-3. Viral stocks were prepared by transfection of 293 T cells (ATCC) with proviral genomes for the wild-type, Nef-defective (ΔNef), or Nef chimeras (all based on NL4-3 backbone) and amplified in the T-cell line, MT2 (NIH AIDS Research and Reference Reagent Program) as previously described [41, 45]. Viral replication was assessed in the U87MG astroglioma cell line engineered to express the HIV-1 co-receptors CD4 and CXCR4 or in the T-lymphoblast cell line, CEM-T4 [41, 45]. 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. HIV-1 infectivity was measured using the reporter cell line TZM-bl, in which the HIV LTR drives transcription of luciferase . Details of the assay conditions are described elsewhere .
Activation of endogenous SFKs by HIV-1 Nef
CEM-T4 cells (1 × 105) were infected with 50 pg p24 equivalents/ml of wild-type HIV-1 NL4-3 or the Nef-defective mutant in a final culture volume of 10 ml. DQBS or the DMSO carrier solvent alone were added followed by incubation for eight days. The infected cells were then lysed in RIPA buffer and endogenous Src-family kinases were immunoprecipitated with a pan-specific antibody and protein-G Sepharose beads as described elsewhere . Kinase activation was assessed by immunoblotting each immunoprecipitate with a phosphospecific antibody against the activation loop phosphotyrosine residue (pY418) common to all Src family members. 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.
MHC-I downregulation assays
H9 T cells were infected with wild-type vaccinia virus or with a vaccinia recombinant expressing Nef-Flag (moi = 10) for 8 h as described previously [22, 42]. Cells were incubated in the presence of DQBS or carrier solvent alone (DMSO) for 4 h prior to harvest. The cells were then fixed in 2% paraformaldehyde, washed and resuspended in FACS buffer (PBS, pH 7.2, containing 0.5% FBS) and incubated with mAb W6/32 (anti-MHC-I, 1:4,000) followed by PE-conjugated donkey anti-mouse IgG (1:1,000; Jackson IR, West Grove, PA). Cells were analyzed by listmode acquisition on a FACSCalibur (BD) flow cytometer using CellQuest acquisition/analysis software (BD) and data analyzed using CellQuest or FCS express (De Novo Software, Los Angeles, CA).
Co-immunoprecipitation of Nef:kinase complexes
To measure the effect of DQBS on the interaction between Nef, Hck and class I PI3K, H9 T cells were co-infected with wild-type vaccinia virus (moi = 10) or a combination of the Nef-Flag (moi = 10) and Hck viruses (moi = 12 total). Cells were treated with 10 μM DQBS at 4 h post-infection and harvested 4 h later by lysis in PBS containing 1% NP40 supplemented with protease and phosphatase inhibitors. Nef-Flag was immunoprecipitated with mAb M2-agarose beads (Sigma) and co-immunoprecipitating Hck (N-30, Santa Cruz) and p85 (Millipore) were detected by immunoblot analysis. Nef-Flag recovery was confirmed by immunoblotting with anti-Nef antibodies (AIDS Reagent and Reference Program). Control blots of cell lysates were performed with actin antibodies (mAb 1501, Millipore). To measure the effect of DQBS on the Nef-dependent activation of Zap-70, H9 cells were co-infected with wild-type vaccinia virus (moi = 6) or the Nef-Flag (moi = 6) and Zap-70 viruses (moi = 10 total). Infected cells were then treated with 10 μM DQBS for 4 h prior to harvest and lysed as described above. The presence of active ZAP-70 was assessed by immunoblotting with a phosphospecific antibody against the activation loop phosphotyrosine site (pY319-ZAP-70; clone 2 F3.2, Millipore). Zap-70 (Cell Signaling) and Nef levels were measured by immunoblotting of the clarified cell lysates.
The structure of DQBS was docked to the crystal structure of HIV-1 Nef  (PDB: 1EFN; without the SH3 domain) using AutoDock Vina . Independent docking routines were performed using the Nef dimer and a single Nef monomer. The three-dimensional structures of the compound and the Nef proteins were first converted from pdb into pdbqt format with MGL Tools . The Nef structures were kept rigid during the docking routine, while rotatable bonds in DQBS imparted ligand flexibility. A grid box was centered on and covered each Nef structure. Nef residues predicted to participate in Nef:DQBS complex formation from the docking results with the lowest binding energies are presented in Table 1.
Synthesis of DQBS
The synthesis of all compounds was performed under a nitrogen atmosphere. Commercially available precursors, solvents and reagents (Aldrich) were used without additional purification. NMR spectra were recorded on a Bruker 600 MHz spectrometer; chemical shifts are given in ppm and are referenced to residual solvent peaks.
4-Chlorobenzenesulfonamide (1.92 g, 10 mmol) was dissolved in anhydrous DMF (50 ml). Potassium carbonate (1.38 g, 10 mmol) was added in one portion, and the reaction mixture was stirred for 10 min. 2,3-Dichloroquinoxaline (1.99 g, 10 mmol) was added, and the reaction mixture was refluxed under N2 for 2.5 h with reaction progress monitored by TLC (hexanes/ethyl acetate 3:1 as mobile phase). The reaction mixture was cooled and added slowly to an aqueous solution of acetic acid (1%, 500 ml) with vigorous stirring. The product precipitated as grey crystals, which were filtered and dried overnight in a desiccator (Drierite). Yield 2.32 g, 66%. Rf = 0.7 (hexanes/ethyl acetate 1:1).
Compound QBS (354 mg, 1 mmol; above) was dissolved in xylenes (20 ml). 6-Amino-1,4-benzodioxane (2 mmol, 246 μl) was added and the reaction mixture was refluxed under N2 for 5 h. The solvent was evaporated under vacuum, and DQBS was isolated and purified by column chromatography (hexanes/ethyl acetate 9:1 as solvent phase). The final product formed yellow crystals with a melting point of 257-258°C. Yield, 61%. Rf = 0.3 (hexanes/ethyl acetate 3:1). 1H NMR (CDCl3, 600 MHz): δ 4.31 (m, 2H), 6.88 (d, J = 9.0 Hz, 1H), 7.15 (dd, J = 9.0 Hz, 2.4 Hz, 1H), 7.29 (dd, J = 1.2 Hz, 1H), 7.36 (td, J = 7.8 Hz, 1.2 Hz, 1H), 7.42 (td, J = 7.8 Hz, 1.2 Hz, 1H), 7.53 (d, J = 9 Hz, 2H), 7.70 (m, 2H), 7.98 (d, J = 8.4 Hz, 2H), 8.19 (br.s, 1H), 11.88 (br.s, 1H). 13C NMR (CDCl3, 150 MHz): δ 64.34, 64.53, 109.36, 113.54, 116.18, 117.28, 124.16, 125.87, 126.60, 126.81, 127.89, 129.38, 131.99, 134.18, 139.41, 140.14, 140.28, 141.24, 143.43, 144.08. HRMS [C22H18ClN4O4S]+: calculated, 469.0732; observed 469.0704.
Differential Scanning Fluorimetry (DSF)
A real-time StepOnePlus qPCR instrument (Applied Biosystems) and software (version 2.3) were used to perform DSF measurements. Recombinant full-length Nef (SF2 allele) and human Hck-YEEI were expressed and purified as described previously [40, 41]. DSF assays (20 μl) were run in triplicate wells in MicroAmp Fast 96-well qPCR plates sealed with optical adhesive covers (Applied Biosystems). Baseline DSF profiles were obtained with recombinant Nef and Hck-YEEI proteins (1 μM) in bicine buffer (10 mM bicine, 150 mM NaCl, pH 8.0) and SYPRO Orange (Sigma) diluted to a 5X working concentration as described [51, 52]. The test compounds DQBS, 2,3-diaminoquinoxaline (ChemDiv) and dasatinib (LC Laboratories) were solubilized in DMSO and diluted into the DSF assays, followed by incubation for 15 min with each protein at 4°C prior to the addition of SYPRO Orange. Parallel reactions were run in the absence of the proteins to correct for background fluorescence. The final DMSO concentration in all reactions was 1.1%. For DSF measurements, the qPCR instrument was set to use the ROX emission filter (≅ 610 nm) without a quencher or passive reference as recommended by the manufacturer. DSF mixtures were allowed to equilibrate to 25°C for 2 min, followed by an increase to 99°C at a 1% temperature ramp rate (1.6°C/min) with continuous data collection. Data were corrected for background (no protein controls) and mean fluorescence intensities were plotted as a function of temperature. The resulting melt curves were fit to the Boltzmann sigmoid function using GraphPad Prism 6, and melt temperature (Tm) values were derived from the midpoint of the melt transition as described previously [51, 52]. ΔTm values were calculated as the difference between the Tm values obtained in the presence and absence of each test compound.
This work was supported by National Institutes of Health Grants CA81398, AI57083 and AI102704 (to T.E.S.) and CA151564 (to G.T.). The authors thank Dr. Frank Boschelli, Wyeth Pharmaceuticals, for generously providing the yeast strain W303a. We also thank Dr. John S. Lazo and Caleb Foster, formerly of the University of Pittsburgh Drug Discovery Institute, for their help with robotic plating of compound libraries for the screening assays. The Nef antagonist DLC27-14 was generously provided by Dr. Xavier Morelli, Cancer Research Center of Marseille, France.
- Fackler OT, Baur AS: Live and let die: Nef functions beyond HIV replication. Immunity. 2002, 16: 493-497. 10.1016/S1074-7613(02)00307-2.View ArticlePubMedGoogle Scholar
- Geyer M, Fackler OT, Peterlin BM: Structure–function relationships in HIV-1 Nef. EMBO Rep. 2001, 2: 580-585. 10.1093/embo-reports/kve141.PubMed CentralView ArticlePubMedGoogle Scholar
- Kestler HW, Ringler DJ, Mori K, Panicali DL, Sehgal PK, Daniel MD, Desrosiers RC: Importance of the nef gene for maintenance of high virus loads and for development of AIDS. Cell. 1991, 65: 651-662. 10.1016/0092-8674(91)90097-I.View ArticlePubMedGoogle Scholar
- Hanna Z, Kay DG, Rebai N, Guimond A, Jothy S, Jolicoeur P: Nef harbors a major determinant of pathogenicity for an AIDS-like disease induced by HIV-1 in transgenic mice. Cell. 1998, 95: 163-175. 10.1016/S0092-8674(00)81748-1.View ArticlePubMedGoogle Scholar
- Hanna Z, Kay DG, Cool M, Jothy S, Rebai N, Jolicoeur P: Transgenic mice expressing human immunodeficiency virus type 1 in immune cells develop a severe AIDS-like disease. J Virol. 1998, 72: 121-132.PubMed CentralPubMedGoogle Scholar
- Carl S, Greenough TC, Krumbiegel M, Greenberg M, Skowronski J, Sullivan JL, Kirchhoff F: Modulation of different human immunodeficiency virus type 1 Nef functions during progression to AIDS. J Virol. 2001, 75: 3657-3665. 10.1128/JVI.75.8.3657-3665.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Kirchhoff F, Easterbrook PJ, Douglas N, Troop M, Greenough TC, Weber J, Carl S, Sullivan JL, Daniels RS: Sequence variations in human immunodeficiency virus type 1 Nef are associated with different stages of disease. J Virol. 1999, 73: 5497-5508.PubMed CentralPubMedGoogle Scholar
- Deacon NJ, Tsykin A, Solomon A, Smith K, Ludford-Menting M, Hooker DJ, McPhee DA, Greenway AL, Ellett A, Chatfield C: Genomic structure of an attenuated quasi species of HIV-1 from a blood transfusion donor and recipients. Science. 1995, 270: 988-991. 10.1126/science.270.5238.988.View ArticlePubMedGoogle Scholar
- Kirchhoff F, Greenough TC, Brettler DB, Sullivan JL, Desrosiers RC: Brief report: absence of intact nef sequences in a long-term survivor with nonprogressive HIV-1 infection. N Engl J Med. 1995, 332: 228-232. 10.1056/NEJM199501263320405.View ArticlePubMedGoogle Scholar
- Zou W, Denton PW, Watkins RL, Krisko JF, Nochi T, Foster JL, Garcia JV: Nef functions in BLT mice to enhance HIV-1 replication and deplete CD4 + CD8+ thymocytes. Retrovirology. 2012, 9: 44-10.1186/1742-4690-9-44.PubMed CentralView ArticlePubMedGoogle Scholar
- Malim MH, Emerman M: HIV-1 accessory proteins–ensuring viral survival in a hostile environment. Cell Host Microbe. 2008, 3: 388-398. 10.1016/j.chom.2008.04.008.View ArticlePubMedGoogle Scholar
- Foster JL, Garcia JV: HIV-1 Nef: at the crossroads. Retrovirology. 2008, 5: 84-10.1186/1742-4690-5-84.PubMed CentralView ArticlePubMedGoogle Scholar
- Saksela K: Interactions of the HIV/SIV pathogenicity factor Nef with SH3 domain-containing host cell proteins. Curr HIV Res. 2011, 9: 531-542. 10.2174/157016211798842107.View ArticlePubMedGoogle Scholar
- Lee CH, Leung B, Lemmon MA, Zheng J, Cowburn D, Kuriyan J, Saksela K: A single amino acid in the SH3 domain of Hck determines its high affinity and specificity in binding to HIV-1 Nef protein. EMBO J. 1995, 14: 5006-5015.PubMed CentralPubMedGoogle Scholar
- Arold S, O’Brien R, Franken P, Strub MP, Hoh F, Dumas C, Ladbury JE: RT loop flexibility enhances the specificity of Src family SH3 domains for HIV-1 Nef. Biochemistry. 1998, 37: 14683-14691. 10.1021/bi980989q.View ArticlePubMedGoogle Scholar
- Moarefi I, LaFevre-Bernt M, Sicheri F, Huse M, Lee C-H, Kuriyan J, Miller WT: Activation of the Src-family tyrosine kinase Hck by SH3 domain displacement. Nature. 1997, 385: 650-653. 10.1038/385650a0.View ArticlePubMedGoogle Scholar
- Briggs SD, Sharkey M, Stevenson M, Smithgall TE: SH3-mediated Hck tyrosine kinase activation and fibroblast transformation by the Nef protein of HIV-1. J Biol Chem. 1997, 272: 17899-17902. 10.1074/jbc.272.29.17899.View ArticlePubMedGoogle Scholar
- Briggs SD, Scholtz B, Jacque JM, Swingler S, Stevenson M, Smithgall TE: HIV-1 Nef promotes survival of myeloid cells by a Stat3-dependent pathway. J Biol Chem. 2001, 276: 25605-25611. 10.1074/jbc.M103244200.View ArticlePubMedGoogle Scholar
- Komuro I, Yokota Y, Yasuda S, Iwamoto A, Kagawa KS: CSF-induced and HIV-1-mediated distinct regulation of Hck and C/EBPbeta represent a heterogeneous susceptibility of monocyte-derived macrophages to M-tropic HIV-1 infection. J Exp Med. 2003, 198: 443-453. 10.1084/jem.20022018.PubMed CentralView ArticlePubMedGoogle Scholar
- Dikeakos JD, Atkins KM, Thomas L, Emert-Sedlak L, Byeon IJ, Jung J, Ahn J, Wortman MD, Kukull B, Saito M, Koizumi H, Williamson DM, Hiyoshi M, Barklis E, Takiguchi M, Suzu S, Gronenborn AM, Smithgall TE, Thomas G: Small molecule inhibition of HIV-1-induced MHC-I down-regulation identifies a temporally regulated switch in Nef action. Mol Biol Cell. 2010, 21: 3279-3292. 10.1091/mbc.E10-05-0470.PubMed CentralView ArticlePubMedGoogle Scholar
- Atkins KM, Thomas L, Youker RT, Harriff MJ, Pissani F, You H, Thomas G: HIV-1 Nef binds PACS-2 to assemble a multikinase cascade that triggers major histocompatibility complex class I (MHC-I) down-regulation: analysis using short interfering RNA and knock-out mice. J Biol Chem. 2008, 283: 11772-11784. 10.1074/jbc.M707572200.PubMed CentralView ArticlePubMedGoogle Scholar
- Hung CH, Thomas L, Ruby CE, Atkins KM, Morris NP, Knight ZA, Scholz I, Barklis E, Weinberg AD, Shokat KM, Thomas G: HIV-1 Nef assembles a Src family kinase-ZAP-70/Syk-PI3K cascade to downregulate cell-surface MHC-I. Cell Host Microbe. 2007, 1: 121-133. 10.1016/j.chom.2007.03.004.View ArticlePubMedGoogle Scholar
- Hanna Z, Weng X, Kay DG, Poudrier J, Lowell C, Jolicoeur P: The pathogenicity of human immunodeficiency virus (HIV) type 1 Nef in CD4C/HIV transgenic mice is abolished by mutation of its SH3-binding domain, and disease development is delayed in the absence of Hck. J Virol. 2001, 75: 9378-9392. 10.1128/JVI.75.19.9378-9392.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Brugge JS, Jarosik G, Andersen J, Queral-Lustig A, Fedor-Chaiken M, Broach JR: Expression of Rous sarcoma virus transforming protein pp 60v-src in Saccharomyces cerevisiae cells. Mol Cell Biol. 1987, 7: 2180-2187.PubMed CentralView ArticlePubMedGoogle Scholar
- Kornbluth S, Jove R, Hanafusa H: Characterization of avian and viral p60src proteins expressed in yeast. Proc Natl Acad Sci U S A. 1987, 84: 4455-4459. 10.1073/pnas.84.13.4455.PubMed CentralView ArticlePubMedGoogle Scholar
- Murphy SM, Bergman M, Morgan DO: Suppression of c-Src activity by C-terminal Src kinase involves the c-Src SH2 and SH3 domains: analysis with Saccharomyces cerevisiae. Mol Cell Biol. 1993, 13: 5290-5300.PubMed CentralView ArticlePubMedGoogle Scholar
- Florio M, Wilson LK, Trager JB, Thorner J, Martin GS: Aberrant protein phosphorylation at tyrosine is responsible for the growth-inhibitory action of pp 60v-src expressed in the yeast Saccharomyces cerevisiae. Mol Biol Cell. 1994, 5: 283-296. 10.1091/mbc.5.3.283.PubMed CentralView ArticlePubMedGoogle Scholar
- Nada S, Okada M, MacAuley A, Cooper JA, Nakagawa H: Cloning of a complementary DNA for a protein-tyrosine kinase that specifically phosphorylates a negative regulatory site of p60c-src. Nature. 1991, 351: 69-72. 10.1038/351069a0.View ArticlePubMedGoogle Scholar
- Trible RP, Emert-Sedlak L, Smithgall TE: HIV-1 Nef selectively activates SRC family kinases HCK, LYN, and c-SRC through direct SH3 domain interaction. J Biol Chem. 2006, 281: 27029-27038. 10.1074/jbc.M601128200.PubMed CentralView ArticlePubMedGoogle Scholar
- Nada S, Yagi T, Takeda H, Tokunaga T, Nakagawa H, Ikawa Y, Okada M, Aizawa S: Constitutive activation of Src family kinases in mouse embryos that lack Csk. Cell. 1993, 73: 1125-1135. 10.1016/0092-8674(93)90642-4.View ArticlePubMedGoogle Scholar
- Superti-Furga G, Fumagalli S, Koegl M, Courtneidge SA, Draetta G: Csk inhibition of c-Src activity requires both the SH2 and SH3 domains of Src. EMBO J. 1993, 12: 2625-2634.PubMed CentralPubMedGoogle Scholar
- Schindler T, Sicheri F, Pico A, Gazit A, Levitzki A, Kuriyan J: Crystal structure of Hck in complex with a Src family-selective tyrosine kinase inhibitor. Mol Cell. 1999, 3: 639-648. 10.1016/S1097-2765(00)80357-3.View ArticlePubMedGoogle Scholar
- Sicheri F, Moarefi I, Kuriyan J: Crystal structure of the Src family tyrosine kinase Hck. Nature. 1997, 385: 602-609. 10.1038/385602a0.View ArticlePubMedGoogle Scholar
- Engen JR, Wales TE, Hochrein JM, Meyn MA, Banu OS, Bahar I, Smithgall TE: Structure and dynamic regulation of Src-family kinases. Cell Mol Life Sci. 2008, 65: 3058-3073. 10.1007/s00018-008-8122-2.View ArticlePubMedGoogle Scholar
- Lee C-H, Saksela K, Mirza UA, Chait BT, Kuriyan J: Crystal structure of the conserved core of HIV-1 Nef complexed with a Src family SH3 domain. Cell. 1996, 85: 931-942. 10.1016/S0092-8674(00)81276-3.View ArticlePubMedGoogle Scholar
- Choi HJ, Smithgall TE: Conserved residues in the HIV-1 Nef hydrophobic pocket are essential for recruitment and activation of the Hck tyrosine kinase. J Mol Biol. 2004, 343: 1255-1268. 10.1016/j.jmb.2004.09.015.View ArticlePubMedGoogle Scholar
- Wilson MB, Schreiner SJ, Choi HJ, Kamens J, Smithgall TE: Selective pyrrolo-pyrimidine inhibitors reveal a necessary role for Src family kinases in Bcr-Abl signal transduction and oncogenesis. Oncogene. 2002, 21: 8075-8088. 10.1038/sj.onc.1206008.View ArticlePubMedGoogle Scholar
- Meyn MA, Schreiner SJ, Dumitrescu TP, Nau GJ, Smithgall TE: SRC family kinase activity is required for murine embryonic stem cell growth and differentiation. Mol Pharmacol. 2005, 68: 1320-1330. 10.1124/mol.104.010231.View ArticlePubMedGoogle Scholar
- Meyn MA, Smithgall TE: Chemical genetics identifies c-Src as an activator of primitive ectoderm formation in murine embryonic stem cells. Sci Signal. 2009, 2: ra64-PubMed CentralPubMedGoogle Scholar
- Emert-Sedlak L, Kodama T, Lerner EC, Dai W, Foster C, Day BW, Lazo JS, Smithgall TE: Chemical library screens targeting an HIV-1 accessory factor/host cell kinase complex identify novel antiretroviral compounds. ACS Chem Biol. 2009, 4: 939-947. 10.1021/cb900195c.PubMed CentralView ArticlePubMedGoogle Scholar
- Narute PS, Smithgall TE: Nef alleles from all major HIV-1 clades activate Src-family kinases and enhance HIV-1 replication in an inhibitor-sensitive manner. PLoS One. 2012, 7: e32561-10.1371/journal.pone.0032561.PubMed CentralView ArticlePubMedGoogle Scholar
- Blagoveshchenskaya AD, Thomas L, Feliciangeli SF, Hung CH, Thomas G: HIV-1 Nef downregulates MHC-I by a PACS-1- and PI3K-regulated ARF6 endocytic pathway. Cell. 2002, 111: 853-866. 10.1016/S0092-8674(02)01162-5.View ArticlePubMedGoogle Scholar
- Collins KL, Chen BK, Kalams SA, Walker BD, Baltimore D: HIV-1 Nef protein protects infected primary cells against killing by cytotoxic T lymphocytes. Nature. 1998, 391: 397-401. 10.1038/34929.View ArticlePubMedGoogle Scholar
- Dikeakos JD, Thomas L, Kwon G, Elferich J, Shinde U, Thomas G: An interdomain binding site on HIV-1 Nef interacts with PACS-1 and PACS-2 on endosomes to down-regulate MHC-I. Mol Biol Cell. 2012, 23: 2184-2197. 10.1091/mbc.E11-11-0928.PubMed CentralView ArticlePubMedGoogle Scholar
- Emert-Sedlak LA, Narute P, Shu ST, Poe JA, Shi H, Yanamala N, Alvarado JJ, Lazo JS, Yeh JI, Johnston PA, Smithgall TE: Effector Kinase Coupling Enables High-Throughput Screens for Direct HIV-1 Nef Antagonists with Antiretroviral Activity. Chem Biol. 2013, 20: 82-91. 10.1016/j.chembiol.2012.11.005.PubMed CentralView ArticlePubMedGoogle Scholar
- Das J, Chen P, Norris D, Padmanabha R, Lin J, Moquin RV, Shen Z, Cook LS, Doweyko AM, Pitt S, Pang S, Shen DR, Fang Q, De Fex HF, McIntyre KW, Shuster DJ, Gillooly KM, Behnia K, Schieven GL, Wityak J, Barrish JC: 2-aminothiazole as a novel kinase inhibitor template. Structure-activity relationship studies toward the discovery of N-(2-chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-1- piperazinyl)]-2-methyl-4-pyrimidinyl]amino)]-1,3-thiazole-5-carboxamide (dasatinib, BMS-354825) as a potent pan-Src kinase inhibitor. J Med Chem. 2006, 49: 6819-6832. 10.1021/jm060727j.View ArticlePubMedGoogle Scholar
- Yamamoto N, Takeshita K, Shichijo M, Kokubo T, Sato M, Nakashima K, Ishimori M, Nagai H, Li YF, Yura T, Bacon KB: The orally available spleen tyrosine kinase inhibitor 2-[7-(3,4-dimethoxyphenyl)-imidazo[1,2-c]pyrimidin-5-ylamino]nicotinamide dihydrochloride (BAY 61–3606) blocks antigen-induced airway inflammation in rodents. J Pharmacol Exp Ther. 2003, 306: 1174-1181. 10.1124/jpet.103.052316.View ArticlePubMedGoogle Scholar
- Trott O, Olson AJ: AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem. 2010, 31: 455-461.PubMed CentralPubMedGoogle Scholar
- Liu LX, Heveker N, Fackler OT, Arold S, Le Gall S, Janvier K, Peterlin BM, Dumas C, Schwartz O, Benichou S, Benarous R: Mutation of a conserved residue (D123) required for oligomerization of human immunodeficiency virus type 1 Nef protein abolishes interaction with human thioesterase and results in impairment of Nef biological functions. J Virol. 2000, 74: 5310-5319. 10.1128/JVI.74.11.5310-5319.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Jia X, Singh R, Homann S, Yang H, Guatelli J, Xiong Y: Structural basis of evasion of cellular adaptive immunity by HIV-1 Nef. Nat Struct Mol Biol. 2012, 19: 701-706. 10.1038/nsmb.2328.PubMed CentralView ArticlePubMedGoogle Scholar
- Niesen FH, Berglund H, Vedadi M: The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat Protoc. 2007, 2: 2212-2221. 10.1038/nprot.2007.321.View ArticlePubMedGoogle Scholar
- Vedadi M, Niesen FH, Allali-Hassani A, Fedorov OY, Finerty PJ, Wasney GA, Yeung R, Arrowsmith C, Ball LJ, Berglund H, Hui R, Marsden BD, Nordlund P, Sundstrom M, Weigelt J, Edwards AM: Chemical screening methods to identify ligands that promote protein stability, protein crystallization, and structure determination. Proc Natl Acad Sci U S A. 2006, 103: 15835-15840. 10.1073/pnas.0605224103.PubMed CentralView ArticlePubMedGoogle Scholar
- Smithgall TE, Thomas G: Small molecule inhibitors of the HIV-1 virulence factor, Nef. Drug Discov Today: Technol. 2013, 10: e523-e529. 10.1016/j.ddtec.2013.07.002.View ArticleGoogle Scholar
- Lugari A, Breuer S, Coursindel T, Opi S, Restouin A, Shi X, Nazabal A, Torbett BE, Martinez J, Collette Y, Parrot I, Arold ST, Morelli X: A specific protein disorder catalyzer of HIV-1 Nef. Bioorg Med Chem. 2011, 19: 7401-7406. 10.1016/j.bmc.2011.10.051.View ArticlePubMedGoogle Scholar
- Betzi S, Restouin A, Opi S, Arold ST, Parrot I, Guerlesquin F, Morelli X, Collette Y: Protein protein interaction inhibition (2P2I) combining high throughput and virtual screening: Application to the HIV-1 Nef protein. Proc Natl Acad Sci U S A. 2007, 104: 19256-19261. 10.1073/pnas.0707130104.PubMed CentralView ArticlePubMedGoogle Scholar
- Gervaix A, West D, Leoni LM, Richman DD, Wong-Staal F, Corbeil J: A new reporter cell line to monitor HIV infection and drug susceptibility in vitro. Proc Natl Acad Sci U S A. 1997, 94: 4653-4658. 10.1073/pnas.94.9.4653.PubMed CentralView ArticlePubMedGoogle Scholar
- Friedrich TC, Piaskowski SM, Leon EJ, Furlott JR, Maness NJ, Weisgrau KL, Mac Nair CE, Weiler AM, Loffredo JT, Reynolds MR, Williams KY, Klimentidis YC, Wilson NA, Allison DB, Rakasz EG: High viremia is associated with high levels of in vivo major histocompatibility complex class I Downregulation in rhesus macaques infected with simian immunodeficiency virus SIVmac239. J Virol. 2010, 84: 5443-5447. 10.1128/JVI.02452-09.PubMed CentralView ArticlePubMedGoogle Scholar
- Sol-Foulon N, Sourisseau M, Porrot F, Thoulouze MI, Trouillet C, Nobile C, Blanchet F, DiB V, Noraz N, Taylor N, Alcover A, Hivroz C, Schwartz O: ZAP-70 kinase regulates HIV cell-to-cell spread and virological synapse formation. EMBO J. 2007, 26: 516-526. 10.1038/sj.emboj.7601509.PubMed CentralView ArticlePubMedGoogle Scholar
- Simmons A, Aluvihare V, McMichael A: Nef triggers a transcriptional program in T cells imitating single-signal T cell activation and inducing HIV virulence mediators. Immunity. 2001, 14: 763-777. 10.1016/S1074-7613(01)00158-3.View ArticlePubMedGoogle Scholar
- Yanagi S, Inatome R, Takano T, Yamamura H: Syk expression and novel function in a wide variety of tissues. Biochem Biophys Res Commun. 2001, 288: 495-498. 10.1006/bbrc.2001.5788.View ArticlePubMedGoogle Scholar
- Chutiwitoonchai N, Hiyoshi M, Mwimanzi P, Ueno T, Adachi A, Ode H, Sato H, Fackler OT, Okada S, Suzu S: The identification of a small molecule compound that reduces HIV-1 Nef-mediated viral infectivity enhancement. PLoS One. 2011, 6: e27696-10.1371/journal.pone.0027696.PubMed CentralView ArticlePubMedGoogle Scholar
- Temesgen Z, Warnke D, Kasten MJ: Current status of antiretroviral therapy. Expert Opin Pharmacother. 2006, 7: 1541-1554. 10.1517/146565220.127.116.111.View ArticlePubMedGoogle Scholar
- Machouf N, Thomas R, Nguyen VK, Trottier B, Boulassel MR, Wainberg MA, Routy JP: Effects of drug resistance on viral load in patients failing antiretroviral therapy. J Med Virol. 2006, 78: 608-613. 10.1002/jmv.20582.View ArticlePubMedGoogle Scholar
- Turner D, Wainberg MA: HIV transmission and primary drug resistance. AIDS Rev. 2006, 8: 17-23.PubMedGoogle Scholar
- Lerner EC, Smithgall TE: SH3-dependent stimulation of Src-family kinase autophosphorylation without tail release from the SH2 domain in vivo. Nat Struct Biol. 2002, 9: 365-369.PubMedGoogle Scholar
- Kushnirov VV: Rapid and reliable protein extraction from yeast. Yeast. 2000, 16: 857-860. 10.1002/1097-0061(20000630)16:9<857::AID-YEA561>3.0.CO;2-B.View ArticlePubMedGoogle Scholar
- Sanner MF: Python: a programming language for software integration and development. J Mol Graph Model. 1999, 17: 57-61.PubMedGoogle Scholar
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