Discovery of a diaminoquinoxaline benzenesulfonamide antagonist of HIV-1 Nef function using a yeast-based phenotypic screen.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Temporal Changes in Microrna Expression in Blood Leukocytes from Patients with the Acute Respiratory Distress Syndrome.

BACKGROUND: MicroRNA (miRNA) control gene transcription by binding to and repressing the translation of messenger RNA (mRNA). Their role in the acute respiratory distress syndrome (ARDS) is undefined. METHODS: Blood leukocytes from 51 patients enrolled in a prior randomized trial of corticosteroids for ARDS were analyzed. After screening eight patients with microarrays for altered miRNA expression, 25 miRNAs were selected for further analysis using RT-PCR in all 51 patients. RESULTS: On day 0, the 51 patients had APACHE III score of 60.4 ±â€Š17.7 and PaO2/FiO2 of 117 ±â€Š49. 21 miRNA were expressed at increased levels in blood leukocytes at the onset of ARDS compared with healthy controls. These miRNA remained elevated at day 3 and increased further by day 7 (log2 fold change from 0.66 to 5.7 fold, P <0.05 compared to day 0). In a subgroup analysis (37 patients treated with corticosteroids and 14 treated with placebo), the interaction of miRNA expression over time and steroid administration was not significant suggesting that systemic corticosteroids had no effect on the miRNA detected in our study. In contrast, corticosteroids but not placebo decreased IL-6 and C-reactive protein at day 3 (P < 0.001) demonstrating an early systemic anti-inflammatory response whereas both treatment arms had decreased values by day 7 (P <0.001). CONCLUSIONS: Expression of miRNA is increased in blood leukocytes of patients with ARDS at day 0 and day 3 and rises further by day 7, when systemic inflammation is subsiding. These effects appear independent of the administration of steroids, suggesting different inflammatory modifying roles for each in the resolving phases of ARDS.

Effector kinase coupling enables high-throughput screens for direct HIV-1 Nef antagonists with antiretroviral activity.

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 submicromolar 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 nonenzymatic 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.

Nef alleles from all major HIV-1 clades activate Src-family kinases and enhance HIV-1 replication in an inhibitor-sensitive manner.

The HIV-1 accessory factor Nef is essential for high-titer viral replication and AIDS progression. Nef function requires interaction with many host cell proteins, including specific members of the Src kinase family. Here we explored whether Src-family kinase activation is a conserved property of Nef alleles from a wide range of primary HIV-1 isolates and their sensitivity to selective pharmacological inhibitors. Representative Nef proteins from the major HIV-1 subtypes A1, A2, B, C, F1, F2, G, H, J and K strongly activated Hck and Lyn as well as c-Src to a lesser extent, demonstrating for the first time that Src-family kinase activation is a highly conserved property of primary M-group HIV-1 Nef isolates. Recently, we identified 4-amino substituted diphenylfuropyrimidines (DFPs) that selectively inhibit Nef-dependent activation of Src-family kinases as well as HIV replication. To determine whether DFP compounds exhibit broad-spectrum Nef-dependent antiretroviral activity against HIV-1, we first constructed chimeric forms of the HIV-1 strain NL4-3 expressing each of the primary Nef alleles. The infectivity and replication of these Nef chimeras was indistinguishable from that of wild-type virus in two distinct cell lines (U87MG astroglial cells and CEM-T4 lymphoblasts). Importantly, the 4-aminopropanol and 4-aminobutanol derivatives of DFP potently inhibited the replication of all chimeric forms of HIV-1 in both U87MG and CEM-T4 cells in a Nef-dependent manner. The antiretroviral effects of these compounds correlated with inhibition of Nef-dependent activation of endogenous Src-family kinases in the HIV-infected cells. Our results demonstrate that the activation of Hck, Lyn and c-Src by Nef is highly conserved among all major clades of HIV-1 and that selective targeting of this pathway uniformly inhibits HIV-1 replication.

HIV-1 Nef interaction influences the ATP-binding site of the Src-family kinase, Hck.

BACKGROUND: Nef is an HIV-1 accessory protein essential for viral replication and AIDS progression. Nef interacts with a multitude of host cell signaling partners, including members of the Src kinase family. Nef preferentially activates Hck, a Src-family kinase (SFK) strongly expressed in macrophages and other HIV target cells, by binding to its regulatory SH3 domain. Recently, we identified a series of kinase inhibitors that preferentially inhibit Hck in the presence of Nef. These compounds also block Nef-dependent HIV replication, validating the Nef-SFK signaling pathway as an antiretroviral drug target. Our findings also suggested that by binding to the Hck SH3 domain, Nef indirectly affects the conformation of the kinase active site to favor inhibitor association. RESULTS: To test this hypothesis, we engineered a "gatekeeper" mutant of Hck with enhanced sensitivity to the pyrazolopyrimidine tyrosine kinase inhibitor, NaPP1. We also modified the RT loop of the Hck SH3 domain to enhance interaction of the kinase with Nef. This modification stabilized Nef:Hck interaction in solution-based kinase assays, as a way to mimic the more stable association that likely occurs at cellular membranes. Introduction of the modified RT loop rendered Hck remarkably more sensitive to activation by Nef, and led to a significant decrease in the Km for ATP as well as enhanced inhibitor potency. CONCLUSIONS: These observations suggest that stable interaction with Nef may induce Src-family kinase active site conformations amenable to selective inhibitor targeting.

On the solution conformation and dynamics of the HIV-1 viral infectivity factor.

Human immunodeficiency virus-1 (HIV-1) has evolved a cunning mechanism to circumvent the antiviral activity of the APOBEC3 family of host cell enzymes. HIV-1 Vif [viral (also called virion) infectivity factor], one of several HIV accessory proteins, targets APOBEC3 proteins for proteasomal degradation and downregulates their expression at the mRNA level. Despite the importance of Vif for HIV-1 infection, there is little conformational data on Vif alone or in complex with other cellular factors due to incompatibilities with many structural techniques and difficulties in producing suitable quantities of the protein for biophysical analysis. As an alternative, we have turned to hydrogen exchange mass spectrometry (HX MS), a conformational analysis method that is well suited for proteins that are difficult to study using X-ray crystallography and/or NMR. HX MS was used to probe the solution conformation of recombinant full-length HIV-1 Vif. Vif specifically interacted with the previously identified binding partner Hck and was able to cause kinase activation, suggesting that the Vif studied by HX MS retained a biochemically competent conformation relevant to Hck interaction. HX MS analysis of Vif alone revealed low deuteration levels in the N-terminal portion, indicating that this region contained structured or otherwise protected elements. In contrast, high deuteration levels in the C-terminal portion of Vif indicated that this region was likely unstructured in the absence of cellular interacting proteins. Several regions within Vif displayed conformational heterogeneity in solution, including the APOBEC3G/F binding site and the HCCH zinc finger. Taken together, these HX MS results provide new insights into the solution conformation of Vif.