CROI 2019 Abstract eBook

Abstract eBook

Poster Abstracts

Methods: Nef-kinase interaction and kinase activation were assessed in transfected 293T cells by combining bimolecular fluorescence complementation (BiFC) with immunofluorescence using phosphospecific antibodies for the activation loop tyrosine of Itk (pY511) and Btk (pY551). Mutants of the Nef dimerization interface, based on previous crystal structures, targeted conserved residues L112, Y115, and F121 either alone or in combination. Endogenous Itk activation was assessed by phosphoflow cytometry in HIV-1 infected Jurkat and SupT1 cells using the phosphospecific antibody for Itk. Results: BiFC analysis showed that wild-type HIV-1 Nef interacted with both Itk and Btk at the cell membrane, resulting in enhanced kinase activation loop phosphorylation. Nef dimerization interface mutants retained interaction with both kinases, but failed to induce kinase activation, supporting a role for Nef homodimer formation in the activation mechanism. Addition of small molecule Nef inhibitors reversed Nef-dependent Itk autophosphorylation, suggesting that these compounds may interfere with Nef dimerization and Itk activation through an allosteric mechanism. HIV-1 infection upregulated endogenous Itk activity in Jurkat and SupT1 cells in a Nef-dependent manner, while HIV-1 with Nef dimerization interface mutations replicated poorly in both T cell lines and donor PBMCs. Conclusion: Our results support a mechanism in which Nef recruits Itk and Btk to the membrane, and drives kinase activation via a dimerization-dependent mechanism. Nef dimerization interface mutants replicate poorly in T cells, and Nef inhibitors interfere with Itk activation, suggesting that suppression of the Nef-Itk pathway may account for part of their antiretroviral mechanism of action. These findings provide a strong rationale to support further antiretroviral drug development targeting Nef homodimerization and the Nef-Itk/Btk signaling pathways. 193 ACTIVATION OF TEC-FAMILY KINASES ITK AND BTK BY HIV-1 AND SIV NEF PROTEINS IN VITRO Manish Aryal , Kindra Whitlatch, Tom E. Smithgall University of Pittsburgh, Pittsburgh, PA, USA Background: The Nef proteins encoded by HIV-1 and SIV are critical for efficient viral replication and AIDS progression. In addition to downregulating cell- surface immune and viral receptors, Nef also induces constitutive activation of host-cell tyrosine kinases of the Src and Tec families. Nef-mediated activation of Itk, a Tec family member expressed in CD4 T cells, is essential for several stages of the HIV-1 life cycle. Nef also interacts with Btk, which is expressed in B cells and macrophages. Itk and Btk share a similar domain organization consisting of PH, SH3, SH2 and kinase domains. Here we tested whether interaction of recombinant purified Btk and Itk proteins with Nef was sufficient for kinase activation in vitro. Methods: Full-length and ‘Src-like’ cores (SH3-SH2-kinase) of Itk and Btk were expressed in Sf9 insect cells and purified. A kinetic kinase assay (ADP Quest; Eurofins) was used to measure the rates of autophosphorylation and peptide substrate phosphorylation in the presence and absence of recombinant purified HIV-1 and SIV Nef. Activation loop autophosphorylation was assessed by immunoblotting with phosphospecific antibodies. Surface plasmon resonance (SPR) was used to measure the interaction of Nef with recombinant purified Itk and Btk regulatory domains. Results: Both HIV-1 and SIV Nef strongly enhanced full-length Btk autophosphorylation and kinase activity in vitro, with autophosphorylation occurring primarily on the activation loop at Tyr551. In contrast, Nef had no effect on the Btk core protein, implying that the PH domain is important for interaction with Nef and kinase activation. Nef induced modest enhancement of full-length Itk autophosphorylation on activation loop Tyr511, but did not affect the ITK core. Interestingly, a mutant of Nef lacking the conserved motif required for SH3 domain binding (PxxP) activated Btk and Itk to the same extent as wild-type Nef. This mutant failed to activate the Src-family kinase Hck, suggesting that Nef activates Tec and Src family kinases by distinct mechanisms. This conclusion is supported by SPR data, which showed that the Itk SH3 domain alone does not bind to Nef. Conclusion: HIV-1 and SIV Nef proteins activate Btk and to a lesser extent Itk in vitro, through an SH3 domain-independent mechanism distinct from Src- family kinases. Small molecules that interfere with this Nef-dependent kinase signaling pathway may provide a new route to antiretroviral drug development.

showed similar trend. Infectivity assays revealed that the patient-derived Nef clones carrying the double 120F/125H mutations exhibited impaired ability to counteract SERINC3/5 and that the observed impairment was restored when reversion mutations (120Y/125Q) were introduced. Corroboratively, Western blot analysis of viral particles revealed that the Nef-mediated inhibition of SRINC5 incorporation to progeny viral particles was impaired in Nef clones harboring the double 120F/125H mutations, compared to those harboring the consensus 120Y/125Q. In contrast, Nef functions of CD4 and HLA class I downregulation remained unchanged regardless of the Nef genotype of 120Y/125Q or 120F/125H. Conclusion: Taken together, these results suggested that the immune-driven Nef mutations 120F/125H located at the highly conserved FPD 121-123 motif impose fitness cost to Nef-mediated counteraction of SERINC5 and thereby viral replication in infected hosts. 191 SYNTHESIS AND EVALUATION OF TIGHT-BINDING HYDROXYPYRAZOLE INHIBITORS OF HIV-1 NEF Haibin Shi 1 , Colin Tice 2 , Lori A. Emert-Sedlak 1 , Li Chen 1 , Wing Fai Li 1 , Jay E. Wrobel 2 , Allen B. Reitz 2 , Thomas E. Smithgall 1 1 University of Pittsburgh, Pittsburgh, PA, USA, 2 Fox Chase Chemical Diversity Center, Inc, Doylestown, PA, USA Background: The HIV-1 Nef accessory factor is critical to the HIV life cycle in vivo and promotes immune escape of HIV-infected cells in part via downregulation of cell-surface MHC-I. Previously we discovered small molecules that bind directly to Nef and block many of its functions, including enhancement of viral infectivity and replication in T cell lines. These compounds, based on a hydroxypyrazole core, also rescue cell-surface MHC-I expression in patient- derived CD4+ T-cells, enabling recognition and killing by autologous CTLs. Nef inhibitors may provide a new approach to antiretroviral therapy that includes a path to eradication of HIV-infected cells. This study focused on medicinal chemistry optimization of hydroxypyrazole Nef inhibitors to improve potency and metabolic stability. Methods: Nef inhibitor analogs in this study are based on a previous diphenylpyrazolodiazene hit compound with a hydroxypyrazole core linked to chlorophenyl, nitrophenyl, and thioamide moieties. The thioamide group was replaced with a variety of heterocyclic moieties, along with multiple substitutions of the other ring systems, for a total of 254 unique compounds. Analogs were screened for interaction with recombinant Nef proteins by surface plasmon resonance (SPR), and for antiretroviral activity in TZM-bl reporter cells infected with HIV-1. Active compounds were then evaluated for antiretroviral activity in donor PBMCs and for metabolic stability against liver microsomes in vitro. Results: Multiple analogs bound tightly to recombinant Nef proteins by SPR, with K D values in the nM to pM range. Several of these compounds suppressed HIV-1 replication in donor PBMCs with IC 50 values in the 1-10 nM range without cytotoxicity, and were resistant to metabolism by mouse liver microsomes. Some analogs also reversed MHC-I downregulation in a Nef-transfected T cell line. Conclusion: HIV-1 Nef inhibitors based on a hydroxypyrazole core are amenable to a wide range of structural modifications and retain inhibitory activity despite addition of bulky heterocyclic substituents. Several analogs displayed tight binding to recombinant Nef in vitro, potent antiretroviral activity in primary cells infected with HIV-1, and the capacity to restore cell-surface MHC-I expression. Future efforts will evaluate pharmacologic properties in vivo with the goal of identifying analogs suitable for testing in humanized mouse models of HIV-1 replication and latency. 192 ACTIVATION OF TEC KINASES BY HIV-1 NEF AT THE CELL MEMBRANE REQUIRES DIMER FORMATION Wing Fai Li , Sherry Shu, Tom E. Smithgall University of Pittsburgh, Pittsburgh, PA, USA Background: The HIV-1 Nef virulence factor supports high-titer viral replication and pathogenicity. Nef interacts with Itk and Btk, two Tec-family kinases expressed in HIV-1 target cells (CD4 T cells and macrophages). Knockdown or pharmacological inhibition of Itk suppresses HIV-1 entry, transcription and egress, suggesting that Nef-mediated Itk activation is required for efficient viral replication in vivo. Here we demonstrate that Nef activates both Itk and Btk at the cell membrane through a mechanism dependent on Nef homodimer formation.

Poster Abstracts

69

CROI 2019