Tight-Binding Hydroxypyrazole HIV-1 Nef Inhibitors Suppress Viral Replication in Donor Mononuclear Cells and Reverse Nef-Mediated MHC-I Downregulation

Abstract

The HIV-1 Nef accessory factor is critical to the viral life cycle in vivo and promotes immune escape of infected cells 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 also restore cell-surface MHC-I expression in HIV-infected CD4 T cells from AIDS patients, enabling recognition and killing by autologous cytotoxic T lymphocytes (CTLs). In this study, we describe the synthesis and evaluation of a diverse set of analogs based on the original hydroxypyrazole Nef inhibitor core. All analogs were screened for the interaction with recombinant HIV-1 Nef by surface plasmon resonance (SPR) and for antiretroviral activity in TZM-bl reporter cells infected with HIV-1. Active analogs were ranked on the basis of an activity score that integrates three aspects of the SPR data (affinity, residence time, and extent of binding) with antiretroviral activity. The top scoring compounds bound tightly to Nef by SPR, with K D values in the low nM to pM range, and displayed very slow dissociation from their Nef target. These analogs also suppressed HIV-1 replication in donor peripheral blood mononuclear cells (PBMCs) with IC 50 values in the 1−10 nM range without cytotoxicity, inhibited Nef-mediated IL-2-inducible tyrosine kinase (Itk) and hematopoietic cell kinase (Hck) activation, and rescued MHC-I downregulation in a Nef-transfected T cell line. The development of Nef inhibitors based on the structure−activity relationships defined here has promise as a new approach to antiretroviral therapy that includes a path to eradication of HIV-infected cells via the adaptive immune response.

Structure, function, and inhibitor targeting of HIV-1 Nef-effector kinase complexes

“…Medicinal chemistry optimization has led to more potent analogs such as FC-8052 which shares a hydroxypyrazole core with B9 (red). FC-8502 binds to recombinant Nef in vitro with a KD value of approximately 10 pM, compared to about 80 nM for B9, and inhibits Nef-dependent HIV-1 replication in PBMCs in the sub-nanomolar range (96).…”

“…B9 demonstrated inhibitory activity against multiple Nef functions including enhancement of viral infectivity, replication, as well as MHC-I downregulation (99,102). Synthesis and characterization of more than 200 analogs of B9 have been reported without the potentially carcinogenic diazene functionality (96,103). Several of these analogs bound to recombinant Nef with K D values in the nM to pM range, and inhibited Nef-mediated enhancement of HIV-1 replication in donor PBMCs with low nM potency.…”

Nef dimerization defect abrogates HIV viremia and associated immune dysregulation in the Bone Marrow-Liver-Thymus-Spleen (BLTS) humanized mouse model

“…Nef enhances HIV infectivity and replication in some cell lines (Crotti et al, 2006; Spina et al, 1994; Yang et al, 2002). Nef also enhances HIV replication in human PBL-derived activated-primary CD4+ T cells in a viral input-dependent manner, with the Nef effect lost at a high viral-input dose (Shi et al, 2020). Here we show that nef -deleted HIV (NL4-3 strain) exhibits reduced replication compared to wild-type HIV in the TZM-bl cell line (Figure 1A).…”

Nef homodimers down-regulate SERINC5 by AP-2–mediated endocytosis to promote HIV-1 infectivity

“…Understanding the molecular determinants of SERINC5 antagonism by Nef may help to identify therapeutic strategies that promote SERINC5 virion incorporation and restriction of viral infectivity. Along these lines, docking studies of small molecule inhibitors of Nefmediated enhancement of viral infectivity have identified potential binding sites within the dimer interface (12,44,45). Disruption of the Nef homodimer by these compounds may explain their inhibitory effects on multiple Nef functions, including not only infectivity but also host cell kinase activation, receptor downregulation, and enhancement of viral replication.…”

“…Both T cell lines were cultured in RPMI 1640 medium supplemented with 10% FBS, L-glutamine, and 2 µg/mL puromycin (CEM-SS cells only). Donor PBMCs were obtained from Vitalant Pittsburgh and isolated by Ficoll gradient centrifugation and activated for 3 days with PHA (Sigma, # L1668) and IL-2 (Thermo Fisher, # CB-40043B) as described elsewhere (44).…”