Screening and Functional Profiling of Small-Molecule HIV-1 Entry and Fusion Inhibitors

Abstract

HIV-1 entry and fusion with target cells is an important target for antiviral therapy. However, a few currently approved treatments are not effective as monotherapy due to the emergence of drug resistance. This consideration has fueled efforts to develop new bioavailable inhibitors targeting different steps of the HIV-1 entry process. Here, a high-throughput screen was performed of a large library of 100,000 small molecules for HIV-1 entry/fusion inhibitors, using a direct virus-cell fusion assay in a 384 half-well format. Positive hits were validated using a panel of functional assays, including HIV-1 specificity, cytotoxicity, and single-cycle infectivity assays. One compound-4-(2,5-dimethyl-pyrrol-1-yl)-2-hydroxybenzoic acid (DPHB)-that selectively inhibited HIV-1 fusion was further characterized. Functional experiments revealed that DPHB caused irreversible inactivation of HIV-1 Env on cell-free virions and that this effect was related to binding to the third variable loop (V3) of the gp120 subunit of HIV-1 Env. Moreover, DPHB selectively inhibited HIV-1 strains that use CXCR4 or both CXCR4 and CCR5 co-receptors for entry, but not strains exclusively using CCR5. This selectivity was mapped to the gp120 V3 loop using chimeric Env glycoproteins. However, it was found that pure DPHB was not active against HIV-1 and that its degradation products (most likely polyanions) were responsible for inhibition of viral fusion. These findings highlight the importance of post-screening validation of positive hits and are in line with previous reports of the broad antiviral activity of polyanions.

The role of IL‐1β during human immunodeficiency virus type 1 infection

“…[189][190][191][192][193] In the context of HIV-1 infection, activation of purinergic receptors enhances HIV-1 infection of target cells through cell-to-cell or cell-virus infection routes and viral replication, and promotes inflammation, in part, through the production of the pro-inflammatory cytokine IL-1β. 189,190,194,195 P2X receptors are highly expressed in immune cells including CD4 + T cells, monocytes/macrophages, and DCs, [196][197][198][199][200][201] all of which are target cells for HIV-1. 1,25 In fact, it has been reported that P2X7 is a key player in promoting the production of IL-1β.…”

Novel Naturally Occurring Dipeptides and Single-Stranded Oligonucleotide Act as Entry Inhibitors and Exhibit a Strong Synergistic Anti-HIV-1 Profile

“…HIV-1 cellular entry is mediated by the interaction between the envelope glycoproteins (gp120, gp41), the cellular CD4 receptor, and the co-receptors CCR5 or CXCR4. Recently, an attachment inhibitor, fostemsavir, and a post-attachment inhibitor ibalizumab have been approved for patients with multi-drug resistance [ 1 ]. In addition, small molecule inhibitors of the viral entry can interfere with CD4/gp120 binding [ 2 , 3 ], co-receptor binding [ 4 , 5 ] or gp41 six-helix bundle formation [ 6 , 7 ] have been available for several years.…”

A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion

“…It has been successfully used to screen the FDA library as well as a small library of purinergic inhibitors 1,6 . Other investigators have also identified purinergic inhibitors in HIV-1 fusion using an adapted and optimized high-throughput fusion assay that reports the transfer of virus-encapsulated beta-lactamase into the cytoplasm 7,8 .…”