Chemical Library Screens Targeting an HIV-1 Accessory Factor/Host Cell Kinase Complex Identify Novel Antiretroviral Compounds

Abstract

Nef is an HIV-1 accessory protein essential for AIDS progression and an attractive target for drug discovery. Lack of a catalytic function makes Nef difficult to assay in chemical library screens. We developed a high-throughput screening assay for inhibitors of Nef function by coupling it to one of its host cell binding partners, the Src-family kinase Hck. Hck activation is dependent upon Nef in this assay, providing a direct readout of Nef activity in vitro. Using this screen, a unique diphenylfuropyrimidine was identified as a strong inhibitor of Nef-dependent Hck activation. This compound also exhibited remarkable antiretroviral effects, blocking Nef-dependent HIV replication in cell culture. Structurally related analogs were synthesized and shown to exhibit similar Nef-dependent anti-viral activity, identifying the diphenylfuropyrimidine substructure as a new lead for antiretroviral drug development. This study demonstrates that coupling non-catalytic HIV accessory factors with host cell target proteins addressable by high-throughput assays may afford new avenues for the discovery of anti-HIV agents.

The Accessory Factor Nef Links HIV-1 to Tec/Btk Kinases in an Src Homology 3 Domain-dependent Manner

“…Recent work has established that selective inhibitors of Nef-induced SFK activation block Nef-dependent HIV infectivity and replication in cell culture (21)(22)(23). These studies support the idea that interaction of Nef with SH3-containing kinases and other proteins is a common event in HIV-infected cells that generates downstream signals essential for viral pathogenesis.…”

Interaction with the Src Homology (SH3-SH2) Region of the Src-family Kinase Hck Structures the HIV-1 Nef Dimer for Kinase Activation and Effector Recruitment

“…The resulting binding constants are summarized below the graphs. Nef⅐Hck complex, which is essential for Nef function and represents a druggable target for HIV-1 (14,16,17,21). Finally, the Nef dimer that results from Hck SH3-SH2 binding allows for surface display of Nef residue Asp-123, which plays a critical role in multiple Nef functions including CD4 and MHC-I down-regulation (32,43,61) as well as enhancement of viral replication (43).…”

Crystal Structure of the Src Family Kinase Hck SH3-SH2 Linker Regulatory Region Supports an SH3-dominant Activation Mechanism

“…VSL12 mass and purity were verified by mass spectrometry. In vitro kinase assays were performed using the Tyr-2 FRET peptide substrate and the Z'-Lyte TM assay (Invitrogen) as described previously (14,17).…”