Drug Discovery Opinion

The first treatments for HIV-1/AIDS targeted the viral enzymes, reverse transcriptase and protease; more recently, compounds that block viral fusion/entry have also been developed. HIV-1 regulatory proteins are considered to be more difficult drug targets, not least because ways to assay for activity are less clear, but researchers at the University of Pittsburgh have now described an assay for HIV-1 Nef and have identified compounds that block the function of this viral protein. The HIV-1 nef gene was originally believed to inhibit transcription and to be of little importance (hence ‘Negative regulatory Factor’), but the protein is now known to play key roles in viral replication and pathogenesis. Nef is one of the first proteins to be detected after infection of the host cell and has three main functions: it increases viral infectivity, down-regulates surface antigens, and alters signalling pathways to enhance survival of infected T-cells.

Nef binds to src family kinases via their SH3 domains and the Pittsburgh team have exploited Nef’s interaction with Hck (hemopoietic cell kinase) to develop an assay system suitable for high-throughput screening. In the assay, Hck activation is coupled to Nef, providing a direct readout of Nef activity. The assay was used to screen a library of 10,000 compounds biased towards kinase and phosphatase inhibitors, but also containing more diverse structures.

The 4-amino substituted diphenylfuropyrimidine, DFP-4AP, which is structurally related to a series of recently described protein-tyrosine kinase inhibitors, was found to be active both in the initial FRET-based assay and in a cell-based assay using U87MG astroglioma cells engineered to express the HIV-1 receptors CD4 and CXCR4. DFP-4AP blocked wild-type HIV replication in U87MG cells with an IC₅₀ value in the low µM range, but had no effect on replication of an HIV-ΔNef mutant, even at 10 µM, indicating a requirement for Nef in the antiviral activity of the compound.

The study, which is published in ACS Chemical Biology, establishes that coupling of Nef to one of its known host cell targets provides a viable high-throughput screen which can be used to identify small molecule inhibitors. Including Hck in the assay may also induce relevant conformations of both Hck and Nef that are essential for small molecule inhibitor binding and function, an idea that is supported by the enhanced potency and efficacy of the inhibitors in the kinase assay when Nef is present. It may be possible to use a similar coupled protein approach to identify compounds that block the function of other HIV virulence factors and compounds which inhibit the function of Nef – or other virulence factors – could eventually become new weapons in the fight against HIV/AIDS.

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This entry was posted on Friday, October 16th, 2009 at 8:30 am and is filed under News. You can follow any responses to this entry through the RSS 2.0 feed. You can leave a response, or trackback from your own site.