Background:  In 2003 we identified the cellular protein, LEDGF/p75, as a strong binding partner of HIV-1 integrase (IN) in eukaryotic cells. RNAi, mutagenesis, and transdominant inhibition corroborated LEDGF/p75 as an important co-factor of HIV integration. Furthermore resistance selection in the transdominant phenotype validated the virus-host interaction as a potential drug target for novel antiviral therapy. To fuel our small-molecule drug discovery program, we have now probed this antiviral target with specific peptides.

Methods:  The recently solved X-ray structure of IN-core complexed with the integrase binding domain (IBD) of p75 shows a well-defined interphase and suggests that peptides based on the p75 “KID”-loop would be effective inhibitors of the interaction. Likewise the apparent stability of the p75 surface is promising for binding IN-derived peptides. Both strategies were applied in designing libraries of peptides targeting the IN-p75 interaction. Essential amino acid residues of the most active peptides were projected on natural occurring stable helical folds in order to improve inhibitory activities. For in vitro hit identification we developed a high throughput Alphascreen assay monitoring protein protein interaction. JPO2 has emerged as the predominant physiological binding partner of LEDGF/p75 in cells. To predict potential cellular toxicity we established a counterscreen to further evaluate identified hits. Antiviral activity and cytotoxicity of initial hits from in vitro screening were evaluated in cell culture by MTT/MT4.

Results:  Purification of recombinant IN, LEDGF/p75, and JPO2 was optimized in order to set up a high-throughput screening assay for hit identification. Thorough evaluation of the LEDGF/p75-IN and LEDGF/p75-JPO2 Alphascreen assays evidences its applicability for antiviral drug discovery. By rational peptide design we generated potent inhibitors of the LEDGF/p75-IN interaction with IC₅₀ in the low to sub-micromolar range, with the strongest inhibition observed for PEA-64 (IC₅₀ = 0.35 µM). In cell culture the identified hits, fused to a transduction domain, demonstrate moderate antiviral activity at non-toxic concentrations.

Conclusions:  The identification of the first inhibitors of LEDGF/p75-IN interaction validates this interaction as a target for antiviral therapy. Peptidomimetics will optimize the pharmacological properties of the inhibitory peptides and fuel our small molecule drug discovery projects.