Peptide arrays are a powerful tool for characterizing protein interactions and for identifying and optimizing peptide ligands. Here we demonstrate the use of peptide arrays for performing detailed SAR studies of lead peptide inhibitors of the interaction between the HIV integrase and Rev proteins. The integration of viral DNA into the genome of the host cell, mediated by the viral integrase (IN) enzyme, is a crucial step in the HIV-1 replication cycle. We have recently found that IN activity is regulated by interactions with the HIV-1 Rev protein and identified three lead peptides derived from the Rev-binding interface in IN. Due to their ability to promote dissociation of the Rev–IN complex in HIV infected cells, these peptides caused IN activation leading to multi-integration, genomic instability and specific eradication of such infected cells. Here we explored the mechanism of action of these three IN-derived peptides as the basis for developing improved anti HIV-1 leads. Using peptide array screening, we found that the IN derived peptides bound IN and Rev in a similar pattern. The Rev-binding sites in IN also mediate IN oligomerization while the IN-binding sites in Rev are also involved in Rev oligomerization. A structural homology was found between the oligomerization domains of Rev and IN residues 171–188 and 211–220. We performed SAR studies of the lead inhibitory peptides using a peptide array containing truncated peptides, alanine scan, D-amino acid scan and N-methylated amino acid scan. We screened IN and Rev for binding this array of modified IN-derived peptides. The screening results showed that C-terminal positively charged residues were essential for the interaction of the IN 118–128 and IN 174–188 peptides with both Rev and IN. The peptides could be shortened and modified without loss of binding to IN and Rev. This provides a basis for the future development of shorter peptides with better pharmacological properties that inhibit the Rev–IN interaction. We conclude that peptide arrays are excellent tools to perform detailed SAR binding studies in one short efficient experiment. The SAR study by the peptide array method is a powerful tool for developing improved inhibitors based on a lead peptide sequence.
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