Isolation of recombinant HIV-1 Rev protein and investigation of a new class of benzimidazole inhibitors capability to disrupt Rev-RRE complex.

In the present study, an efficient method for the expression and purification of recombinant HIV Rev protein with a C-terminal hexahistidine tag was proposed. Noteworthy, this method circumvents the precipitation of the protein into inclusion bodies and their subsequent aggregation during purification. It does not necessitate denaturing isolation conditions, in contrast to currently widely used protocols. As a result, protocols for HIV Rev isolation have been developed allowing the production of non-aggregated Rev protein in a good yield, high purity, and free of bacterial RNA impurities. This high-purity result became possible due to high salt extraction buffer usage. Complementary [α-P]-labeled Rev response element (RRE) RNA has been synthesized and an inhibitor test system was developed based on Rev-RRE complex formation. We were able to reveal a novel class of potential Rev-RRE inhibitors based on dimeric benzimidazole derivatives and used those results to validate the testing system. The proposed protocols for screening and structure-activity relationship for new inhibitors of Rev binding to viral RNA broaden the scope of potential candidates for anti-HIV drug development.