Background:  We have determined a 3-dimensional structure of HIV-1 reverse transcriptase (RT) complexed with an inhibitor that may become the prototype for development of a new class of anti-AIDS drugs:  RNase H inhibitors (RNHI). HIV-1 RT uses 2 enzymatic activities, polymerase and RNase H, to convert the single-stranded viral genomic RNA into double-stranded DNA. RNase H degrades viral RNA after it has been copied into DNA, removes the tRNA used to initiate minus-strand DNA synthesis, and generates and removes the PPT primer used to initiate plus-strand DNA synthesis. RNase H is essential for virus replication; however, very few small-molecule inhibitors targeting this function have been reported and there are no crystal structures of HIV RT in a complex with an RNHI.

Methods:  A variety of N-acyl hydrazones were synthesized and evaluated for their ability to inhibit the polymerase and RNase H activities of HIV-1 RT. We have identified an N-acyl hydrazone derivative, KMMP05, that specifically inhibits the RNase H (IC₅₀ of 0.5 µM) but not the polymerase activity of RT. We have used X-ray crystallography to determine a 3.0 Å resolution structure of a complex of HIV-1 RT with KMMP05.

Results:  Although KMMP05 is primarily an RNHI, it binds more than 40 Å away from the RNase H active site in the palm of the p66 subunit between the primer grip and the polymerase active site. The inhibitor partially overlaps the non-nucleoside inhibitor (NNRTI) binding pocket. The inhibitor appears to interact with the conserved residues D186 and W229, as well as with Y188, K223, D224, P226, F227, and L228. Substitutions on KMMP05 could enhance the interactions in the NNRTI binding pocket leading to inhibition of the polymerase activity of RT. Preliminary structure-activity analyses show that derivatives with increased steric bulk at the 4-position of the 3,4-dihydroxybenzoyl ring have an increased ability to inhibit the polymerase.

Conclusions:  Our results suggest that KMMP05 binding may affect RNase H activity by altering the trajectory of the nucleic acid or enzyme processivity. These studies may help in the design of inhibitors that target a novel binding site and that should have little or no cross-resistance with existing NRTI (nucleoside RT inhibitors) and NNRTI families of antivirals, thus providing options for novel therapeutic strategies in the treatment of AIDS.

Keywords: RNase H Inhibitor; Novel site on HIV-1 RT; Anti-AIDS drug design