Alizarine derivatives as new dual inhibitors of the HIV-1 reverse transcriptase-associated DNA polymerase and Ribonuclease H activities effective also on non-nucleoside resistant RTs

Background. The HIV-1 reverse transcriptase (RT) has two associated activities, DNA polymerase and RNase H, both essential for viral replication and validated drug targets. While all RT inhibitors approved for therapy target the DNA polymerase activity, there is the pressing need of new RT inhibitors possibly targeting the RNase H function and also active on RTs resistant to the known non-nucleoside inhibitors (NNRTI). Methods. The newly synthesized alizarine analogues were tested in biochemical and cellular assays and a docking study was performed to identify their binding pocket. Results. Several alizarine derivatives were synthesised and tested on both HIV-1 reverse transcriptase (RT)-associated activities in biochemical assays. Alizarine analogue K49 was the most potent derivative showing IC50 values for both RT-associated functions around 10 µM, while derivative KNA53 was active at around 20 µM. When tested on the K103N RT, K49 and KNA53 inhibited both RNase H and DNA polymerase functions, while when tested on the Y181C RT only KNA53 was found to be active. Mechanism of action studies showed that these derivatives do not intercalate into DNA and do not chelate the divalent cofactor Mg2+. Kinetic studies demonstrated that they are non-competitive inhibitors and that they do not bind to the classical NNRTI binding site. Docking studies were also performed and confirmed these observations. Conclusions. Alizarine derivatives represents the proof of a new class of compounds which inhibits both RT-associated functions of wt and NNRTI mutant enzymes binding to a new RT pocket.