Structure of HIV-1 Reverse Transcriptase with the Inhibitor β-Thujaplicinol Bound at the RNase H Active Site

Abstract

Summary Novel inhibitors are needed to counteract the rapid emergence of drug-resistant HIV variants. HIV-1 reverse transcriptase (RT) has both DNA polymerase and RNase H (RNH) enzymatic activities, but approved drugs that inhibit RT target the polymerase. Inhibitors that act against new targets, like RNH, would be effective against all of the current drug-resistant variants. Here, we present 2.80 Å and 2.04 Å resolution crystal structures of an RNH inhibitor, β-thujaplicinol, bound at the RNH active site of both HIV-1 RT and an isolated RNH domain. β-thujaplicinol chelates two divalent metal ions at the RNH active site. We provide biochemical evidence that β-thujaplicinol is a slow-binding RNH inhibitor with non-competitive kinetics and suggest that it forms a tropylium ion that interacts favorably with RT and the RNA:DNA substrate.

Inhibition of the Ribonuclease H Activity of HIV-1 Reverse Transcriptase by GSK5750 Correlates with Slow Enzyme-Inhibitor Dissociation

“…For the ␤-thujaplicinol anion, we speculate that its negative charge may not be as readily available for coordinate bond formation due to its delocalization across the tropolone ring, carbonyl, and hydroxyl groups. This is generally consistent with the perspective of Himmel et al (22), with the exception that they suggest that ␤-thujaplicinol binds as a neutral molecule.…”

“…The existence of such a complex has been postulated in the context of ␤-thujaplicinol (22). It is conceivable that the benzothiophene moiety of GSK5750 contributes to this interaction by directly binding to the substrate, perhaps by intercalation.…”

“…As such, these compounds are generally referred to as active site RNase H inhibitors. Crystal structures of HIV-1 RT RNase H with a bound active site inhibitor confirmed that these compounds are anchored at the RNase H active site and interact with the bound divalent metal ions (22)(23)(24)(25). Allosteric RNase H inhibitors can bind in the vicinity of the polymerase active site as shown for N-acyl hydrazones (26,27), or in the vicinity of the p51 thumb subdomain as shown for vinylogous ureas (28,29).…”

“…These findings suggested that ␤-thujaplicinol rapidly dissociates from the complex and rebinding of the inhibitor is blocked by the nucleic acid substrate. The structure of RT in complex with ␤-thujaplicinol, along with a modeled RNA/DNA hybrid indeed point to a steric conflict between the inhibitor and the intact RNA/DNA substrate (22). Notably, ␤-thujaplicinol and derivatives appear to be able to bind to the nicked product of the primary cleavage reaction as subsequent secondary, polymerase-independent cuts are effectively inhibited (31).…”

Targeting Divalent Metal Ions at the Active Site of the HIV-1 RNase H Domain: NMR Studies on the Interactions of Divalent Metal Ions with RNase H and Its Inhibitors

“…-thujaplicinol, a selective inhibitor of HIV RT, is a typical metal chelator [33]. Our study demonstrated that it binds to two Mg [21].…”