Structure of a Dihydroxycoumarin Active-Site Inhibitor in Complex with the RNase H Domain of HIV-1 Reverse Transcriptase and Structure–Activity Analysis of Inhibitor Analogs

Abstract

HIV encodes four essential enzymes: protease, integrase, reverse transcriptase (RT) associated DNA polymerase, and RT-associated ribonuclease H (RNase H). Current clinically approved anti-AIDS drugs target all HIV enzymatic activities except RNase H, which has proven to be a very difficult target for HIV drug discovery. Our high-throughput screening activities identified the dihydroxycoumarin compound F3284-8495 as a specific inhibitor of RT RNase H, with low micromolar potency in vitro. Optimization of inhibitory potency can be facilitated by structural information about inhibitor-target binding. Here, we report the crystal structure of F3284-8495 bound to the active site of an isolated RNase H domain of HIV-1 RT at a resolution limit of 1.71 Å. From predictions based on this structure, compounds were obtained that showed improved inhibitory activity. Computational analysis suggested structural alterations that could provide additional interactions with RT and thus improve inhibitory potency. These studies established proof-of-concept that F3284-8495 could be used as a favorable chemical scaffold for development of HIV RNase H inhibitors.

Studies on Cycloheptathiophene‐3‐carboxamide Derivatives as Allosteric HIV‐1 Ribonuclease H Inhibitors

“…Q500 has been reported as one of the key residues responsible for RNA:DNA hybrid binding, [35] and it was recently proposed as a possible druggable binding site for allosteric RNase H inhibitors. [36][37][38] Theoretically, compounds interacting with residue Q500 (Figure 3), or residues in its vicinity that are important for RNA:DNA substrate positioning, could potentially interfere with duplex accommodation in the active site of RNase H. [36] On the basis of these results, we hypothesized that the binding of cHTC derivatives to the allosteric site of RNase H could be affected by competition with the RNA:DNA duplex, especially for compounds that were weaker inhibitors. To test this hypothesis, the RNase H activity of compounds 31 and 33 was re-assayed by modifying the order of addition of the RNA:DNA duplex substrate.…”

Design, Synthesis, and Biological Evaluations of Hydroxypyridonecarboxylic Acids as Inhibitors of HIV Reverse Transcriptase Associated RNase H

“…hydroxyisoquinolinedione (HID, 1 ), 10 β-thujaplicinol ( 2 ), 11 and dihydroxycoumarin ( 3 ). 12 More potent and selective RNase H inhibition was achieved with structurally more elaborate chemotypes, such as diketoacid 4 , 13 pyrimidinol carboxylic acid 5 , 14 hydroxynaphthyridine 6 15 and pyridopyrimidone 7 , 16 all featuring a hydrophobic aromatic moiety seemingly important for antiviral activity. Compounds 6–7 are among the very few RNase H inhibitors with reported antiviral activity.…”

Ribonuclease H, an unexploited target for antiviral intervention against HIV and hepatitis B virus

“…Dihydroxycoumarins were identified by virtual screening and emerged as a promising scaffold for the development of HIV-1 RNase H inhibitors when compound F3284-8495 ( Figure 12), 7,8-dihydroxy-2-oxo-2H-chromen-4-yl)acetic acid, was co-crystallized within the HIV-1 RNase H active site (Himmel et al, 2014). The compound coordinated the two metal ions within the active site, while making additional interactions with His539.…”

“…First, when compounds are reported to have a high potency in biochemical assays, such as in the low nanomolar range, in most cases there is not a correspondent potency in inhibiting viral replication, raising some concerns about the reliability of the enzymatic assay. To optimize the outcome, the assay should, for example, use conditions that reflect better the physiological ones (Himmel et al, 2014). Second, in the case of active site inhibitors, most of the currently identified compounds often exhibit, together with increased potency, higher toxicity, probably related to inhibition of host counterparts (e.g.…”