Synthesis, Activity, and Structural Analysis of Novel α-Hydroxytropolone Inhibitors of Human Immunodeficiency Virus Reverse Transcriptase-Associated Ribonuclease H

Abstract

The α-hydroxytroplone, manicol (5,7-dihydroxy-2-isopropenyl-9-methyl-1,2,3,4-tetrahydro-benzocyclohepten-6-one) potently and specifically inhibits ribonuclease H (RNase H) activity of human immunodeficiency virus reverse transcriptase (HIV RT) in vitro. However, manicol was ineffective in reducing virus replication in culture. Ongoing efforts to improve the potency and specificity over the lead compound led us to synthesize 14 manicol derivatives that retain the divalent metal-chelating α-hydroxytropolone pharmacophore. These efforts were augmented by a high resolution structure of p66/p51 HIV-1 RT containing the nonnucleoside reverse transcriptase inhibitor (NNRTI), TMC278 and manicol in the DNA polymerase and RNase H active sites, respectively. We demonstrate here that several modified α-hydroxytropolones exhibit antiviral activity at non-cytotoxic concentrations. Inclusion of RNase H active site mutants indicated that manicol analogs can occupy an additional site in or around the DNA polymerase catalytic center. Collectively, our studies will promote future structure-based design of improved α-hydroxytropolones to complement the NRTI and NNRTI currently in clinical use.

Identification of Highly Conserved Residues Involved in Inhibition of HIV-1 RNase H Function by Diketo Acid Derivatives

“…This is a singular pattern that is interesting for the large number of residues involved and is in agreement with recent observations on the interaction of RDS1643 and the PFV domain of RNase H (32). In fact, all of the crystal structures of RT/RNase H prototypes solved in complexes with active site RHIs show the ligands coordinating the metal cofactors (25,47) while exhibiting an orientation of binding that does not allow extended secondary interactions with amino acid side chains. The only exceptions are represented by hydroxytropolones and naphthyridinones (25,48), with the latter being found to be sandwiched by a loop containing residues A538 and H539 on one side and N474 on the opposite side (12).…”

“…In fact, all of the crystal structures of RT/RNase H prototypes solved in complexes with active site RHIs show the ligands coordinating the metal cofactors (25,47) while exhibiting an orientation of binding that does not allow extended secondary interactions with amino acid side chains. The only exceptions are represented by hydroxytropolones and naphthyridinones (25,48), with the latter being found to be sandwiched by a loop containing residues A538 and H539 on one side and N474 on the opposite side (12). Interestingly, our modeling studies propose different binding orientations for ester and acid DKAs in the RNase H domain, due to the steric hindrance of the alkyl substituent on the DKA branch of ester derivatives, which mainly influence the interactions with Q475 and R448.…”

“…In fact, efforts aimed to develop Mg 2ϩ -binding RHIs have been hampered by the topology of the RNase H active site, which is relatively large and shallow in comparison with closely related virus-encoded polynucleotidyl transferases such as HIV-1 DNA polymerase and integrase (IN) (23,24), hampering the identification of a suitable binding pocket near the catalytic region. In fact, in all cocrystallized structures of HIV-1 RT/RNase H and active site RHIs (12,24,25), the RHIs exhibited binding poses showing a large part of their structures extending out of the protein domain. Therefore, besides the coordination with the two catalytic Mg 2ϩ ions, the inhibitors established very few contacts with RNase H binding site residues (26), providing poor information about the binding interactions.…”

Inhibition of Foamy Virus Reverse Transcriptase by Human Immunodeficiency Virus Type 1 RNase H Inhibitors

“…For the analysis of FV PR-RT inhibition, we chose the diketo acid derivative 6-[1-(4-fluorophenyl)methyl-1H-pyrrol-2-yl)]-2,4-dioxo-5-hexenoic acid ethyl ester (RDS1643) (28), 3-hydroxy-4-propionyl-3,4-dihydroquinolin-2(1H)-one (DHQ) (30), and the natural product 2,7-dihydroxy-4-isopropyl-cyclohepta-2,4,6-triene (␤-thujaplicinol) (31), which were reported to inhibit HIV-1 RNase H by binding the two divalent Mg 2ϩ ions in the catalytic site. We also chose the vinylogous urea derivative 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide (VU6), which was reported to inhibit HIV-1 RNase H by interacting with a pocket located at the junction between the p51 subunit thumb subdomain and the p66 RNase H domain, possibly affecting the conformation of the adjacent p66 RNase H domain (32), and the hydrazonoindolin-2-one de- rivative, (Z)-3-(2-(4-(3,4-dihydroxyphenyl)thiazol-2-yl)hydrazono)indolin-2-one (NSC657589), which was shown to inhibit both HIV-1 RT-associated RDDP and RNase H activities, possibly by binding to a site located in a region between the polymerase catalytic aspartate triad (Asp110, Asp185, Asp186) and the NNRTI pocket, hence contiguous to the NNRTI pocket but different from it (33).…”

Structural and Inhibition Studies of the RNase H Function of Xenotropic Murine Leukemia Virus-Related Virus Reverse Transcriptase

“…Such drugs would likely be active against all current drug-resistant viral strains, because the RNase H active site is located at the opposite end of the enzyme from the polymerase domain (ϳ50 Å away) that is currently targeted by nucleoside and nonnucleoside RT inhibitors (79). Several compounds have been found in recent years to effectively inhibit the RNase H activity of HIV-1 RT, including ␣,␥-diketo acids and derivatives (27,77,78,91), pyrimidinol carboxylic acids (43,47), hydroxytropolones (including ␤-thujaplicinol) (6,12,15,21,34), dimeric lactones (19), 1,3,4,5-tetragalloylapiitol (86), phenolic glycosides (10), vinylogous ureas (16,94), N-hydroxyimides (33,44), 2-hydroxyisoquinoline-1,3(2H,4H)-diones (8,9), acylhydrazones (11,30,35,80), and naphthyridinones (84,95). Although these inhibitors have been studied for the ability to inhibit HIV-1 RT, little is known about their effectiveness against other retroviral RTs, such as the gammaretroviral Moloney murine leukemia virus (MoMLV) and xenotropic murine leukemia virus-related virus (XMRV) RTs.…”