A 2-Hydroxyisoquinoline-1,3-Dione Active-Site RNase H Inhibitor Binds in Multiple Modes to HIV-1 Reverse Transcriptase

Abstract

The RNase H (RNH) function of HIV-1 reverse transcriptase (RT) plays an essential part in the viral life cycle. We report the characterization of YLC2-155, a 2-hydroxyisoquinoline-1,3-dione (HID)-based active-site RNH inhibitor. YLC2-155 inhibits both polymerase (50% inhibitory concentration [IC 50 ] ϭ 2.6 M) and RNH functions (IC 50 ϭ 0.65 M) of RT but is more effective against RNH. X-ray crystallography, nuclear magnetic resonance (NMR) analysis, and molecular modeling were used to show that YLC2-155 binds at the RNH-active site in multiple conformations.KEYWORDS RNase H, human immunodeficiency virus, inhibitor, reverse transcriptase H IV-1 reverse transcriptase (RT) plays a critical role in virus replication. It has multiple functions, including RNA-dependent DNA synthesis, RNase H (RNH) activity, and DNA-dependent DNA synthesis to convert the viral single-stranded RNA genome into double-stranded DNA for downstream incorporation into the host cell genome (1). At least one component of highly active antiretroviral therapy (HAART) administered to patients includes RT polymerase inhibitors, either a nucleoside RT inhibitor(s) or a nonnucleoside RT inhibitor, or both. Prolonged use of antivirals can lead to side effects or drug resistance (2, 3). Hence, new antivirals that act by novel mechanisms of action are needed. No currently approved therapeutics target the RNH function of HIV-1 RT. Hence, RNH is an attractive target for future antiviral therapies.RNH inhibitors of several different chemotypes have been identified and characterized for their effectiveness against HIV-1 (4). These include acylhydrazones (5-7), diketo acids (8, 9), ␣-hydroxytropolones (10), vinylogous ureas (11), naphthyridinones (12), pyridopyrimidinones (13,14), pyrimidinol carboxylic acids (15), hydroxypyridonecarboxylic acids (16), 3-hydroxypyrimidine-2,4-diones (17, 18), and 2-hydroxyisoquinoline-1,3-diones (HIDs) (19). Notably, many 2-hydroxyisoquinoline-1,3-diones inhibit both RT polymerase and RNH functions of RT (19). In order to further understand the mechanism of RT inhibition by HIDs, we further characterized YLC2-155, which is substituted at the C7 position of the 2-hydroxyisoquinoline-1,3-dione with a furan ring (Fig. 1).

Design, synthesis and biological evaluations of N-Hydroxy thienopyrimidine-2,4-diones as inhibitors of HIV reverse transcriptase-associated RNase H

“…The asymmetric unit comprises two RT molecules (Figure 3, middle panel), and therefore we were able to capture two distinct RNase H active sites in the same crystal structure, similar to previous reports [15, 38]. Importantly, analogue 11b is bound at both of these sites and adopts a different conformation in each active site (Figure 3, top and bottom panels).…”

“…In RNase H active site 1 (Figure 3, top panel), the thieno group points in the direction of H539, while in RNase H active site 2 (Figure 3, bottom panel), the same group points in the opposite direction. Multiple binding modes of an inhibitor at the HIV-1 RT RNase H active site have also recently been observed for a 2-hydroxyisoquinoline-1,3-dione analogue [38]. The lack of additional protein-inhibitor interactions and the observed multiple modes of inhibitor binding at the RNase H active site may contribute to the decreased antiviral activity of the N -hydroxy thienopyrimidine-2,3-dione compounds.…”

Targeting HIV-1 RNase H: N’-(2-Hydroxy-benzylidene)-3,4,5-Trihydroxybenzoylhydrazone as Selective Inhibitor Active against NNRTIs-Resistant Variants

“…In the second binding mode ( Figure 3C,D) compound 13 coordinates the two Mn 2+ cofactors with the pyrogallol moiety serving as a chelating motif, while the remaining atoms of the molecule accommodate between alpha-helix E (in yellow) and beta-sheet 1 (in green), interacting with charged and polar residues Asp443, Glu449, Glu478, Arg557, Asn474, Arg557, establishing further hydrogen bonds with the side chain of Gly444, Ala446 and Arg448 ( Figure 4B). This last orientation of the chelating portion is different from the one reported for the selective RNase H inhibitor RDS1759, but similar to the chelating portion of other compounds as beta-tuhjaplicinol (pdb 3I1G) [55] and 2-hydroxyisoquinoline-1,3-dione (pdb code 5UV5) [56], while the binding mode exhibited by the tail portion of compound 13 significantly differs from all of them.…”

Binding thermodynamics of metal ions to HIV-1 ribonuclease H domain

“…The isolated RNH domain of RT, folded by itself, is not catalytically active, due to lack of the substrate handle region, but exhibits a native-like protein fold and has been used to test RNH-inhibitor interactions for therapeutic applications [13][14][15][16][17]. The weak but critical binding of Mg 2+ to isolated RNH has been analyzed using NMR titration experiments, showing the K a value to be 3.1 -3.7 × 10 2 M -1 for the A site and ~29 M -1 for the B site [18].…”