Derivatives of mesoxalic acid block translocation of HIV-1 reverse transcriptase

Abstract

The pyrophosphate mimic and broad spectrum antiviral phosphonoformic acid (PFA, foscarnet) was shown to freeze the pre-translocational state of the reverse transcriptase (RT) complex of the human immunodeficiency virus type 1 (HIV-1). However, PFA lacks a specificity domain, which is seen as a major reason for toxic side effects associated with the clinical use of this drug. Here, we studied the mechanism of inhibition of HIV-1 RT by the 4-chlorophenylhydrazone of mesoxalic acid (CPHM) and demonstrate that this compound also blocks RT translocation. Hot spots for inhibition with PFA or CPHM occur at template positions with a bias toward pre-translocation. Mutations at active site residue Asp-185 compromise binding of both compounds. Moreover, divalent metal ions are required for the formation of ternary complexes with either of the two compounds. However, CPHM contains both an anchor domain that likely interacts with the catalytic metal ions and a specificity domain. Thus, although the inhibitor binding sites may partly overlap, they are not identical. The K65R mutation in HIV-1 RT, which reduces affinity to PFA, increases affinity to CPHM. Details with respect to the binding sites of the two inhibitors are provided on the basis of mutagenesis studies, structure-activity relationship analyses with newly designed CPHM derivatives, and in silico docking experiments. Together, these findings validate the pre-translocated complex of HIV-1 RT as a specific target for the development of novel classes of RT inhibitors.

Keywords: Antiviral Agent; Drug Action; Drug Resistance; Human Immunodeficiency Virus (HIV); Reverse Transcription.

FIGURE 1.

Sequence dependence of PFA- and CPHM-mediated inhibition of DNA synthesis. A, structures of PFA and CPHM. B, DNA synthesis in the absence of inhibitor (left panel), 20 μm PFA (middle panel), and 200 μm CPHM (right panel) was observed over a time period of 10 min. The concentration of CPHM was higher to aim at similar levels of inhibition. As a negative control, MgCl₂ was omitted from the reaction. Full-length product (fl) and unextended primer (p) are indicated on the gel. The radiolabeled 5′ end of the primer is indicated with an asterisk on the sequence. Arrows indicate sequence positions where PFA and CPHM inhibition is strongest (+3 and +16). The radiolabel is indicated by the black star.

FIGURE 2.

PFA and CPHM trap the pre-translocated complex of RT. Site-specific Fe²⁺ footprinting of complexes using a sequence showing post-translocational bias. A, translocational status of WT HIV-1 RT was monitored in the presence of increasing concentrations of PFA. Cleavage fragments corresponding to the pre- and post-translocated complexes are indicated with arrows. B, translocational status of WT HIV-1 RT was monitored in the presence of increasing concentrations of CPHM.

FIGURE 3.

Role of Mg²⁺ in PFA and CPHM binding. Electrophoretic mobility shift assays using a sequence position, which show a bias toward formation of a pre-translocated complex (PPT20), were performed. Pre-formed binary complexes were incubated with increasing concentrations of PFA (A) and CPHM (B), in the presence or absence of 6 mm MgCl₂. The complexes were subsequently challenged with heparin. The controls are as follows: 1) negative RT control; 2) negative heparin control + 100 μm inhibitor; 3) heparin pre-incubation control + 100 μm inhibitor.

FIGURE 4.

Effect of PFA and CPHM on primary RNase H cleavages. A, inhibition of RNase H activity under steady-state conditions. Time course reaction (0–32 min) was in the absence and presence of PFA (100 μm) and CPHM (100 μm). RNase H cleavages at positions −18 and −19 are marked post- and pre-translocation, respectively. B, time course experiments (0.05–30 s) under pre-steady-state conditions in the presence and absence of CPHM (50 μm) and PFA (50 μm). C, graphs of data shown in A and B with no inhibitor (black circle), PFA (open square), and CPHM (black triangle).

FIGURE 5.

Effect of CPHM and β-thujaplicinol on primary and secondary RNase H cleavages. A, inhibition of RNase H activity on a chimeric DNA-RNA/DNA substrate in the presence of increasing concentrations of CPHM and β-thujaplicinol. The primary RNase H cleavage occurs at a single nucleotide downstream of the DNA/RNA junction, followed by multiple secondary cuts that are more sensitive to RNase H inhibition with β-thujaplicinol, but not with CPHM. The radiolabel is indicated by the black star. B, graphs of data shown from A representing primary (black circle) and secondary (open square) cleavages.

FIGURE 6.

PFA versus CPHM inhibition of DNA synthesis during multiple nucleotide incorporation events by HCMV UL54 and HIV-1 RT. Reactions were monitored in the presence of constant concentrations of dNTPs and increasing concentrations of PFA or CPHM. The reaction conditions were chosen such that the maximum of the available primer-template substrate was used in the absence of inhibitor. PFA- and CPHM-mediated inhibition of DNA synthesis is illustrated by the disappearance of a band corresponding to the migration pattern of a full-length product (fl), which is indicated by the corresponding arrow. The migration pattern of the 5′ end radioactively labeled primer (p) is illustrated by the corresponding arrow.

FIGURE 7.

Critical structural elements required for CPHM activity determined by SAR analysis. A, compounds used in this gel-based SAR analysis are shown. B, DNA synthesis in the absence of inhibitor and in the presence of either 20 μm PFA, 200 μm CPHM, or 200 μm of a CPHM derivative was observed over a time period of 10 min. A negative control lane is included for each panel where MgCl₂ is omitted from the reaction. Full-length product (fl) and unextended primer (p) are shown on the gel. Arrows indicate sequence positions, and where CPHM inhibition is strongest (+3 and +16).

FIGURE 8.

Molecular models of PFA and CPHM binding to the polymerase active site of HIV-1 RT. The different binding orientations of docked PFA and CPHM are highlighted by their proximity to the two active site magnesium ions (red spheres) and Lys-65. The “primer terminus” of the pseudo pre-translocated complex generated for the docking simulation (see under “Experimental Procedures”) is shown with an arrow.