Article Figures & Data
Figures
- FIG. 1.
Identification of INDOPY-1 from a high-throughput screening campaign. The triangle shows the screening cascade that resulted in the identification of a cluster of four active indolopyridones from a library of over 200,000 commercially available synthetic compounds with drug-like properties (Lipinsky rule of 5). Starting from an anti-HIV activity and a toxicity assay, in which molecules were tested at up to 32 μM, over 600 selective HIV inhibitors where identified using the indicated criteria (SI, selectivity index). The majority showed a loss of activity on the NNRTI-resistant strain (K103N Y181C), and these compounds were therefore not further pursued. From the remaining 230, four were found to be RT inhibitors in an enzymatic assay. This set of compounds, which included INDOPY-1, had a very similar chemical scaffold, the indolopyridone core.
- FIG. 2.
Chemical structure of INDOPY-1. 5-Methyl-1-(4-nitro-phenyl)-2-oxo-2,5-dihydro-1H-pyrido[3,2-b]indole-3-carbonitrile.
- FIG. 3.
Time of drug addition. Parallel cultures of MT4-LTR-EGFP cells with synchronized HIV-1 IIIB infection were treated with HIV inhibitors at the indicated time points. Final compound concentrations were at least 10-fold higher than the EC50s: 1 μM AMD3100, 1 μM T20, 10 μM zidovudine (ZDV), 1 μM nevirapine (NVP), 10 μM INDOPY-1, and 10 μM L-870810.
- FIG. 4.
Fitted curves for the competitive model for INDOPY-1 inhibition of HIV-1 RT. HIV-1 RT was incubated for 30 min at 37°C with various concentrations of INDOPY-1 (•, 0 μM; ○, 0.25 μM; ▪, 0.5 μM; □, 1 μM; ▴, 2 μM) and dTTP concentrations as indicated.
- FIG. 5.
Sequence dependence of INDOPY-1-mediated inhibition of DNA synthesis. (A) Inhibition of DNA synthesis was monitored in time course experiments in the absence (−) and presence (+) of INDOPY-1, using a heteropolymeric DNA/RNA primer/template substrate. The sequence ahead of the primer 3′ end is shown on the right. Lane P, control in the absence of Mg2+; lanes T and C, reaction mixtures in the presence of dideoxythymidine triphosphate and ddCTP, respectively. Lanes 1 to 6 show different reaction times: 30 s and 1, 2, 5, 10, and 30 min, respectively. (B) Sequence parameters that affect inhibition by NRTIs and INDOPY-1. Primer/template sequences are shown as light blue rectangles. Binding of NRTIs or the next nucleotide (blue rectangle) is determined by the nature of the base of the template (N) ahead of the primer. Inhibition by INDOPY-1 (orange oval) follows predominantly the incorporation of a thymidine.
- FIG. 6.
Stabilization of preformed RT-DNA/DNA complexes with INDOPY-1. Preformed RT-DNA/DNA complexes were incubated with increasing concentrations of INDOPY-1 (left), the next complementary nucleotide (middle), or nevirapine (right). These complexes were challenged with heparin to trap dissociated RT molecules before the samples were analyzed on a nondenaturing gel. Lane 1 shows the labeled primer, and lanes 2, 3, and 4 are controls in the absence of enzyme, preincubation with heparin, and in the absence of heparin, respectively.
- FIG. 7.
Effect of the 3′ end of the primer on INDOPY-1 binding. (A) Preformed RT-DNA/DNA complexes containing variable residues at the 3′ end of the primer and the complementary template position were incubated with increasing concentrations of INDOPY-1. Complexes were challenged with heparin to trap dissociated RT molecules, and the samples were analyzed on a nondenaturing gel as shown in Fig. 6. The relevant sequences are shown below the band shift experiment. (B) Graphic representation of the data shown in panel A. T, thymidine; C, deoxycytidine; G, deoxyguanosine; A, deoxyadenosine. The lower concentration range of the graph on the top is also shown with an expanded x axis (below).
- FIG. 8.
Effect of INDOPY-1 on the translocational equilibrium of HIV-1 RT. (A) We employed a site-specific footprinting approach to determine the ratio of complexes that exist pre- or posttranslocation. The translocation state was monitored in the presence of increasing concentrations of INDOPY-1, the next nucleotide (middle), and nevirapine. (B) Schematic representation of results shown in panel A. Open arrows show cleavage positions in the absence of inhibitors. Filled arrows show cleavage positions at highest concentrations of inhibitors. The size indicates the relative cleavage intensity. (C) Model of INDOPY-1 binding. The position of the primer/template is shown relative to the nucleotide binding site. The lower complex represents the pretranslocational state, in which the N-site is occupied by the 3′ end of the primer terminus. The presence of the next nucleotide (blue rectangle) or the presence of INDOPY-1 (orange oval) can trap the complex in its posttranslocational state (top complex). nt, nucleotide.
- FIG. 9.
Competition between INDOPY-1 and the nucleotide substrate. (A) Wild-type (WT) RT and the M184V Y115F double mutant were incubated with a DNA/DNA substrate and a radiolabeled nucleotide (dTTP). The DNA primer contained a dideoxycytidine at its 3′ end to prevent incorporation of the nucleotide. The complex was challenged with increasing concentrations of INDOPY-1, and the samples were analyzed on nondenaturing gels. The relevant sequences are shown below the band shift experiment. (B) Graphic representation of the data shown in panel A. The values shown are averages ± standard deviations (error bars) from three independent experiments.
Tables
- TABLE 1.
Inhibition of DNA synthesis with different template/primers
Drug IC50 (μM)a Poly(rA) · poly(dT) Poly(rI) · poly(dC) Poly(rC) · poly(dG) Poly(rU) · poly(dA) INDOPY-1 0.29 ± 0.07 1.10 ± 0.15 >200 >100 Zidovudine-TP 0.011 ± 0.010 >100 >100 >100 Efavirenz 0.24 ± 0.08 0.22 ± 0.06 0.022 ± 0.012 0.11 ± 0.11 ↵ a IC50, 50% inhibitory concentration. Average and standard deviation of at least three independent measurements.
- TABLE 3.
Median change of INDOPY-1 susceptibility of HIV-1 strains derived from clinical isolates
Mutation pattern Median fold change in EC50 compared to HIV-1 IIIB Interquartile range (Q1-Q3) No. of samples Y115F M184Va 16 6.5-25 40 M184Va 3.6 2.0-5.8 566 K65Ra 0.5 0.3-0.9 38 K65R M184Va 1.3 0.8-1.8 17 M41L L210W T215Yb 1.3 0.9-1.8 36 D67N K70R K219Qb 1.4 1.1-3.1 18 D67N K70R T215F K219Qb 1.2 0.9-2.8 12 ↵ a For each determination, viruses with additional mutations at positions 65, 115, or 184 were excluded.
↵ b Viruses with mutations at positions 65, 115, or 184 were excluded from the analysis. Also for each determination, the viruses with additional mutations at positions 41, 67, 70, 210, 215, or 219 were excluded.
- TABLE 4.
Influence of different NRTI resistance-associated mutations on the susceptibility of INDOPY-1
HIV-1 HXB2 mutation(s) in RT Fold change in EC50 ± SDa compared to HIV-1 IIIB TAMs (M41L D67N K70R T215Y) 3.0 ± 1.2 T69 insertion complex (T69S ins69-70S-S L210W T215Y) 0.9 ± 0.33 Q151M complex (A62V V75I F77L F116Y Q151M) 1.0 ± 0.27 Y115F M184V >100 M184V 5.0 ± 2.6 Y115F 7.9 ± 1.6 K65R 0.5 ± 0.2 K65R M184V 0.9 ± 0.2 ↵ a Average and standard deviation of at least three independent measurements.
