Article Figures & Data
Figures
- Figure 1.
Comparative PARP catalytic inhibition of BMN 673. A, chemical structures of BMN 673, olaparib (AZD2281), rucaparib (AG-014699), and NAD+. The nicotinamide moiety is outlined in dotted lines. Arrows in the BMN 673 structure indicate chiral centers involved in drug activity (see Supplementary Fig. S1). B, catalytic PARP inhibition potency of BMN 673 in comparison with olaparib and rucaparib. Total PAR levels in wild-type DT40 cells were examined by Western blotting against PAR 30 minutes after the indicated drug treatments. The asterisk represents a nonspecific band. C, PAR levels in drug-treated wild-type DT40 and DU145 cells measured by ELISA. Cells were incubated with the indicated concentrations of PARP inhibitors for 2 hours. PAR levels without drug treatment were set as 100% in each cell line.
- Figure 2.
BMN 673 is markedly more cytotoxic than olaparib and rucaparib while requiring PARP1/2 for activity. For all experiments, viability curves were derived after continuous treatment for 72 hours with the indicated PARP inhibitors in the indicated cell lines. Cellular ATP concentration was used to measure cell viability. The viability of untreated cells was set as 100%. Error bars represent SD (n ≥ 3). A, viability curves of wild-type, PARP1−/−, and BRCA2tr/− DT40 cells. Drug IC90 values are tabulated at the bottom. B, viability curves of DU145 (human prostate cancer) and EW8 (Ewing's sarcoma) cells. Drug IC90 values are tabulated at the bottom. C, viability curves of PARP1−/− DT40 cell line to high concentrations of the PARP inhibitors. D, viability curves of MDA-MB231 (human breast cancer) cells.
- Figure 3.
Comparison of the sensitivity patterns in the three PARP inhibitors across the NCI60 cell lines. The IC50 obtained from the NCI60 databases (refs. 35, 41; http://discover.nci.nih.gov/cellminer) is plotted for each cell line. Cell lines are colored according to tissue of origin (41). NA, data not available.
- Figure 4.
Comparative trapping of PARP1- and PARP2–DNA complexes by BMN 673, olaparib, and rucaparib. PARP–DNA complexes were determined by Western blot analyses of chromatin-bound fractions from drug-treated DT40 cells (A) and DU145 cells (B). DT40 and DU145 treatments were for 30 minutes and 4 hours, respectively. Blots were probed with the indicated antibodies. Histone H3 was used as positive markers for chromatin-bound fractions and as loading control. The blots of lanes 1 to 4 are identical to the blots of lanes 8 to 11 for PARP2 in B. The blots are representative of multiple experiments.
- Figure 5.
Biochemical trapping of PARP1 by BMN 673. A, scheme of the fluorescence anisotropy (FA) binding assay. The star indicates the site labeled on the DNA substrate with Alexa Fluor 488. Unbound nicked DNA substrate rotates fast and gives low fluorescence anisotropy. PARP1 binding to the substrate slows the rotation and gives high fluorescence anisotropy. Addition of NAD+ leads to PARP1 dissociation from DNA due to autoPARylation. B, concentration-dependent PARP1–DNA association in the presence of BMN 673 or olaparib. Fluorescence anisotropy was measured 40 minutes after adding NAD+. C, time-course of PARP1–DNA dissociation in the presence of BMN 673 and olaparib (0.12 μmol/L each). Addition of NAD+ in the absence of PARP inhibitor immediately reduces PARP1–DNA complexes (no drug, DMSO control). In the absence of NAD+, PARP1–DNA complexes remain stable for at least 180 minutes (no NAD+). Data are mean ± SEM (n = 3).
- Figure 6.
BMN 673 enhances the cytotoxicity of alkylating agents more efficiently than olaparib and rucaparib. A, survival curves of wild-type DT40 cells treated with MMS alone (upper curves labeled “0”) or with the indicated concentrations of PARP inhibitors (at the concentration shown beside each curve in micromolar units). The viability of untreated cells was set as 100%. Data are mean ± SD (n ≥ 3). B, PARP1−/− cells are hypersensitive to MMS (compare with upper curves in A) and resistant to the PARP inhibitors. C, viability curves of the indicated human cancer cells treated with MMS in combination with the indicated PARP inhibitors (the concentration of each PARP inhibitor is shown beside each curve). The viability of untreated cells was set as 100%. Data are mean ± SD (n ≥ 3). D, same as C but using temozolomide instead of MMS in prostate cancer DU145 cells.
Tables
- Table 1.
Summary of the IC50 and IC90 PAR level inhibitions for each drug in DT40 and DU145 cells
IC50, nmol/L IC90, nmol/L Drug DT40 DU145 DT40 DU145 BMN 673 4 11 30 71 Olaparib 6 18 80 120 Rucaparib 21 18 100 120 - Table 2.
Pearson correlation coefficient analyses of Fig. 3 between the drugs
BMN 673 Olaparib Rucaparib BMN 673 1.00 Olaparib 0.52 1.00 Rucaparib 0.04 0.16 1.00 NOTE: Numbers in italic indicate highly significant correlations (P < 0.001).
Supplementary Data
Files in this Data Supplement:
- Supplementary Figures 1 - 4 - PDF file - 1641K, Supplementary figure 1. Stereospecific targeting of PARP by BMN 673. Supplementary figure 2. Lack of effect of PARP inhibition on ATP concentration. Supplementary figure 3. Flow cytometry analyses after treatment with different PARP inhibitors. Supplementary figure 4. Stereospecific poisoning of PARP by BMN 673.
Volume 13, Issue 2
