Journal: Biochemistry

Article Title: Slow Dissociation from the PARP1–HPF1 Complex Drives Inhibitor Potency

doi: 10.1021/acs.biochem.3c00243

Figure Lengend Snippet: Olaparib associates with PARP1 in a concentration-dependent manner. (A–C) Representative graphs showing the changes in the FP signal over time following the addition of NAD + to a complex of PARP1, p18mer*, and olaparib (10 nM in A, 20 nM in B, and 40 nM in C) at varying delay times (1.5–150 s) following addition of olaparib. Each line in the graph corresponds to the indicated delay time after addition of olaparib. The replots (measured linear rates vs delay times) are shown on the right for each graph of the primary data and reveal the apparent rate of association ( k obs ) under the experimental conditions. (D) The graph shown is a re-replot of the k obs derived from the replots shown in the insets of (A–C) vs the concentration of olaparib. The linear dependence of these k obs demonstrates a simple one-step mechanism of association. The other PARPi that were subjected to this concentration-dependent experiment (fluzoparib, talazoparib, and saruparib) are shown in Figures S1–S3 .

Article Snippet: Veliparib (S1004), olaparib (S1060), fluzoparib (S9712), rucaparib (S1098), niraparib (S2741), and talazoparib (S7048) were purchased from Selleck.

Techniques: Concentration Assay, Derivative Assay

Journal: Biochemistry

Article Title: Slow Dissociation from the PARP1–HPF1 Complex Drives Inhibitor Potency

doi: 10.1021/acs.biochem.3c00243

Figure Lengend Snippet: Rates of association of PARPi to PARP1 or the PARP1–HPF1 complex. Representative graphs demonstrate that veliparib (A) has a significantly faster rate of association with PARP1 ( k on ) compared to AZD9574 (B). The raw data in the left graphs show the loss in FP signal after the addition of NAD + to a mixture of PARP1, p18mer, and 20 nM PARPi at different delay times between 10 and 250 s. The replot graphs on the right (measured linear rates vs delay times) are used to calculate the actual k on values for veliparib and AZD9574. (C) Summary of rates of association of PARPi to PARP1 or PARP1–HPF1 complex. Rates of association for each PARPi with PARP1 (lighter shade, left bar of each pair) and PARP1–HPF1 complex (darker shade, right bar of each pair) as averages with standard deviations. Pairwise statistical comparison demonstrates differences ( p < 0.05) following the addition of HPF1 for fluzoparib, niraparib, and saruparib. Values for k on and number of replicates are shown in Table , and representative data for each PARPi are shown in Figures A,B, S4, and S6 .

Article Snippet: Veliparib (S1004), olaparib (S1060), fluzoparib (S9712), rucaparib (S1098), niraparib (S2741), and talazoparib (S7048) were purchased from Selleck.

Techniques: Comparison

Journal: Biochemistry

Article Title: Slow Dissociation from the PARP1–HPF1 Complex Drives Inhibitor Potency

doi: 10.1021/acs.biochem.3c00243

Figure Lengend Snippet: Measuring the binding affinity of fluzoparib, saruparib, and AZD9574 for PARP1 or PARP1 ± HPF1. Representative data measuring the release of p18mer* from PARP1 in the presence of varying concentrations (as indicated on the right) of the fluzoparib (A), saruparib (B), and AZD9574 (C). The lines through the points reflect fitting to first-order kinetics, and the data were processed to derive K i values as previously described. Results of replicate experiments are shown in Table .

Article Snippet: Veliparib (S1004), olaparib (S1060), fluzoparib (S9712), rucaparib (S1098), niraparib (S2741), and talazoparib (S7048) were purchased from Selleck.

Techniques: Binding Assay

Journal: Biochemistry

Article Title: Slow Dissociation from the PARP1–HPF1 Complex Drives Inhibitor Potency

doi: 10.1021/acs.biochem.3c00243

Figure Lengend Snippet: Measured Rates of Association ( k on ) for PARPi to PARP1 ± HPF1 a

Article Snippet: Veliparib (S1004), olaparib (S1060), fluzoparib (S9712), rucaparib (S1098), niraparib (S2741), and talazoparib (S7048) were purchased from Selleck.

Techniques: