Fig. 1.

Non-hydrolyzable NAD⁺ analog binding and inhibition of PARP-1. a Schematic representation of PARP-1 domains. b Chemical structure of key compounds used in this study: NAD⁺, non-hydrolyzable NAD⁺ analogs carba-NAD⁺ and benzamide adenine dinucleotide (BAD), benzamide, and ADP-ribose (ADPr). c SDS-PAGE PARP-1 activity assay (1 μM DNA, 1 μM protein, 50 μM NAD⁺) in the presence of carba-NAD⁺ and BAD. An image of the entire gel is included in Supplementary Fig. 10. d, e Differential scanning fluorimetry (DSF) experiment using PARP-1 CAT domain WT or ΔHD (5 μM) and various amounts of carba-NAD⁺, BAD, benzamide, and ADP-ribose. ΔT_M is calculated by subtracting the T_M and ΔT_M obtained in the absence of compound from the T_M obtained in the presence of compound. The experiments were all performed in triplicate for which averages and standard deviations are shown. f Colorimetric assay of full-length PARP-1 DNA-dependent activity in the presence of ADP-ribose, benzamide, and BAD using 20 nM PARP-1, 40 nM DNA (18-bp duplex) and 50 μM NAD⁺ (99:1, NAD⁺:bio-NAD⁺). Experiments were performed in quadruplicate and the averages and standard deviations for each compound concentration are shown, as well as the average IC50 values for benzamide and BAD