Fig. 6: A PARP1 biophysical perturbagen screen enabled by a L713F-nLuc dual luminescence assay format.

a The PARP1 L713F-nLuc biosensor (em: 460 nm) was paired with akaLuc (em: 650 nm) to enable sequential dual luciferase analyses. b Time-resolved detection of PARP1 L713F-nLuc stabilization following veliparib treatment and normalized to akaLuc signal. n = 2 with line of best fit shown. c Dose-dependent veliparib stabilization of different PARP1 L713F-nLuc abundances (DOX gradient) after 24 hours and normalized to akaLuc. n = 2 with line of best fit shown. d The MedChemExpress Epigenetics and Selleck Nordic Oncology libraries were screened (10 µM, 24 hours) with the L713F-nLuc/akaLuc system. Compounds was excluded if akaLuc intensity differed > 4 SDs from controls, leaving 840 compounds for further analysis. e Ranked, log₂-transformed L713F-nLuc/akaLuc ratios from 840 screening compounds (dark gray). Negative (light gray, DMSO) and positive controls (blue, 10 µM veliparib) are shown for reference. Hits were defined as at least 2 (orange) or 3 standard deviations (σ, red) from the screening library mean. Annotated PARPi are indicated with black borders and trapping DNMT compounds are labeled. f Detailed overview of positive screening hits (n = 47). Non-PARPi were triaged by target class, contextualized by hit rate within the general target class, and by anecdotally defined primary target/compound class. g Hit rates of PARPi within the screening library by increasing stringency (general PARPi → PARP1i → PARP1i [IC₅₀ < 1 µM]) and numbers of qualifying compounds. Hit proportions are shown in blue, while non-hits are gray. * – PJ34 missed the akaLuc cut-off. h Hit confirmation of PARPi (orange) and non-PARPi (yellow) positive screening hits. Identical positive (blue) and negative controls (gray) are used from the screen, and means of n = 24 (negative, positive control), n = 3 (linifanib to fluzoparib), or n = 6 (pamiparib to AZD5305) data points are shown ± SD. Names of statistically significant compounds in red, and confirmed non-PARPi are summarized by primary target hit rate (final target share). P values are shown for one-way ANOVA analysis with comparisons to DMSO control (Dunnett’s test; F_Treatment [DFn, DFd] = 200.9 [48, 202]). FC fold change.