This work was supported by grants from NIH (GM122932 to Y.Y.) and Welch foundation (I-1800 to Y.Y.). Competing interests Y.Y. of these two pathways in mediating the cytotoxicity of PARPi, however, is incompletely understood. Here we designed a series of small molecule PARP degraders. Treatment with one such compound iRucaparib results in highly efficient and specific PARP1 degradation. iRucaparib blocks the enzymatic activity of PARP1 in vitro, and PARP1-mediated PARylation signaling in intact cells. This strategy mimics PARP1 genetic depletion, which enables the pharmacological decoupling of PARP1 inhibition from PARP1 trapping. Finally, by depleting PARP1, iRucaparib protects muscle cells and primary cardiomyocytes from DNA damage-induced energy crisis and cell Stearoylcarnitine death. In summary, these compounds represent non-trapping PARP1 degraders that block both the catalytic activity and scaffolding effects of PARP1, providing an ideal approach for the amelioration of the various pathological conditions caused by PARP1 hyperactivation. Introduction PARP1 is an abundant nuclear protein that is critically involved in a number of biological processes linked to cellular stress responses1C3. The enzymatic function of PARP1 is to catalyze a protein posttranslational modification Stearoylcarnitine called Poly-ADP-ribosylation (PARylation)4. PARylation is tightly connected to DNA damage response (DDR)5, 6. PARP1 becomes activated upon sensing DNA strand breaks, leading to the generation of a large number of PARylated proteins, including itself. PAR polymers then recruit various DNA repair factors that contain PAR-binding motifs (e.g., OB-fold, WWE, PBZ, BRCT and macrodomain). These PBMs (PAR-binding motifs) bind to different topological units within PAR (e.g., ADP-ribose and iso-ADP-ribose), and thereby trigger downstream DDR signaling6. < 0.01, *** < 0.001. values were 1.010?4 and 0.0022. To characterize the trapped proteome in an unbiased manner, we performed multiplexed quantitative mass spectrometric analyses of Rabbit Polyclonal to PARP (Cleaved-Gly215) the chromatin-bound fractions isolated from HeLa cells treated with (1) MMS+DMSO; (2) MMS+Rucaparib; and Stearoylcarnitine (3) MMS+iRucaparib (Figure 5B). We included two biological replicates for each condition, and from this TMT6-plex sample, we were able to identify and quantify a total of 3,392 proteins (protein FDR = 1%). Consistent with our biochemical results, we found that PARP1 and PARP2 were two of the proteins that are most enriched in the chromatin-bound fraction, consistent with the robust trapping activity of Rucaparib. These two proteins were, however, depleted (compared to MMS treatment) from the chromatin after iRucaparib treatment (Figure 5B and Supplementary Figure 9C). The trapped PARP1/DNA protein complex is known to impair replication fork progression and subsequently, induce a DNA damage response9, 10. To determine the contribution of trapped PARP1 in mediating the cytotoxicity of PARP1 inhibitors under the basal conditions, we treated HeLa cells with DMSO, Rucaparib or iRucaparib for 72 hrs. Cell cycle analyses revealed that Rucaparib, but not iRucaparib, induced G2 accumulation (Supplementary Figure 9D and 9E). In addition, treatment of Rucaparib but not iRucaparib caused DNA damage (as shown by the accumulation of H2AX) (Figure 5C) and impaired cell proliferation (Figure 5D). These data are consistent with a model where spontaneously generated base lesions are recognized by PARP1, which, upon Rucaparib treatment, leads to the formation of trapped PARP1 and causes cell death. These toxic PARP1/DNA complexes, however, are abrogated by PARP1 degraders, resulting reduced suppression of cell survival (Figure 5D). We also repeated these experiments using iRucaparib-TP3 and obtained very similar results (Supplementary Figure 9A and 9FC9K). Protection against genotoxic stress-induced cell death We sought to test whether iRucaparib treatment mimics PARP1 genetic deletion, and therefore protects cells against genotoxic stress-induced cell death. Similar to our previous observation in primary cardiomyocytes, we found that iRucaparib treatment also resulted in efficient PARP1 degradation in mouse C2C12 myoblasts, and fully differentiated C2C12 myotubes (Supplementary Figure 10A and 10B). Besides iRucaparib, we found that the treatment of C2C12 myotubes and primary cardiomyocytes with iRucaparib-AP5 also led to robust Stearoylcarnitine and specific PARP1 degradation (Supplementary Figure 10CC10F). Importantly, MMS- and H2O2-induced PARP1 activation was completely blocked by pretreating C2C12 myotubes with either Rucaparib or iRucaparib (Supplementary Figure 10G). Furthermore, we also tested peroxynitrite, which is a reactive nitrogen species generated during ischemia-reperfusion, and a critical contributor to IR-dependent tissue injury29. We showed that peroxynitrite treatment robustly activated PARP1, leading to profound accumulation of PAR. Peroxynitrite-induced PARP1 activation was completely inhibited by either Rucaparib or iRucaparib (Supplementary Figure 10G). Next we examined.