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. 2023 Oct 25;9(43):eadg7752. doi: 10.1126/sciadv.adg7752

Fig. 4. Nimbolide traps PARylated-PARP1 and PAR-dependent DNA repair factors.

Fig. 4.

(A) Structure of nimbolide. The total synthesis of nimbolide is reported in (46, 47). (B) Nimbolide blocks the auto-ubiquitination of RNF114. (C) Nimbolide treatment induces potent PARP1 trapping. The indicated green fluorescent protein (GFP)–tagged PARP1-expressing HeLa cells were pretreated with olaparib (1 μM) or nimbolide (1 μM) for 1 hour. (D) Nimbolide, but not olaparib, treatment induces the trapping of XRCC1. HeLa cells expressing GFP-tagged XRCC1 were pretreated with olaparib or nimbolide (1 μM for 1 hour). (E) Nimbolide treatment induces potent PARP1 and XRCC1 trapping. HCT116 cells were treated with nimbolide or PARPi (1 μM for 24 hours). (F) RNF114 deletion abolishes the nimbolide-induced PARP1 trapping. The indicated HCT116 cells were treated with nimbolide (1 μM for 24 hours). (G) RNF114 deletion rescues cells from nimbolide-induced cell death. The indicated HeLa cells were treated with nimbolide (1 μM for 24 hours). (H and I) RNF114 (H) or PARP1 (I) deletion rescues cells from nimbolide-induced cell death. The indicated HeLa cells were treated with nimbolide (1 μM for 24 hours). The deletion of RNF114 and PARP1 was confirmed as shown in fig. S3H. (J) Structure of a nimbolide analog. The compound as shown was inactive against UWB1 cells (IC50 > 10 μM). Additional nimbolide analogs are shown in fig. S4. (K) Nimbolide treatment is synthetic lethal with respect to BRCA1 deficiency. UWB1 and UWB1 + BRCA1 cells were subjected to nimbolide treatment for 96 hours. (L) Nimbolide induces stronger cytotoxicity in UWB1 cells compared to PARPi. UWB1 cells were treated with nimbolide or PARPi (1 μM for 96 hours). (M and N) Nimbolide overcomes intrinsic and acquired resistance to PARPi. HCC1937 (M) or UWB1 (SYr12) (N) cells were treated with nimbolide or PARPi (1 μM for 96 hours). (O and P) Body weight (O) and tumor volume (P) of HCC1937-bearing mice that were treated with vehicle, olaparib, or nimbolide. Scale bars, 10 μm. DMSO, dimethyl sulfoxide.