Fig 1: NVB inhibits POLθ but not HSP90 or TOP2 in human cells. NVB inhibits POLθ but not HSP90 or TOP2 in human cells.a, HSP90 client degradation assay in RPE1 and RPE1-BRCA1−/− cells. Cells were treated with a potent HSP90 inhibitor PU-H71 or NVB for 48 hours, and then cells were collected for Western blot analysis of the HSP90 client AKT1. b-c, HSP90 client degradation assay in MCF7 cells. Cells were treated with the potent HSP90 inhibitor PU-H71 or NVB for 24 hours, and then cells were collected for Western blot analysis of the HSP90 client proteins AKT1, CDK6 (b) and BRCA1 (c). Levels of HSP90 and HSP70 were also analyzed after NVB or PU-H71 treatment (c). d-e, Combination effect of etoposide and novobiocin in killing TOV21G cells (d) and CAPAN1 cells (e), showing their non-epistatic effects. Mean ± SD of n=3 independent experiments are shown. f, CellTiter-Glo cell viability assay of empty vector (EV) and FANCF-complemented TOV21G cells, in the presence of olaparib, novobiocin or both. Mean ± SD, n = 4 biological replicates are shown. g, IC50 values of olaparib in TOV21G cells with or without NVB, derived from data in f. Mean ± SD of IC50 were shown, from n = 4 biological replicates.
Fig 2: A small-molecule screen identifies Novobiocin (NVB) as a specific POLθ ATPase inhibitor that kills HR-deficient tumors.a, The domain structures of full length (FL) and ATPase domain (ΔPol, a.a. 1–987) of POLθ, a Coomassie-stained gel of the purified POLθ ATPase domain used for screen, and a schematic of the small molecule screen. b, Results of the small-molecule screen. Shown is POLθ ATPase activity in the presence of small-molecule libraries (enriched with bioactive compounds). Four top hits verified in the secondary screen (Extended Data Fig. 1b) were labeled: reactive blue 2 (RB), suramin (SUR), novobiocin (NVB) and aurintricarboxylic acid (ATA). Data are mean from n = 2 replicates. c, Quantification of the ATPase activity of POLθ, SMARCAL1, CHD1, BLM, TRIP13, RAD51 and HSP90AA1 with increasing concentration of NVB. ATPase activity was determined by ADP-Glo assay (Promega). Activities were normalized to DMSO (0.1%). Mean of 6 replicates from n = 2 two independent experiments are shown. d, The NVB binding tunnel in POLθ ATPase domain (shown in surface - PDB 5AGA) was predicted by extra precision glide docking and lowest binding free energy from prime MM-GBSA calculations to have multiple hydrogen bonds and close hydrophobic packing. Top and side views showing NVB docking into the tunnel. Binding modes of NVB (green sticks) and AMP-PNP (magenta sticks) in POLθ helicase domain (PDB 5AGA) with green sphere showing active site Mg2+ ion. e, Quantification and representative images of POLθ-GFP accumulation at sites of laser micro-irradiated DNA damage in RPE1-POLQ−/− cells overexpressing GFP-tagged POLQ WT, polymerase mutant, ATPase/Helicase mutant, or double mutant. GFP-POLQ WT expressing cells treated with DMSO (0.1%) or 100 μM of NVB were shown. Mean ± SEM were shown. WT(+DMSO), n = 7 cells; WT(+NVB), n = 8 cells; Pol dead, n = 24 cells; ATPase dead, n = 13 cells; Pol/ATPase dead, n= 14 cells, from three independent experiments. GFP only, n = 4, from two independent experiments. f, MMEJ and HR repair reporter assays in U2OS cells treated with increasing concentration of NVB. Percentage of GFP positive cells are shown as pathway efficiency. Mean ± SD, from n = 6 (for MMEJ) and n = 3 (for HR) from three independent experiments, ordinary one-way ANOVA. g, Tumor growth of the GEMM model (Brca1−/− TNBC) after treatment with vehicle (PBS) or NVB. Tumor bearing FVB/129P2 were treated with PBS or 100 mg/kg NVB via IP injection twice a day for 5 weeks. Mean ± SEM is shown, n = 6 mice for PBS group and n = 7 mice for NVB group. h, Survival plot of the experiment shown in (g). Median survival and p value are shown, Statistical analyses in g and h were two-tailed t-test. *, p < 0.05. **, p < 0.01.
Fig 3: The results of molecular docking.(A) Isoscopoletin-Hsp90AA1 (5njx). (B) Isoscopoletin-GPI (7w72). (C) Isoscopoletin-GPD2 (1x0v). (D) Isoscopoletin-PGK2 (2paa).
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