Development of an efficient NUPR1 inhibitor with anticancer activity

Figures

• 

  Fig. 1. In vitro molecular screening of compounds based on thermal denaturations of NUPR1. (A) Thermal denaturation profiles of selected compounds capable of interacting with NUPR1, and altering its thermal denaturation profile. (B) Regarding the subsequent assays and for a more detailed look, the most promising compound of the screening is shown (with the thermal denaturation profile of AJO14 indicated as a dashed line). The typical thermal denaturation profiles obtained from control samples (NUPR1 with no compound) are also shown (continuous line). (C) Calorimetric titration for AJO14 interacting with NUPR1: thermogram (upper panel) and binding isotherm (lower panel). Non-linear fit according to a model considering a single ligand binding site (continuous line) is shown. (D) Structure of the synthesized AJO14 compound.

• 

  Fig. 2. AJO14 inhibited PDAC cells proliferation in vitro due to interaction with NUPR1. (A) Viability upon a treatment for 72 h with AJO compounds in MIA PaCa-2 cells. (B) Viability of AJO14 on one NUPR1-WT clone and two NUPR1-KO clones treated for 72 h. (C) Viability of 11 primary patients-derived PDAC cell lines treated with AJO14 for 72 h. (D) Viability of cell lines HepG2, MDA-MB-231, H358, A375, HT29, U87, U2OS and PC-3 at increasing concentrations of AJO14 (n = 3).

• 

  Fig. 3. AJO14 and AJO14-derivatives had low affinity for the off-target receptor hERG. (A) Representative graph of the concentration–response curve of E-4031, ZZW-115 and AJO14 binding to hERG, obtained by using a fluorescence polarization assay. Data are presented as mean ± SD, (n = 3). (B) Half-maximal inhibitory concentration (IC50) values on hERG channel inhibition by E-4031, ZZW-115 and AJO14. (C) Representative graph of the concentration–response curve of E-4031 and selected eight AJO14-derivatives binding to hERG, obtained by using a fluorescence polarization assay. Data are presented as mean ± SD, (n = 3).

• 

  Fig. 4. AJO14 induced cancer cell death by necroptosis, apoptosis, and parthanatos. (A) LDH release and (B) caspase 3/7 activity measured on MIA PaCa-2 cells treated with increasing concentrations of AJO14 for 24, 48 and 72 h. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (2-way ANOVA, Dunnett’s test) (n = 3). (C) Flow cytometry analysis of annexin/PI staining following 24, 48 and 72 h of treatment with AJO14. A representative experiment of the dot plot profile of cells is shown (n = 3). (D) Apoptotic rate, as the sum of the early apoptotic proportion and the late apoptotic/necrotic proportion were calculated *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (2-way ANOVA, Dunnett’s test). (E) Viability upon a 24 h period of treatment with increasing concentrations of AJO14 in MIA PaCa-2 cells in the presence or absence of Z-VAD-FMK (20 µM), Nec-1 (40 µM), or Olaparib (25 μM). **p < 0.01, ***p < 0.001, ****p < 0.0001 (1-way ANOVA, Dunnett’s test) (n = 3).

• 

  Fig. 5. AJO14 treatment induced mitochondrial metabolic failure. (A) Total ATP content measured in MIA PaCa-2 cells incubated with AJO14 for 24 h. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (2-way ANOVA, Dunnett’s test) (n = 3). (B and C) Oxygen consumption rate measured in MIA PaCa-2 cells using a Seahorse XF bioanalyzer after 24 h of treatment with increasing concentrations of (B) AJO14, and in the presence of AJO14 (10 μM) with increasing concentrations of (C) Olaparib. A representative experiment is shown (n = 3).

• 

  Fig. 6. Antitumoral effect induced by AJO14 treatment in vivo. Results on Crl:NU(Ico)-Foxn1nu mice bearing pancreatic cancer MIA PaCa-2 cell lines xenograft treated daily with the vehicle or 5, 10, 20 or 50 mg/kg AJO14 via intraperitoneal injection (n = 5). (A) Tumor volume and (B) mice body weight were measured twice per week. *p < 0.05, ***p < 0.001, ****p < 0.0001 (1-way ANOVA, Dunnett’s test). (C) Representative images of tissues sections after TUNEL, Ki-67 or cleaved caspase-3 IHC staining of tumor tissues (n = 5) (scale bar: 100 μm).

• 

  Fig. 7. Calorimetric titration for LZX-2-73 interacting with NUPR1. (A) Thermogram (upper panel) and binding isotherm (lower panel). Non-linear fit according to a model considering a single ligand binding site (continuous line) is shown. Chemical structure of the synthesized LZX-2-73 compound. (B) Interaction parameters of the NUPR1-LZX-2-73 complexes obtained by ITC at 15 °C and pH 7.

• 

  Fig. 8. Antitumoral effect induced by LZX-2-73 treatment in vivo. Results on Crl:NU(Ico)-Foxn1nu mice bearing pancreatic cancer MIA PaCa-2 cell lines xenograft treated daily with the vehicle or 5 or 25 mg/kg LZX-2-73 via intraperitoneal injection (n = 3). Tumor volume and mice body weight were measured twice per week. **p < 0.01 (1-way ANOVA, Dunnett’s test).

• 

  Fig. 9. Molecular docking of compound LZX-2-73 to key 5-residue fragments of NUPR1. (A) Most favorable pose among those predicted in the docking to the sole fragment S31LAHS35. (B) Most favorable pose in the simultaneous docking of both fragment S31LAHS35 and L66VTKL70.