Discovery of an orally active small-molecule irreversible inhibitor of protein disulfide isomerase for ovarian cancer treatment

Abstract

Protein disulfide isomerase (PDI), an endoplasmic reticulum chaperone protein, catalyzes disulfide bond breakage, formation, and rearrangement. The effect of PDI inhibition on ovarian cancer progression is not yet clear, and there is a need for potent, selective, and safe small-molecule inhibitors of PDI. Here, we report a class of propynoic acid carbamoyl methyl amides (PACMAs) that are active against a panel of human ovarian cancer cell lines. Using fluorescent derivatives, 2D gel electrophoresis, and MS, we established that PACMA 31, one of the most active analogs, acts as an irreversible small-molecule inhibitor of PDI, forming a covalent bond with the active site cysteines of PDI. We also showed that PDI activity is essential for the survival and proliferation of human ovarian cancer cells. In vivo, PACMA 31 showed tumor targeting ability and significantly suppressed ovarian tumor growth without causing toxicity to normal tissues. These irreversible small-molecule PDI inhibitors represent an important approach for the development of targeted anticancer agents for ovarian cancer therapy, and they can also serve as useful probes for investigating the biology of PDI-implicated pathways.

Fig. 1.

BODIPY conjugation of PACMA 31. (A) Structures of 31, 57, 58, and 59; (B) 31 and 57 but not 58 and 59 inhibited growth of OVCAR-8 cells as measured by MTT assay after 72 h treatment. Curves were generated from mean values (BAR, SEM). (C) BODIPY labeling of 57, 58, and 59 displays significant concentration-dependent fluorescence (λ_ex = 492 nm, λ_em = 535 nm). (D) PACMA 57 covalently binds to specific cellular proteins. Whole-cell lysates of OVCAR-8 cells treated with 57, 58, and 59 at 2 μM for 30 min were subjected to SDS/PAGE followed by fluorescence scan of BODIPY (Left) and SYPRO Ruby (Right). Arrow indicates a fluorescent band in 57-treated cells. One of three representative experiments is shown.

Fig. 2.

PACMA 57 covalently binds to PDI. (A) Identification of PDI as the cellular protein target for 57. OVCAR-8 cells were incubated with 2 μM 57 for 30 min. Whole-cell lysates were prepared as described for 2D gel electrophoresis (SI Materials and Methods), analyzed by isoelectric focusing/SDS/PAGE, scanned for BODIPY fluorescence (λ_ex = 488 nm, λ_em = 526 nm), and silver stained. The fluorescently tagged protein spot (arrow; ∼57 kDa) was excised from the gel and analyzed by MS. (B) Immunoprecipitated cellular PDI was selectively and covalently bound by 57. OVCAR-8 cells were incubated with 57, 58, and 59 at 2 μM for 30 min. Whole-cell lysates were subjected to immunoprecipitation using monoclonal anti-PDI antibody. Precipitated proteins were analyzed by SDS/PAGE and scanned for BODIPY fluorescence (Upper) followed by Western blotting with anti-PDI antibody (Lower). (C) Subcellular colocalization of PDI and 57. OVCAR-8 cells were treated with 2 μM 57 for 30 min followed by fixation and permeabilization. Cellular PDI was stained with anti-PDI mAb. Subcellular localization of PDI and 57 was analyzed using confocal fluorescence microscopy. Red, PDI; green, 57; yellow, merge. One of five representative microscope fields is shown. (D) Competition between 57 and 31 on PDI; 100 ng/μL recombinant PDI protein was incubated with 100 μM 31 or DMSO in sodium phosphate buffer (pH 7.0) for 1 h at 37 °C followed by 1 h incubation with 20 μM of 57. Solutions were mixed with 5× SDS sample buffer and analyzed by SDS/PAGE, fluorescence scanning (Upper), and Coomassie Blue stain (Lower). (E) Kinetics study of covalent interaction between PDI and 57; 100 ng/μL recombinant PDI was incubated with 20 μM 57 and/or 100 μM DTT for the indicated time at 37 °C followed by analysis using SDS/PAGE, fluorescence scanning (Upper), and Coomassie Blue staining (Lower). (F) PACMA 57 selectively bound to PDI; 20 μM 57 was incubated with 100 ng/μL recombinant PDI (lane 1; with 100 ng/μL BSA as a carrier protein), BSA (lane 2), or the core domain of GRP78 (lane 3) for 30 min at 37 °C followed by analysis using SDS/PAGE, fluorescence scan (Upper), and Coomassie Blue stain (Lower).

Fig. 3.

PACMA 31 covalently binds to Cys397/Cys400 in PDI active site. Covalent docking of 31 (red) against PDI (blue; Protein Data Bank ID code 3UEM) with a covalent bond between the terminal carbon atom of 31’s propynoic moiety and the sulfur atom of (A) Cys397 or (B) Cys400. Genetic Optimization for Ligand Docking fitness values are 40.73 and 33.53 for Cys397 and Cys400, respectively.

Fig. 4.

Active PACMAs inhibit PDI activity. (A) PACMA 31 significantly inhibited the activity of PDI in a dose- and time-dependent manner in the insulin aggregation assay. Curves were generated from mean values (BAR, SEM). *P < 0.05; **P < 0.01; ***P < 0.001. (B) Comparison of the inhibitory activity of 31 and PAO. (C) PACMA 56 did not exhibit significant effects on the enzymatic activity of PDI. Experiments were performed in triplicate.

Fig. 5.

Silencing of PDI inhibits cell growth of OVCAR-8 cells. (A) Representative Western blot of 24–96 h PDI silencing in OVCAR-8 cells. (B) PDI siRNA showed significant cytotoxicity as measured by MTT assay. The histogram shows the mean values of growth inhibition (%). (C) PDI knockdown significantly inhibited colony formation in OVCAR-8 cells. The histogram shows the mean number of colonies. (D) The 24-h treatment of 31 significantly inhibited the formation of OVCAR-8 colonies at indicated doses. The histogram shows the mean number of colonies (BAR, SEM). **P < 0.01; ***P < 0.001.

Fig. 6.

PACMA 31 suppresses tumor growth in a mouse xenograft model of human OVCAR-8 ovarian cancer. (A) Mice were treated with 57, 58, and 59 at 10 mg/kg for 3 d with two injections per day; 6 h after the last injection, tumor, liver, and brain tissues were prepared for frozen sections and fluorescent microscopy analysis. (Left) Representative fluorescence images of indicated tissue sections captured using fluorescence microscopy and phase contrast. (Right) Gel fluorescence and Coomassie blue analysis of homogenized tumor samples from the 57- and 58-treated mice. An ∼57-kDa fluorescent band in the 57-treated tumor is indicated by a red arrow. (B) Growth curves of s.c. tumors in mice treated with 31 through i.p. (red; n = 4) or per os administration (yellow; n = 4) or treated with vehicle (blue; n = 5). Treatment schedules are described in Fig. S8. Results are presented as mean tumor volume (BAR, SEM). (Inset) Comparison of tumor volumes between control and 31 i.p. (**P < 0.01) or 31 per os treatment group (*P < 0.05) on day 62 (BAR, SEM). (C) PACMA 31 treatment induced extensive areas of necrosis in OVCAR-8 tumors. Representative images of H&E-stained tumor sections from control (Left) and 31-treated (Center, i.p.; Right, per os) mice are shown. Arrows indicate areas of necrosis.