Validation of the 2nd Generation Proteasome Inhibitor Oprozomib for Local Therapy of Pulmonary Fibrosis

Abstract

Proteasome inhibition has been shown to prevent development of fibrosis in several organs including the lung. However, effects of proteasome inhibitors on lung fibrosis are controversial and cytotoxic side effects of the overall inhibition of proteasomal protein degradation cannot be excluded. Therefore, we hypothesized that local lung-specific application of a novel, selective proteasome inhibitor, oprozomib (OZ), provides antifibrotic effects without systemic toxicity in a mouse model of lung fibrosis. Oprozomib was first tested on the human alveolar epithelial cancer cell line A549 and in primary mouse alveolar epithelial type II cells regarding its cytotoxic effects on alveolar epithelial cells and compared to the FDA approved proteasome inhibitor bortezomib (BZ). OZ was less toxic than BZ and provided high selectivity for the chymotrypsin-like active site of the proteasome. In primary mouse lung fibroblasts, OZ showed significant anti-fibrotic effects, i.e. reduction of collagen I and α smooth muscle actin expression, in the absence of cytotoxicity. When applied locally into the lungs of healthy mice via instillation, OZ was well tolerated and effectively reduced proteasome activity in the lungs. In bleomycin challenged mice, however, locally applied OZ resulted in accelerated weight loss and increased mortality of treated mice. Further, OZ failed to reduce fibrosis in these mice. While upon systemic application OZ was well tolerated in healthy mice, it rather augmented instead of attenuated fibrotic remodelling of the lung in bleomycin challenged mice. To conclude, low toxicity and antifibrotic effects of OZ in pulmonary fibroblasts could not be confirmed for pulmonary fibrosis of bleomycin-treated mice. In light of these data, the use of proteasome inhibitors as therapeutic agents for the treatment of fibrotic lung diseases should thus be considered with caution.

Fig 1. Toxicity and inhibitory profile of bortezomib and oprozomib in alveolar epithelial cells.

MTT assay after 72 hours of treatment with (A) BZ or (B) OZ (Data represent mean ± SEM. n = 3 per group. *P ≤ 0.05, **P < 0.01, ***P < 0.001. 1way ANOVA Dunnett‘s Multiple Comparison Test). (C) Proteasome activity 24 hours after treatment with BZ or (D) OZ (Data represent mean ± SEM. n = 3 per group. *P ≤ 0.05, **P < 0.01, ***P < 0.001. 1way ANOVA Dunnett‘s Multiple Comparison Test). (E) and (F) MTT assay of primary murine ATII cells after 52 hours of treatment with OZ or BZ (Data represent mean ± SEM. n = 4 per group. *P ≤ 0.05, **P < 0.01, ***P < 0.001. 1way ANOVA Dunnett‘s Multiple Comparison Test).

Fig 2. Inhibition profile of oprozomib in primary mouse lung fibroblasts.

(A) Proteasome activity and (B) Luciferase activity of ODD-Luc FVB-LF 24 hours after treatment with OZ (Data represent mean ± SEM. n = 3 per group. *P ≤ 0.05, **P < 0.01, ***P < 0.001. 1way ANOVA Dunnett‘s Multiple Comparison Test). (C) Native gel of ODD-Luc FVB-LF 24 hours after OZ treatment.

Fig 3. Antifibrotic effects of oprozomib in primary mouse lung fibroblasts.

(A) Immunofluorescence staining for Coll-I (green), F-Actin (red) and nuclei (blue) after 72 hours of treatment with OZ. (B) BrdU proliferation assay of primary lung fibroblasts treated with OZ for 72 hours (Data represent mean ± SEM. n = 4 per group. *P ≤ 0.05, **P < 0.01, ***P < 0.001. 1way ANOVA Dunnett‘s Multiple Comparison Test). (C) Immunofluorescence staining for Coll-I (green), F-Actin (red) and nuclei (blue) after treatment with TGF-β and OZ. (D) and (E) RT-qPCR analysis of mRNA expression of Coll-I and αSMA after treatment with TGF-β and OZ (Data represent mean ± SEM. n = 3 per group. *P ≤ 0.05, **P < 0.01, ***P < 0.001. Paired t-Test).

Fig 4. Dose response to local pulmonary application of 0.5, 1, and 5 mg/kg OZ or Pluronic F-127 0.1% solvent control after 24 hours or 96 hours.

(A) Treatment scheme: local pulmonary application of OZ, (B) CT-L proteasome activity after 24 hours, (C) percent of PMNs to total BAL count after 24 hours, (D) CT-L proteasome activity after 96 hours, and (E) percent of PMNs to total BAL count after 96 hours (Data represent mean ± SEM. n = 5 per group. *P ≤ 0.05, **P < 0.01, ***P < 0.001. 1way ANOVA Dunnett‘s Multiple Comparison Test).

Fig 5. Local pulmonary application of oprozomib does not provide antifibrotic effects in the bleomycin mouse model.

(A) Treatment scheme: local pulmonary application of OZ. (B) and (C) mRNA levels of Coll-I and fibronectin (Fn) (Data represent mean ± SEM. n = 6 per group. *P ≤ 0.05, **P < 0.01, ***P < 0.001. 1way ANOVA Bonferroni‘s Multiple Comparison Test). (D) H&E staining of lung slices. (E) CT-L proteasome activity and (F) luciferase activity of whole lung tissue. (G) Treatment scheme: repeated local pulmonary application of OZ. (H) Weight loss of animals at different time points (Data represent mean ± SEM. n = 6 per group. *P ≤ 0.05, **P < 0.01, ***P < 0.001. 1way ANOVA Bonferroni‘s Multiple Comparison Test).

Fig 6. Oral application of oprozomib does not reduce proteasome activity in fibrotic lungs and is not well tolerated in bleomycin challenged animals.

(A) Treatment scheme: repeated oral application of OZ. (B) CT-L proteasome activity (Data represent mean ± SEM. n = 5–6 per group. *P ≤ 0.05, **P < 0.01, ***P < 0.001. Mann Whitney t-test) and (B) weight loss of animals at different time points (Data represent mean ± SEM. n = 5–6 per group. *P ≤ 0.05, **P < 0.01, ***P < 0.001. 1way ANOVA Bonferroni‘s Multiple Comparison Test).

Fig 7. Oral application of oprozomib provides no antifibrotic therapeutic effects.

(A) and (B) mRNA levels of Coll-I and Fn (Data represent mean ± SEM. n = 5–6 per group. *P ≤ 0.05, **P < 0.01, ***P < 0.001. 1way ANOVA Bonferroni‘s Multiple Comparison Test). (C) H&E staining of lung slices and immunofluorescence (IF) staining for Coll-I (red) and αSMA (green).