Background/Purpose:

Prolyl-tRNA synthetase (PRS), a member of aminoacyl tRNA synthetases (ARS), is an enzyme that conjugates amino acid proline to its tRNA to generate prolyl-tRNA to be used in protein synthesis. Since proline is one of major components of collagen, we hypothesized that suppression of PRS would down-regulate collagen synthesis, which could be beneficial in fibrosis. Systemic sclerosis (SSc) is an autoimmune disease that is characterized by progressive thickening of skin, caused by excessive accumulation of collagen.

Methods:

Inhibitory activity of DWN12088 against PRS was assessed by aminoacylation assay. Fibroblasts were treated with DWN12088 either before or after treatment of transforming growth factor beta (TGFβ), and then collagen and pro-fibrotic markers were assessed using western blot. 3D skin organoid was constructed using systemic sclerosis patient-derived dermal fibroblast and keratinocyte. For in vivo study, constructed patient-derived 3D skin organoid was engrafted to SCID mouse using tie-over dressing. After 2 weeks administration of DWN12088, degree of dermal thickness and collagen I (Col I) expression were determined by histological analysis.

Results:

To validate our hypothesis, we investigated the expression levels of pro-fibrotic markers by overexpression or knockdown of PRS in vitro. We showed that PRS is closely related to the expression of Col I and alpha smooth muscle actin (αSMA). Interestingly, PRS protein expression is significantly increased in lung tissue of idiopathic pulmonary fibrosis (IPF) patients. We developed a novel selective inhibitor of PRS, DWN12088, which has IC₅₀ value of 74 nM against PRS. DWN12088 reduced TGFβ-induced Col I and pro-fibrotic marker expression in various cell-lines and primary fibroblasts. This suggests that PRS may be a critical contributor of excessive accumulation of collagen, a main pathological hallmark of fibrosis.

Using systemic sclerosis patient-derived dermal fibroblast, we developed 3D skin organoid in order to investigate efficacy of DWN12088. In this model, DWN12088 successfully decreased skin thickness, Col I, and αSMA. In a mouse xenograft model using the patient-derived 3D skin organoid, treatment of 10 mg/kg DWN12088 also showed decrease in skin thickness and collagen expression compared to control group.

Conclusion:

PRS is an enzyme required during protein translation involving proline amino acid. In fibrotic condition, we observed that PRS is overexpressed, which may be responsible for over-production of collagen. Inhibition of PRS successfully inhibited formation of collagen and expression of pro-fibrotic markers in various cellular systems, systemic sclerosis patient-derived skin organoid model, and its mouse xenograft model. Therefore, DWN12088, a novel PRS inhibitor, may serve as a potential therapeutic agent in systemic sclerosis and other fibrotic diseases.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/inhibition-of-prolyl-trna-synthetase-as-a-novel-therapeutic-target-for-systemic-sclerosis/