Key Laboratory of Preclinical Study for New Drugs of Gansu Province, School of Basic Medical Sciences & Research Unit of Peptide Science, Chinese Academy of Medical Science, 2019RU066 (L.C., D.W., B.D., J.L., J.Z., X.G., T.Y., X.Y., Y.A., B.Z., J.X.) and School of Life Sciences (L.C., D.W.), Lanzhou University, Lanzhou, China.
Key Laboratory of Preclinical Study for New Drugs of Gansu Province, School of Basic Medical Sciences & Research Unit of Peptide Science, Chinese Academy of Medical Science, 2019RU066 (L.C., D.W., B.D., J.L., J.Z., X.G., T.Y., X.Y., Y.A., B.Z., J.X.) and School of Life Sciences (L.C., D.W.), Lanzhou University, Lanzhou, China
J Pharmacol Exp Ther. 2022 Aug;382(2):100-112. doi: 10.1124/jpet.121.001031. Epub 2022 Jun 30.
Pulmonary fibrosis (PF), which is characterized by enhanced extracellular matrix (ECM) deposition, is an interstitial lung disease that lacks an ideal clinical treatment strategy. It has an extremely poor prognosis, with an average survival of 3-5 years after diagnosis. Our previous studies have shown that the antioxidant peptide DR8 (DHNNPQIR-NH), which is extracted and purified from rapeseed, can alleviate PF and renal fibrosis. However, natural peptides are easily degraded by proteases , which limits their potency. We have since synthesized a series of DR8 analogs based on amino acid scanning substitution. DR7dA [DHNNPQ (D-alanine) R-NH] is an analog of DR8 in which L-isoleucine (L-Ile) is replaced with D-alanine (D-Ala), and its half-life is better than that of DR8. In the current study, we verified that DR7dA ameliorated tumor growth factor (TGF)-β1-induced fibrogenesis and bleomycin-induced PF. The results indicated that DR7dA reduced the protein and mRNA levels of TGF-β1 target genes in TGF-β1-induced models. Surprisingly, DR7dA blocked fibrosis in a lower concentration range than DR8 in cells. In addition, DR7dA ameliorated tissue pathologic changes and ECM accumulation in mice. BLM caused severe oxidative damage, but administration of DR7dA reduced oxidative stress and restored antioxidant defense. Mechanistic studies suggested that DR7dA inhibits ERK, P38, and JNK phosphorylation and All results indicated that DR7dA attenuated PF by inhibiting ECM deposition and oxidative stress via blockade of the mitogen-activated protein kinase (MAPK) pathway. Hence, compared with its parent peptide, DR7dA has higher druggability and could be a candidate compound for PF treatment in the future. SIGNIFICANCE STATEMENT: In order to improve druggability of DR8, we investigated the structure-activity relationship of it and replaced the L-isoleucine with D-alanine. We found that the stability and antifibrotic activity of DR7dA were significantly improved than DR8, as well as DR7dA significantly attenuated tumor growth factor (TGF)-β1-induced fibrogenesis and ameliorated bleomycin-induced fibrosis by inhibiting extracellular matrix deposition and oxidative stress via blockade of the MAPK pathway, suggesting DR7dA may be a promising candidate compound for the treatment of PF.
肺纤维化（PF）的特征是细胞外基质（ECM）沉积增加，是一种间质性肺病，缺乏理想的临床治疗策略。它的预后极差，诊断后平均存活 3-5 年。我们之前的研究表明，从油菜籽中提取和纯化的抗氧化肽 DR8（DHNNPQIR-NH）可以缓解 PF 和肾纤维化。然而，天然肽很容易被蛋白酶降解，这限制了它们的效力。此后，我们根据氨基酸扫描取代合成了一系列 DR8 类似物。DR7dA[DHNNPQ（D-丙氨酸）R-NH]是 DR8 的类似物，其中 L-异亮氨酸（L-Ile）被 D-丙氨酸（D-Ala）取代，其半衰期优于 DR8。在本研究中，我们验证了 DR7dA 可改善转化生长因子（TGF）-β1 诱导的纤维化和博来霉素诱导的 PF。结果表明，DR7dA 降低了 TGF-β1 诱导模型中 TGF-β1 靶基因的蛋白和 mRNA 水平。令人惊讶的是，DR7dA 在细胞中的浓度范围比 DR8 更低，从而阻断了纤维化。此外，DR7dA 改善了小鼠的组织病理变化和 ECM 积累。BLM 导致严重的氧化损伤，但给予 DR7dA 可减轻氧化应激并恢复抗氧化防御。机制研究表明，DR7dA 抑制 ERK、P38 和 JNK 磷酸化。所有结果表明，DR7dA 通过抑制细胞外基质沉积和氧化应激来减轻 PF，从而阻断丝裂原活化蛋白激酶（MAPK）途径。因此，与母体肽相比，DR7dA 具有更高的成药性，可能成为未来 PF 治疗的候选化合物。
为了提高 DR8 的成药性，我们研究了它的结构-活性关系并用 D-丙氨酸取代了 L-异亮氨酸。我们发现，DR7dA 的稳定性和抗纤维化活性明显优于 DR8，DR7dA 还通过阻断 MAPK 途径抑制细胞外基质沉积和氧化应激，显著减轻 TGF-β1 诱导的纤维化和博来霉素诱导的纤维化，这表明 DR7dA 可能是治疗 PF 的一种有前途的候选化合物。
