Tackling the MARCKS-PIP3 Circuit to Attenuate Chronic Pulmonary Fibrosis

项目摘要 肺纤维化是正常肺损伤修复过程中的重要环节。然而，不受控制的损伤和修复， 肺实质胶原过度沉积是慢性肺纤维化的病理标志 纤维化，例如特发性肺纤维化（IPF）。这种疾病的平均生存时间只有3到5年 从诊断之日起的几年内。目前，除了两种药物外，没有合适的药物用于治疗： FDA批准的Nepal和吡非尼酮。然而，不良和脱靶效应，以及未能 治疗患者寿命延长表明迫切需要新的和更好的治疗方法， 治疗这种毁灭性疾病的药物。上皮-间充质（EM）和成纤维细胞-肌成纤维细胞（FM） 这些转变与肺纤维化发病机制的发生和发展有关。EM和 FM转换现象是与癌症恶性相关的重要致病事件， 主要由受体介导的酪氨酸激酶（RTK）和PI 3 K-AKT信号通路介导。我们已经表明 在与EM转变相关的肺癌组织/细胞中磷酸-MARCKS升高之前， 肽抑制剂MPS（MARCKS PSD/艾德序列）抑制EM转换和肺癌 通过解决与癌症发病机制相关的异常MARCKS-PIP 3回路来治疗恶性肿瘤。的 在组织切片中也观察到升高的磷酸-MARCKS现象，并且分离的成纤维细胞来源于 IPF肺，但在任何正常、非纤维化肺中未见。我们最近发表的文章显示， MPS肽在博来霉素诱导的纤维化小鼠肺中抑制纤维化病变的潜力。在 在体外，MPS处理异常MARCKS-PIP 3回路以抑制MARCKS磷酸化，并且还选择性地 抑制EM/FM转换和肌成纤维细胞纤维化，以及M1/M2巨噬细胞的改变 极化选择性只发生在IPF衍生的成纤维细胞和活化的巨噬细胞上，而不发生在 正常和失活的单核细胞。通过肽优化，我们进一步开发了一种稳定的， 生物安全性和高效力的新型MPS衍生肽MPS-6413 DTM。初步研究表明， 这种肽的抑制博莱霉素诱导的肺纤维化病变和死亡的小鼠，但没有 对照组我们假设MPS-6413 D是一种有效的抗肺纤维化药物，可抑制纤维化。 通过处理MARCKS-PIP 3回路来控制慢性肺纤维化的进展。为了验证这一假设， 这种肽的治疗潜力，提出了两个目标。目的1是进一步确定治疗效果 MPS-6413 D肽对32周龄（相当于42岁）博莱霉素诱导的肺纤维化病变的作用 老年人）小鼠。治疗效力将根据对促纤维化蛋白表达的抑制来确定。 标记蛋白及其RNA，以及MARCKS的抑制及其在总肺匀浆中的磷酸化， 基质沉积，肺功能，以及这些年龄小鼠的总体存活率和健康状况， 博莱霉素暴露。目的2是评价MPS-6413 D肽对IPF纤维化活性的抑制作用 人源化小鼠模型中的肺成纤维细胞。为了更好地反映临床情况，IPF人肺成纤维细胞 将多克隆抗体（HLF）过继转移至C.B-17 SCID/bg小鼠，以测试MPS在小鼠中的治疗潜力。 肺纤维化这些研究的成功将导致提交针对动物的II期SBIR研究， 自发性肺纤维化，药效学/药代动力学分析，向FDA提交的基于IND的研究， 未来的临床药物开发，以减轻慢性肺纤维化。

Project Summary Lung fibrosis is an important step of normal lung injury-repair process. However, uncontrolled injury and repair, and excessive deposition of collagen in the lung parenchyma is the pathological hallmark of chronic pulmonary fibrosis, such as idiopathic pulmonary fibrosis (IPF). The disease exhibits a median survival time of only 3 to 5 years from the time of diagnosis. Currently, there is no suitable drug for the treatment, except two drugs: Nintedanib and Pirfenidone, approved by FDA. However, adverse and off-target effects, and failure to demonstrate increased longevity in treated patients indicate the urgent need for new and better therapeutic agent(s) to treat this devastating disease. Epithelial-mesenchymal (EM) and fibroblast-myofibroblast (FM) transitions have been implicated in the initiation and the progression of fibrotic lung pathogenesis. The EM and FM transition phenomena, important pathogenic events associated with cancer malignancy, are primordially and mainly mediated by receptor-mediated tyrosine kinase (RTK) and PI3K-AKT signaling pathways. We have shown before the elevation of phospho-MARCKS in lung cancer tissues/cells associated with EM transition and the use of a peptide inhibitor, MPS (MARCKS PSD/ED Sequence), to suppress EM transition and lung cancer malignancy through tackling the aberrant MARCKS-PIP3 circuit associated with cancer pathogenesis. The elevated phospho-MARCKS phenomenon is also seen in tissue sections and isolated fibroblasts derived from IPF lungs, but not seen in any normal, non-fiberotic ones. Our recent publication had shown the therapeutic potential of MPS peptide in the suppression of the fibrotic lesions in bleomycin-induced fibrotic mouse lungs. In vitro, MPS tackles the aberrant MARCKS-PIP3 circuit to suppress MARCKS phosphorylation and also selectively inhibits the EM/FM transition and myofibroblast fibrogenesis, as well as the alteration of M1/M2 macrophage polarization. The selectivity occurs only on IPF-derived fibroblasts and activated macrophage, but not on the normal and inactivated monocytes. Through peptide optimization, we have developed further a stable, more biosafe, and high potency of a novel MPS-derived peptide, MPS-6413DTM. Initial studies have shown the efficacy of this peptide on the suppression of bleomycin-induced lung fibrotic lesions and deceased in mice, but not on the control ones. We hypothesize that MPS-6413D is a potent anti-fibrotic lung drug on the inhibition of fibrogenic progression of chronic lung fibrosis through tackling the MARCKS-PIP3 circuit. To test this hypothesis and the therapeutic potential of this peptide, two aims are proposed. Aim 1 is to determine further the therapeutic effects of MPS-6413D peptide on bleomycin induced lung fibrotic lesions in aged 32-week old (equivalent to 42 years old human) mice. The therapeutic potency will be determined on the inhibition of the expression of profibrogenic marker proteins and their RNA, and the inhibition of MARCKS and its phosphorylation in total lung homogenates, matrix deposition, pulmonary function, and also the overall survival and well-being in these age mice after bleomycin exposure. Aim 2 is to evaluate an inhibitory effect of MPS-6413D peptide on fibrogenic activity of IPF lung fibroblasts in humanized mouse model. To better reflect clinical scenarios, IPF human lung fibroblasts (HLFs) will be adoptively transferred to C.B-17 SCID/bg mice in order to test the therapeutic potential of MPS in lung fibrosis. Success of these studies will lead to the submission of the Phase II SBIR study on animals with spontaneous lung fibrosis, the pharmacodynamic/pharmacokinetic analysis, an IND-based study to FDA for future clinical drug development to attenuate chronic pulmonary fibrosis.