Preclinical development of CFTR stabilizers targeting the CAL PDZ domain

描述（由申请人提供）：囊性纤维化跨膜传导调节因子（CFTR）是一种在囊性纤维化（CF）患者中突变的离子通道，破坏多种上皮组织中的液体和离子平衡。在肺中，粘膜纤毛清除失败 促进耐药细菌生物膜的建立，尽管先进的抗生素和 肺清除策略。因此，慢性肺部感染和炎症目前是CF发病率和死亡率的主要原因，将寿命限制在40岁以下。约90%的CF患者携带一个或两个拷贝的<$F508等位基因，其编码的蛋白质折叠效率低，显示出有限的通道活性，并迅速降解。已经鉴定出解决折叠和通道缺陷的化合物。两者都不能作为单一疗法提供显著的益处，但在25%的<$F508纯合子患者中，它们联合使用可显著改善肺功能（<$FEV1>10%）。为了接触更多的患者并增加功能反应，我们提出了一种新的翻译策略的早期药理学验证，以解决剩余的缺陷-拯救的F508-CFTR的降解。没有临床试验包括专门设计用于增加顶膜处CFTR稳定性的化合物。在鉴定了CFTR相关配体（CAL）作为CFTR降解的关键介质后，我们定位了一个关键的结合界面，设计了阻断它的肽，并表明它们在极化CF支气管上皮细胞中作为功能性<$F508-CFTR的一流“稳定剂”。与目前临床试验中的联合治疗相比，我们的CAL通道（iCAL）方法的临床前进展目前受到铅亲和力、递送和关于额外救援程度的有限数据的限制。在此，我们建议利用所有三个领域的初步进展。我们的新数据证实了与VX-809一致的细胞渗透性iCAL的显著加和性。通过一个经过验证的目标，多种铅化学，一套强大的功能测定，以及结构和生化专业知识，我们提出了一个综合的结构-活性方法来优化CAL抑制剂，并确认其治疗潜力。在目标1中，使用现有的肽抑制剂与最先进的离体CF患者肠电流测量和体外粘膜纤毛转运测定，我们将建立一个功能管道来评估CAL抑制剂与批准的增效剂和校正剂分子的组合或在后期临床试验中。在目标2中，我们提出验证和优化用于全身或吸入疗法的iCAL肽的生物活性。在目标3中，建立在一套生化测定的基础上，我们描述了基于结构的策略来提高小分子抑制剂的亲和力和选择性。这些研究将共同确定控制CFTR救援的阈值参数。在联手为CFTR稳定剂提供概念验证后，我们的跨学科和紧密协调的合作能够很好地获得具有证明疗效和生物耐受性的第二代CAL抑制剂，同时为这种新型治疗靶点的进一步药理学开发引人注目的产品组合。

DESCRIPTION (provided by applicant): The cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel that is mutated in patients with cystic fibrosis (CF), disruptin fluid and ion balance in multiple epithelial tissues. In the lung, failure of mucociliary clearance facilitates the establishment of drug-resistant bacterial biofilms, despite advanced antibiotic and pulmonary clearance strategies. As a result, chronic lung infection and inflammation are currently the major causes of CF morbidity and mortality, limiting lifespan to <40 years. ~90% of CF patients carry one or two copies of the ¿F508 allele, which encodes a protein that is inefficiently folded, shows limited channel activity, and is rapidly degraded. Compounds have been identified that address the folding and channel defects. Neither provides significant benefit as a monotherapy, but in combination they produce significant improvement in lung function (¿FEV1 >10%) in 25% of ¿F508 homozygous patients. To reach more patients and increase the functional response, we propose the early-stage pharmacological validation of a novel translational strategy to address the remaining defect - the degradation of rescued ¿F508-CFTR. No clinical trials include compounds specifically designed to increase CFTR stability at the apical membrane. Having identified the CFTR-Associated Ligand (CAL) as a key mediator of CFTR degradation, we have localized a critical binding interface, designed peptides that block it, and shown that they act as first-in-class 'stabilizers' of functional ¿F508-CFTR in polarized CF bronchial epithelial cells. Preclinical advancement of our inhibitor-of- CAL (iCAL) approach is currently limited by lead affinity, delivery, and limited data on the extent of additioal rescue compared to combination therapies currently in clinical trials. Here, we propose to leverage preliminary advances in all three areas. Our new data confirm substantial additivity for a cell-permeable iCAL in concert with VX-809. With a validated target, multiple lead chemistries, a strong suite of functional assays, and structural and biochemical expertise, we propose an integrated structure-activity approach to optimize CAL inhibitors and confirm their therapeutic potential. In Aim 1, using existing peptide inhibitors with state-of-the-art ex vivo CF patient intestinal current measurement and in vitro mucociliary transport assays, we will establish a functional pipeline to evaluate combinations of CAL inhibitors with potentiator and corrector molecules approved or in late-stage clinical trials. In Aim 2, we propose to validate and optimize the bioactivity of iCAL peptides for systemic or inhaled therapies. In Aim 3, building on a suite o biochemical assays, we describe structure-based strategies to improve the affinity and selectivity of small-molecule inhibitors. Together these studies will determine threshold parameters governing CFTR rescue. Having joined forces to produce proof of concept for CFTR stabilizers, our interdisciplinary and tightly coordinated collaboration is well positioned to obtan second-generation CAL inhibitors with demonstrated efficacy and biological tolerability, while developing a compelling portfolio for the further pharmacological development of this novel therapeutic target.