Restoring Ocrl1 function in Lowe Syndrome and Dent-2 disease

洛威综合征(LS)是一种由OCRL1基因突变引起的疾病，不幸的是，这种突变会导致 受影响的儿童过早死亡，无法治愈。然而，这个项目旨在改变这种情况。此外， 由于OCRL1突变也会导致一种被称为Dent-2(D2)病的相关肾脏疾病，这项提议将 也使D2患者受益。 LS患者表现为智力低下、眼睛(如青光眼、白内障)和肾脏(如肾结石、 低分子蛋白尿)异常，而D2患者几乎只表现出肾脏症状。尽管大多数 在患者中发现的OCRL1错义突变改变了编码蛋白OcrL1的磷酸酶结构域，大约 这些变化中有一半不影响底物结合或修饰所涉及的残基。事实上，我们的 结果表明，相当多的患者表达完整结合/催化的Ocrl1突变蛋白 序列但被锁定在一种构象中，不能处理脂质底物。因此，我们假设一个 Ocr11患者突变蛋白亚群可通过能够稳定的药物作用重新获得功能 具有酶活性的构象(变构激活剂)。事实上，由于一系列小分子 在我们实验室进行的筛查中，我们鉴定出一组化合物(包括FDA批准的药物)能够 恢复不同Ocr11患者突变体的催化活性并抑制读出的LS/D2细胞表型。 为了验证我们的假设，4个变构激活剂候选者将与一组LS和D2 Ocrl1一起使用 患者为了追求以下专注于肾功能的特定目标而进行突变： 目的1.确定选定的候选药物对小组生物化学活性的体外影响 Ocrl1例LS/D2患者发生突变。 目的2.确定选定候选药物对肾脏中观察到的多种LS表型的影响 细胞系、肾脏器官和携带LS/D2患者OCRL1突变的斑马鱼动物模型。 目的3.确定选定的候选药物对Ocrl1稳定性和结构的影响 LS/D2突变突变体与底物结合或不结合。 这个项目具有创新性，因为它引入了LS和D2的概念，它们是具有 一些患者表现出构象/错误折叠的疾病成分，并提出了一种新的治疗方法 使用变构激活剂的方法。重要的是，这个项目具有很高的意义，因为它将解决缺乏 旨在抑制一种影响数万人的疾病的上游病因的治疗方法 美国和世界各地的儿童。此外，FDA批准的候选药物目前用于改善其他 条件可以很容易地改变为LS/D2。因此，这个项目的翻译影响非常高。

Lowe Syndrome (LS) is a disease caused by mutations in the OCRL1 gene that unfortunately leads to the early death of affected children and has no cure. However, this project aims to change such scenario. Further, since OCRL1 mutations also cause a related renal condition known as Dent-2 (D2) disease, this proposal will also benefit D2 patients. LS patients display mental retardation, ocular (e.g., glaucoma, cataracts) and renal (e.g., kidney stones, LMW proteinuria) abnormalities, while D2 patients show renal symptoms almost exclusively. Although most OCRL1 missense mutations found in patients alter the phosphatase domain of the encoded protein Ocrl1, about half of these changes do not affect residues involved in binding or modification of the substrate. In fact, our results indicate that a substantial number of patients express Ocrl1 mutated proteins with intact binding/catalytic sequences but locked in a conformation unable to process lipid substrates. Therefore, we HYPOTHESIZE that a subset of Ocrl1 patient mutated proteins can re-acquire functionality by action of drugs able to stabilize the enzymatically active conformer (allosteric activators). Indeed, as a result of a series of small molecule screens performed in our lab, we identified a group of compounds (including FDA-approved drugs) as able to restore catalytic activity of different Ocrl1 patient mutants and to suppress a readout LS/D2 cellular phenotype. To test our hypothesis, 4 allosteric activator candidates will be used along with a panel of LS and D2 Ocrl1 patient mutated variants to pursue the following specific aims focused on kidney function: AIM 1. To determine the in vitro effect of selected candidate drugs ON THE BIOCHEMICAL ACTIVITY of a panel of Ocrl1 LS/D2 patient mutated variants. AIM 2. To determine the effect of selected candidate drugs ON MULTIPLE LS PHENOTYPES observed in kidney cell lines, kidney organoids and a zebrafish animal model bearing LS/D2 patient OCRL1 mutations. AIM 3. To determine the effect of selected candidate drugs ON THE STABILITY AND STRUCTURE of the Ocrl1 LS/D2 mutated variants bound or not to substrate. This project is INNOVATIVE because it introduces the concept of LS and D2 as heterogenous conditions with some patients displaying a conformational/misfolding disease component and proposes a novel therapeutic approach using allosteric activators. Importantly, this project has high SIGNIFICANCE as it will address the lack of therapeutic approaches designed to suppress the upstream cause of a disease that affects tens of thousands of children in the US and worldwide. Further, FDA-approved candidate drugs currently used to ameliorate other conditions can be readily repurposed to LS/D2. Therefore, the translational IMPACT of this project is very high.