Restoring Ocrl1 function in Lowe Syndrome and Dent-2 disease

Lowe综合征（LS）是一种由OCRL1基因突变引起的疾病，不幸的是， 受影响的儿童过早死亡，无法治愈。然而，该项目旨在改变这种情况。此外，本发明还 由于OCRL1突变也会引起一种称为Dent-2（D2）病的相关肾脏疾病，因此该提案将 也有利于D2患者。 LS患者表现出精神发育迟滞、眼部（例如，青光眼，白内障）和肾（例如，肾结石， 低分子量蛋白尿）异常，而D2患者几乎只显示肾脏症状。虽然大多数 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 这些变化的一半不影响参与底物结合或修饰的残基。其实我们 结果表明，相当数量的患者表达具有完整结合/催化活性的Ocrl1突变蛋白， 序列，但锁定在不能处理脂质底物的构象。因此，我们假设， Ocrl1患者突变蛋白的子集可以通过能够稳定 具有酶活性的构象异构体（变构活化剂）。事实上，由于一系列小分子 在我们实验室进行的筛选中，我们确定了一组化合物（包括FDA批准的药物）， 恢复不同Ocrl1患者突变体的催化活性并抑制读出的LS/D2细胞表型。 为了检验我们的假设，将沿着使用4种变构激活剂候选物以及一组LS和D2 Ocrl 1 患者突变的变体，以追求以下专注于肾功能的特定目标： AIM 1.确定所选候选药物对一组生物化学活性的体外影响 Ocrl1 LS/D2患者突变变体。 AIM 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、肾类器官和携带LS/D2患者OCRL 1突变的斑马鱼动物模型。 AIM 3.确定所选候选药物对Ocrl 1稳定性和结构的影响 LS/D2突变变体结合或不结合底物。 这个项目是创新的，因为它引入了LS和D2的概念作为异质条件， 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， 方法使用变构激活剂。重要的是，该项目具有很高的意义，因为它将解决缺乏 治疗方法旨在抑制影响数万人的疾病的上游原因， 在美国和世界各地的孩子。此外，FDA批准的候选药物目前用于改善其他 条件可以很容易地重新用于LS/D2。因此，该项目的翻译影响力非常高。

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.