Developing new active compounds to correct mutant BEST1 chloride conductance – Establishing a targeted therapy for Best vitelliform macular dystrophy

besstrophin 1 （BEST1）形成一个钙激活的氯离子通道，由五个同质的BEST1亚基组成。BEST1基因突变与视网膜营养不良症（Best vitelliform macular dystrophy， BVMD）的临床谱相关。迄今为止，BVMD或任何与best1相关的疾病没有治疗选择，因此对有效治疗的医疗需求很高。在我们最近的工作中，我们已经确定了两种属于异黄酮亚类的物质，即生物茶素A和蒙都隆，它们具有在体外恢复突变受损的best1介导的阴离子电导的能力。为了在临床前环境中进一步开发这些活性物质，下一步是通过生成生物油豆素a和蒙都龙的衍生物来最初扩大活性物质的范围，然后在细胞培养（体外）和动物模型（体内）中测试这些化合物的安全性和有效性。为此，我们委托生产了总共41种生物油豆素a和蒙都龙衍生物，其特征是，例如，用（羧基）酯取代单个醋酸酯基，用磷酸盐、氨基甲酸酯或碳酸盐残基取代其他官能团。目前的应用旨在实现三个特定目标，其中包括(1)在MDCKII细胞中使用称为YFP卤化物转运试验的功能测试来分析新合成的化合物。有希望的候选人将通过膜片夹紧患者源性视网膜色素上皮（RPE）细胞来恢复氯离子电导率；(2)在体外分析已验证候选物的吸收、分布、代谢、消除和毒性特性；最后(3)在纯合子Best1（Y227N）敲入小鼠中测试最有希望的化合物，以证明在体内恢复Best1功能的可行性。该项目的总体目标集中在从异黄酮亚类中鉴定活性先导化合物，然后进行广泛和成本密集的优化。

Bestrophin 1 (BEST1) forms a calcium-activated chloride channel composed of five homomeric BEST1 subunits. Mutations in the BEST1 gene are associated with a clinical spectrum of retinal dystrophies among others the Best vitelliform macular dystrophy (BVMD). To date, there is no treatment option for BVMD or any of the BEST1-linked conditions leaving a high medical need for an effective therapy. In our recent work, we have identified two substances belonging to the sub-class of isoflavonoids, namely Biochanin A and Mundulone, which have the ability to restore mutation-impaired BEST1-mediated anion conductance in vitro. To further develop these active substances in a preclinical setting, the next step is to initially expand the repertoire of active substances by generating derivatives of Biochanin A and Mundulone, followed by testing these compounds for safety and efficacy in cell cultures (in vitro) and animal models (in vivo). To this end, we have commissioned the manufacturing of a total of 41 Biochanin A and Mundulone derivatives, which are characterized, for example, by replacing individual acetate ester groups with (carboxy) ester and other functional groups with phosphate, carbamate, or carbonate residues. The current application now aims at achieving three specific objectives which include (1) analyzing the newly synthesized compounds using a functional test known as YFP halide transport assay in MDCKII cells. Promising candidates will be verified for restoring chloride conductivity via patch clamping of patient-derived retinal pigment epithelium (RPE) cells; (2) analysis of absorption, distribution, metabolism, elimination, and toxicity properties of the verified candidates in vitro and finally (3) testing of the most promising compounds in homozygous Best1(Y227N) knock-in mice to demonstrate feasibility of restoring Best1 function in vivo. The overall goal of the project is focused on identifying active lead compounds from the sub-class of isoflavonoids which will then undergo extensive and cost-intensive optimization.