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

Bestrophin 1（BEST 1）是一种钙激活的氯离子通道，由5个同聚体BEST 1亚基组成。BEST 1基因的突变与视网膜营养不良的临床谱相关，其中包括Best卵黄状黄斑营养不良（BVMD）。迄今为止，BVMD或任何BEST 1相关疾病都没有治疗选择，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物质属于亚类的真菌，即鹰嘴豆素A和Mundulone，它们有能力恢复突变受损的BEST 1介导的阴离子电导在体外。为了在临床前环境中进一步开发这些活性物质，下一步是通过产生鹰嘴豆芽素A和蒙杜龙的衍生物来初步扩大活性物质的库，然后在细胞培养物（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，我们已经委托生产了总共41种鹰嘴豆素A和芒杜龙衍生物，其特征在于，例如，用（羧基）酯取代单个乙酸酯基团，用磷酸盐、氨基甲酸盐或碳酸盐残基取代其他官能团。本申请现在旨在实现三个具体目标，其包括（1）在MDCKII细胞中使用称为YFP卤化物转运测定的功能测试来分析新合成的化合物。有前途的候选人将通过患者来源的视网膜色素上皮（RPE）细胞的膜片钳恢复氯电导率进行验证;（2）分析吸收、分布、代谢、消除，最后（3）在纯合Best 1（Y227 N）敲除中测试最有希望的化合物。以证明在体内恢复Best 1功能的可行性。该项目的总体目标是重点确定活性铅化合物从子类的砷，然后将进行广泛的和成本密集型的优化。

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.