Development of convenient crystallization inhibition assays for structure-activity relationship studies in the discovery of crystallization inhibitors.
Development of convenient crystallization inhibition assays for structure-activity relationship studies in the discovery of crystallization inhibitors.
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开发方便的结晶抑制测定法，用于结晶抑制剂发现中的结构-活性关系研究。
DOI：
10.1007/s00044-023-03061-7
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Hu,Longqin
中科院分区：
文献类型：
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作者：
Yang,Jeffrey;Albanyan,Haifa;Wang,Yiling;Yang,Yanhui;Sahota,Amrik;Hu,Longqin
肾结石疾病在全球范围内的患病率和复发率正在增加，这表明迫切需要开发可以预防结石形成的新疗法。我们一直在研究的一种方法是开发小分子抑制剂，可以干扰形成结石的化学物质的结晶过程。对于这些药物发现的努力，这是至关重要的，有可用的容易获得的测定方法，以评估潜在的抑制剂，并将它们的结构-活性关系的研究。在这里，我们报告了一个方便的，中到高通量的检测平台，作为一个例子，使用的L-胱氨酸结晶抑制剂的筛选和评价，以预防肾结石的胱氨酸尿症。该测定包括制备过饱和溶液，然后将小体积（<1 mL）的过饱和溶液与试验抑制剂孵育72小时，最后使用比色法或荧光法测量离心后上清液中的L-胱氨酸浓度。与传统的研究结晶抑制剂的技术相比，这种小型化的多孔测定格式易于实施，具有成本效益，并且广泛适用于测定和区分抑制结晶的化合物的活性。该测定法已成功用于测定L-胱氨酸二酰胺作为L-胱氨酸结晶的高效抑制剂，如LH 708，其EC 50为0.058 µM，比L-CDME（EC 50 = 4.31 µM）强70倍。
Kidney stone diseases are increasing globally in prevalence and recurrence rates, indicating an urgent medical need for developing new therapies that can prevent stone formation. One approach we have been working on is to develop small molecule inhibitors that can interfere with the crystallization process of the chemical substances that form the stones. For these drug discovery efforts, it is critical to have available easily accessible assay methods to evaluate the potential inhibitors and rank them for structure-activity relationship studies. Herein, we report a convenient, medium-to-high throughput assay platform using, as an example, the screening and evaluation of inhibitors ofL-cystine crystallization for the prevention of kidney stones in cystinuria. The assay involves preparing a supersaturated solution, followed by incubating small volumes (<1 mL) of the supersaturated solution with test inhibitors for 72 hours, and finally measuringL-cystine concentrations in the supernatants after centrifugation using either a colorimetric or fluorometric method. Compared to traditional techniques for studying crystallization inhibitors, this miniaturized multi-well assay format is simple to implement, cost-effective, and widely applicable in determining and distinguishing the activities of compounds that inhibit crystallization. This assay has been successfully employed to discoverL-cystine diamides as highly potent inhibitors ofL-cystine crystallization such as LH708 with an EC50of 0.058 µM, 70-fold more potent thanL-CDME (EC50= 4.31 µM).Graphical Abstract