Development of convenient crystallization inhibition assays for structure-activity relationship studies in the discovery of crystallization inhibitors.

Abstract

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 of L-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 measuring L-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 discover L-cystine diamides as highly potent inhibitors of L-cystine crystallization such as LH708 with an EC50 of 0.058 μM, 70-fold more potent than L-CDME (EC50 = 4.31 μM).