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

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Abstract

Kidney stone diseases are increasing globally in prevalence and recurrence rates, indicating an urgent medical need for develo** 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).

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Abbreviations

L-CDME:

L-cystine dimethyl ester

CDNMP:

L-cystine bis(N′-methylpiperazide) (LH708)

DTT:

dithiothreitol

EC2x :

the inhibitor concentration required to double the apparent aqueous solubility

EC50 :

the inhibitor concentration required to achieve 50% of the maximal apparent aqueous solubility

ESWL:

extracorporeal shockwave lithotripsy

HPLC:

high performance liquid chromatography

ISE:

ion-selective electrodes

NBC:

N-Boc-L-cysteine

OPA:

o-phthaldialdehyde

PBS:

phosphate buffered saline

Slc3a1 :

solute carrier family 3 member 1

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Acknowledgements

LH conceived of the idea for the development of the crystallization inhibition assay and the application of the colorimetric and fluorometric methods for measuring L-cystine concentration. YY developed and optimized the fluorometric assay. HA applied the colorimetric assay in evaluating inhibitor potency. JY and YW were responsible for further optimization of the assay protocol to ensure assay reproducibility. HA, YY, JY, and YW performed the statistical analyses. JY wrote the initial manuscript draft. JY and LH worked on revising subsequent manuscript drafts. All authors critically reviewed and approved the final manuscript draft. This work was supported by grant R01DK112782 from the National Institutes of Health. The assay protocol has been successfully applied in the following original articles: Hu et al. 2016 [9] and Yang et al. 2018 [10].

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Authors and Affiliations

  1. Department of Medicinal Chemistry, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ, 08854, USA

    Jeffrey Yang, Haifa Albanyan, Yiling Wang, Yanhui Yang & Longqin Hu

  2. Department of Genetics, Rutgers, The State University of New Jersey, Piscataway, NJ, 08854, USA

    Amrik Sahota

  3. The Cancer Institute of New Jersey, New Brunswick, NJ, 08901, USA

    Longqin Hu

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  1. Jeffrey Yang
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Correspondence to Longqin Hu.

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Some authors are inventors of patents on compounds discussed or discovered using the assay described in this paper.

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Yang, J., Albanyan, H., Wang, Y. et al. Development of convenient crystallization inhibition assays for structure-activity relationship studies in the discovery of crystallization inhibitors. Med Chem Res 32, 1391–1399 (2023). https://doi.org/10.1007/s00044-023-03061-7

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