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. Author manuscript; available in PMC: 2024 Jul 1.
Published in final edited form as: Med Chem Res. 2023 Apr 22;32(7):1391–1399. doi: 10.1007/s00044-023-03061-7

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

Jeffrey Yang 1, Haifa Albanyan 1, Yiling Wang 1, Yanhui Yang 1, Amrik Sahota 2, Longqin Hu 1,3
PMCID: PMC10482073  NIHMSID: NIHMS1903585  PMID: 37681210

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).

Keywords: Urolithiasis, Kidney stone disease, Crystallization inhibition assay, Crystallization inhibitors, L-Cystine, Cystinuria

Graphical Abstract

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Introduction

Kidney stone diseases, also referred to as nephrolithiasis or urolithiasis, are characterized by the formation of crystal aggregates due to the accumulation of certain naturally occurring chemical substances such as oxalate, phosphate, uric acid, and amino acids to concentrations above their aqueous solubility in the urinary tract. The number of people afflicted by kidney stones globally has been steadily growing. In the United States, the prevalence of kidney stone diseases increased from 3.2% in 1980 to 11.0% in 2018 [1, 2]. More troubling is that recurrence is common. The recurrence rate ranges from 27.4% to 100% depending on the morphological type of the stone [3]. The likelihood of symptomatic recurrence also increases with each subsequent kidney stone episode [4]. The economic burden of urolithiasis is quite high, costing the United States at least $5.3 billion in 2000 [5]. Some potential complications of kidney stones include chronic kidney disease, end-stage renal failure, cardiovascular diseases, diabetes, and hypertension [6]. It is thus imperative to discover and develop new therapies to prevent stone formation and recurrence, especially considering that current preventative (e.g., fluid intake, diet modification) and therapeutic (e.g., surgery, extracorporeal shockwave lithotripsy or ESWL) measures have been unsuccessful in most cases.

Kidney stones are commonly composed of calcium oxalate, calcium phosphate, struvite or magnesium ammonium phosphate, uric acid, or cystine [6]. When these minerals, salts, and amino acids reach high levels of supersaturation in the urine, they begin to precipitate out and form nuclei that grow into crystals which eventually aggregate together to produce stones. One therapeutic strategy has been to prevent the crystallization process at supersaturation by developing small molecule inhibitors, acting as molecular “imposters”, that can bind to crystal surfaces and retard further crystal growth [7]. For instance, in the case of cystinuria, L-cystine crystallization inhibitors such as L-cystine dimethyl ester (L-CDME) and L-cystine diamides (e.g., LH708) (Fig. 1) reduced the growth velocity of L-cystine crystals as shown by atomic force microscopy studies and increased the apparent aqueous solubility of L-cystine [8–10]. Furthermore, in a Slc3a1 knockout mouse model of cystinuria, LH708 demonstrated effective inhibition of L-cystine stone formation [9], demonstrating the promise of such a therapeutic modality in treating urolithiasis.

Fig. 1.

Fig. 1

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Structures of two small molecule L-cystine crystallization inhibitors for cystinuria.

The behavior and inhibitory activity of crystal growth modifiers has commonly been studied using bulk crystallization. The bulk crystallization method involves the preparation of a metastable supersaturated solution of the stone-forming substance. Additives are then added into the supersaturated solution, often tens or hundreds of milliliters in volume to acquire adequate amounts of crystals for analysis. The mixture is then sealed and stored at room temperature for a period of time without agitation. Crystals that precipitate out during the incubation period can be collected by vacuum filtration, air dried, weighed, and analyzed by high-resolution microscopy (e.g., atomic force microscopy) to qualitatively and quantitatively determine how additives affect crystal morphology and growth [8]. Alternative methods exist that do not require such physical separation of crystals from solutions to quantify the effects of crystallization inhibitors. These methods can directly analyze for components in the incubation solutions and include, but are not limited to, ion depletion assays using ion-selective electrodes (ISE) [11–14] and turbidimetry [15–17]. Ion depletion assays utilize in situ ISE to measure the concentration of free ions (e.g., calcium or oxalate ions in calcium oxalate crystal studies) to determine the extent of crystallization—that is, high measured free calcium concentration would suggest little crystallization. Turbidimetric assays assess the turbidity or “cloudiness” of a solution by passing light through a sample and measuring the intensity of transmitted light, with low transmitted light intensity indicating a high degree of crystallization.

While these methods have been used to identify compounds with the ability to modify crystal growth, they have low throughput which makes it difficult to evaluate more than a few compounds at once. In addition, the large-scale bulk crystallization process can be time- and labor-intensive, heavily resource-draining, and expensive to perform when screening large compound libraries. We aimed to develop and report herein a miniaturized, medium-to-high-throughput crystallization inhibition assay to support early drug discovery and medicinal chemistry efforts. Our assay was originally designed for inhibitors that suppress L-cystine crystallization, but it could be easily translated to inhibitors of other kidney stones such as calcium oxalate, calcium phosphate, and uric acid. Additionally, a modified assay format could be implemented, expanding the utility of this assay to crystallization inhibitors against nonurinary diseases—as examples, hematin crystals in malaria [18, 19] and cholesterol crystals in atherosclerosis [20]. In this paper, we use our L-cystine crystallization inhibition assay as a model to illustrate how this convenient assay platform was used to evaluate and rank the potency of L-cystine crystallization inhibitors.

Assay Principle and Design

Cystinuria is a genetic disorder characterized by defective renal transport and high urinary excretion of L-cystine and dibasic amino acids ornithine, L-lysine, and L-arginine [21]. Unlike the dibasic amino acids, L-cystine has relatively low solubility in urine. L-Cystine stone formation is the result of the accumulation of L-cystine to supersaturated concentrations in the urine retained within the kidneys [6, 22]. At supersaturation, the free L-cystine molecules begin to form microscopic clusters, leading to the formation of nuclei. These nuclei serve as the foundation on which L-cystine crystals stick together in the urine to facilitate crystal growth and the formation of L-cystine stones. The premise behind the L-cystine crystallization inhibition assay is to simulate the supersaturated L-cystine state by generating a metastable supersaturated aqueous solution of L-cystine [8]. The supersaturated solution of L-cystine is then incubated in the presence of varying concentrations of test inhibitors for 72 hours at a controlled temperature of 20 °C. During the 72-hour incubation period, in the absence of inhibitors, L-cystine slowly crystallizes out, eventually reaching an equilibrium concentration reported to be 0.7 mM at 25 °C and pH 7 [23, 24]. In the incubation mixtures containing crystallization inhibitor, the inhibitors bind to the surface of the nuclei that begin to form in solution and sterically block additional L-cystine molecules from adhering to the surface, thereby increasing the apparent L-cystine aqueous solubility [8–10]. In our assay, the apparent aqueous solubility of L-cystine is represented by the concentration of L-cystine in solution measured after a simple and quick centrifugation to remove crystals formed during incubation. It is expected that the concentration of free or uncrystallized L-cystine molecules present in the incubation mixtures containing the inhibitors will be higher than that in the untreated control (i.e., no inhibitor, water only). With increasing concentrations of the crystallization inhibitor, there would be a corresponding increase in the solubility of L-cystine up to the maximum L-cystine concentration in the initially prepared supersaturated solution (i.e., 2.9 mM based on 70 mg L-cystine dissolved in 100 mL water). A sigmoidal relationship is observed between the concentration of the crystallization inhibitor and L-cystine solubility, enabling calculation of either an EC2x (i.e., the inhibitor concentration required to double the apparent aqueous solubility of L-cystine) or EC50 (i.e., the inhibitor concentration required to achieve 50% of the maximal apparent aqueous solubility of L-cystine) value. These values are compared and used to guide subsequent drug design decisions. L-CDME and LH708 have been confirmed to reduce the growth rate of L-cystine crystals by specifically binding to the crystal surface using real-time in situ atomic force microscopy and are appropriate positive controls for the assay [9, 8].

To measure the L-cystine concentrations in the supernatant after centrifugation, either of the following two detection methods that are highly specific for L-cystine can be used: the well-established cyanide-nitroprusside colorimetric assay [25–27] or a fluorometric assay employing OPA/NBC derivatization [9, 10, 28]. Briefly, the cyanide-nitroprusside colorimetric assay has been commonly used in laboratory screening for cystinuria as it is able to quickly detect for the presence of L-cystine in urinary samples. Cyanide converts L-cystine to L-cysteine, which in turn reacts with sodium nitroprusside to produce a red-colored complex in solution. To convert the qualitative results to a quantitative measurement of L-cystine, the absorbance of this solution can be measured at 530 nm [26]. The L-cystine concentrations are calculated from a standard curve of absorbance at 530 nm vs. concentration of L-cystine. The fluorometric assay requires L-cystine to be first reduced using dithiothreitol (DTT) to L-cysteine, which is subsequently alkylated by iodoacetic acid at the thiol group. Finally, o-phthaldialdehyde (OPA)/N-Boc-L-cysteine (NBC) derivatization is performed to obtain a fluorescent compound. This fluorescent derivative emits fluorescence at 460 nm when excited with 355 nm light. The measured fluorescence emission at 460 nm is then converted to L-cystine concentration using a standard curve of fluorescence emission vs. L-cystine concentration. Nonlinear regression of L-cystine concentration measured in the supernatant with either method vs. inhibitor concentration provides a measure of inhibitor potency, expressed as either an EC2x or EC50 value [9, 10].

Results and Discussion

Representative examples of the standard curves using either the colorimetric or fluorometric method and the dose-response curves for L-CDME and LH708 from the L-cystine crystallization inhibition assay are illustrated in Fig. 2 and Fig. 3, respectively. LH708 exhibited an EC50 value of 0.058±0.002 μM, demonstrating more than 70-fold greater potency in inhibiting L-cystine crystallization when compared to L-CDME (EC50 = 4.31±0.14 μM). Compared to our previously reported potencies [9], LH708 displayed a higher potency (EC50 = 0.058 vs 0.26 μM), while L-CDME remained relatively unchanged (EC50 = 4.31 vs 6.37 μM). This discrepancy was resolved after optimization of the assay conditions. The critical step leading to the enhancement in potency of LH708 and ensuring assay reproducibility was to not vortex or vigorously mix the incubation mixture after transferring aliquots of the prepared supersaturated L-cystine solution into the Eppendorf tubes containing the inhibitors (Procedure Step C3). With vortexing, this disruption would cause premature L-cystine crystallization, resulting in lower L-cystine concentration measured in supernatants across all inhibitor concentrations. This would translate to a rightward shift in the dose-response curve of both L-CDME and LH708 (i.e., increased EC50 values). LH708 appeared to be more sensitive to this disturbance as compared to L-CDME. We reasoned that the potency of L-CDME remained relatively unchanged because it is a weak binder to the surface of the L-cystine crystals (binding energy = ‒316 kcal/mol [9]). Hence, any external disruptions would have minimal effect on its already poor binding. However, the binding of LH708 (binding energy = ‒429 kcal/mol [9])) could be more significantly affected as such disruption could hinder the necessary intermolecular hydrogen bond and S-S interactions with the L-cystine crystal surface, thereby reducing its potency.

Fig. 2.

Fig. 2

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Representative standard curves from the cyanide-nitroprusside assay (A) and fluorometric assay (B) for use in the determination of L-cystine concentration in test compound solutions

Fig. 3.

Fig. 3

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Representative dose-response curves for L-CDME and LH708 used for determination of inhibitor potency as EC50 values

The factors that influence the lower and upper limits of the dose-response curve were explored during assay optimization to improve assay reproducibility and sensitivity. Theoretically, the lower limit reflects the equilibrium L-cystine concentration achieved after 72-hour incubation (i.e., 0.7 mM), while the upper limit equals the L-cystine concentration in the prepared supersaturated solution (i.e., 2.9 mM). In our laboratory, the lower limit generally ranged between 0.7 mM and 1.1 mM, averaging 0.92±0.26 mM (N = 24), corresponding to the minimal inhibitory effect of the crystallization inhibitors at low concentrations and is close to the equilibrium concentration. Additional time for incubation could lower the measured concentration to the equilibrium concentration but may not be necessary. The dynamic range using current assay conditions was sufficient as long as errors at the lower and upper ends of the dose-response curve were not large. Improper sample centrifugation after incubation period (i.e., insufficient centrifugation speed and/or duration) and improper withdrawal of supernatant (i.e., accidental disturbance of L-cystine crystal pellet) could also lead to a higher-than-expected lower limit. The upper limit commonly fell between 2.7 mM and 2.9 mM, with an average of 2.81±0.14 mM (N = 24), reflecting the maximal inhibitory effect of the crystallization inhibitors at high concentrations. Previously published data had the upper limit well above 2.9 mM (i.e., ~3.5 mM) [9, 10]. This higher-than-expected concentration could reflect potential issues when preparing the L-cystine standard solutions such as not being careful in weighing out an accurate quantity of L-cystine and/or not using proper glassware or equipment to measure and deliver the appropriate volume of Millipore deionized water for the dissolution of L-cystine. For instance, weighing less than the desired amount of L-cystine or measuring out and delivering more Millipore deionized water would dilute the concentration of the initial standard solution (i.e., <0.4 mM), resulting in a downward shift of the standard curve. Consequently, the L-cystine concentration corresponding to the absorbance measured for a specific sample would be overestimated, leading to the higher-than-expected upper limit. Therefore, it is very important that standard solutions are prepared properly to make certain that assay results are reliable and reproducible.

Many factors could affect the upper limit of the dose-response curve (i.e., mostly a decrease in the upper limit) because of the intrinsic instability of the metastable supersaturated L-cystine solution. Some of these factors may include unclean glassware and apparatus used in the assay, improper refluxing of the supersaturated solution leading to lower-than-intended L-cystine dissolution, rapid rather than gradual cooling of the supersaturated solution after reflux, vigorous mixing during preparation of incubation mixtures, unintentional disturbances during the 72-hour incubation period, and improper preparation of the L-cystine standard solutions. The key to successful execution of this assay is to prepare and handle the supersaturated solution and the incubation mixtures delicately and carefully to avoid premature crystallization. It is imperative that investigators wishing to employ a similar assay format in their research have their laboratory personnel practice preparing and working with the supersaturated solution and carrying out the assay procedure multiple times until reproducible results are obtained. Slight variations in the lower limit and upper limit are expected when the assay is performed by different people and on different days. It is critical to standardize the assay protocol to ensure reproducible data. For comparison of the EC50 values for the inhibitors tested in our assay on different days, a ratio of the fold improvement in potency over the control L-CDME would provide a relative sense of the potency ranking of our inhibitors.

It should be noted that the assay conditions do not emulate the in vivo setting. For example, the incubation of the supersaturated L-cystine solutions with the inhibitors takes place at 20 °C rather than at the physiologic temperature of 37 °C. Running the assay at 20 °C ensures a higher dynamic range than running at 37 °C. The higher temperature of 37 °C increases the equilibrium L-cystine concentration, raising the lower limit of the dose-response curve. A higher dynamic range would provide more reliable EC50 values considering the existence of experimental errors at the lower and upper limits of the dose-response curve. Furthermore, maintaining the temperature at 20 °C would eliminate the need for an incubator as long as room temperature did not fluctuate significantly. The incubation medium used in our assay is also not representative of the composition of physiologic urine, which contains urea, creatinine, chloride, sodium, potassium, and other dissolved ions, inorganic and organic compounds (e.g., proteins, hormones, metabolites). These urinary substances can affect L-cystine solubility in the in vivo environment. However, the sole purpose of our assay was to determine and rank, in a fast and easy manner, the potencies of our small molecule crystallization inhibitors by measuring aqueous L-cystine solubility, which would provide some indication of the inhibition of crystallization in the absence of other substances in the urine. Follow-up confirmatory studies in vitro (e.g., atomic force microscopy studies) and in vivo (e.g., efficacy studies in a genetic mouse model of cystinuria) would certainly be necessary. Our lab has demonstrated that our crystallization inhibition assay can identify effective crystallization inhibitors [9, 10]. LH708 was discovered through the use of this assay platform as a highly potent nanomolar inhibitor of L-cystine crystallization capable of decreasing the proportion of mice with L-cystine stones relative to the control L-CDME (14% vs. 50%, respectively) in Slc3a1 knockout mice [9].

Overall, our crystallization inhibition assay is relatively straightforward to perform and designed to have the throughput for screening large numbers of test compounds. The assay was developed with the intent to simplify and miniaturize the bulk crystallization setup by: (1) performing the crystallization process at different inhibitor concentrations in 1.5-mL Eppendorf tubes at a volume of 0.5 mL, making it technically easier to handle multiple inhibitors at once; (2) removing precipitated L-cystine crystals via quick centrifugation after the 72-hour incubation period; and (3) measuring L-cystine concentrations in the supernatants using either a 96-well, medium-throughput colorimetric assay to monitor cysteine-nitroprusside complex formation or our unique 384-well, high-throughput fluorometric assay to monitor the fluorescence of OPA/NBC derivatives of L-cysteine upon reduction of L-cystine. The colorimetric and fluorometric assays are specific for L-cystine and would be amenable to automation, enhancing the speed and ease with which measurements of L-cystine concentration could be done. However, laboratories could also choose to perform the assays manually and still obtain reliable and reproducible data like we have done in our lab.

Conclusion

In summary, we have presented a simple, convenient, cost-effective, and medium-to-high-throughput crystallization inhibition assay for screening and determining the potency of crystallization inhibitors. The assay was designed with the intent to be highly accessible and applicable to any research group studying crystallization inhibitors. Such an assay will greatly facilitate medicinal chemistry efforts in discovering and developing small molecule crystallization inhibitors for the treatment of nephrolithiasis.

Experimental Procedure

Reagents and Equipment

All reagents and solvents used in the assay were purchased from commercial sources and were of ACS grade. For the cyanide-nitroprusside colorimetric assay, all reagents were made as follows: the potassium cyanide (30% w/v) aqueous solution was made by dissolving 30 grams of potassium cyanide in 100 mL Millipore deionized water; the sodium nitroprusside (20% w/v) aqueous solution was prepared by dissolving 20 grams of sodium nitroprusside in 100 mL Millipore deionized water; the 1x phosphate buffered saline (PBS) solution was created by dissolving 2 grams of sodium chloride, 50 mg of potassium chloride, 360 mg of sodium phosphate dibasic, and 61 mg of potassium phosphate monobasic in 100 mL Millipore deionized water, adjusted to pH 7.4, and made up to a volume of 250 mL with Millipore deionized water. For the fluorometric assay, all reagents were made as follows: DTT (12.5 mM) aqueous solution was prepared by dissolving 193 mg of DTT in 100 mL Millipore deionized water; the sodium phosphate dibasic (0.1 M) aqueous solution was created by dissolving 1.42 grams of sodium phosphate dibasic in 100 mL Millipore deionized water; the iodoacetic acid (100 mM) aqueous solution was made by dissolving 1.86 grams of iodoacetic acid in 100 mL Millipore deionized water; the OPA (100 mM) solution was made by dissolving 1.34 grams of OPA in HPLC-grade methanol; the NBC (100 mM) solution was prepared by dissolving 2.21 grams of NBC in 100 mL HPLC-grade methanol. All solutions prepared for this assay were filtered through a 0.45 micron nylon membrane filter (Cytiva, formerly GE Healthcare Life Sciences, catalog number: 7404–004) to remove all particulate matter to prevent premature L-cystine crystallization. The potassium cyanide (30% w/v), sodium nitroprusside (20% w/v), and PBS (1x, pH 7.4) aqueous solutions were stored at room temperature for at most two weeks. The sodium nitroprusside (20% w/v) aqueous solution must be wrapped in aluminum foil to avoid degradation upon exposure to light. The 1:7 aqueous solution of potassium cyanide (30% w/v) in PBS (Procedure Step F1b) was made fresh in a reservoir prior to use in the assay. The DTT (12.5 mM) and sodium phosphate dibasic (0.1 M) aqueous solutions used in the fluorometric assay were stored at ‒20°C and room temperature, respectively, for at most three months. All other solutions used in the fluorometric assay were prepared fresh.

The SpectraMax Plus 384 microplate reader (Molecular Devices, San Jose, CA) was used to measure the absorbance of the solutions in the 96-well plate at 530 nm for the cyanide-nitroprusside colorimetric assay. The Wallac Victor 3V multi-label microplate reader (Perkin Elmer, Waltham, MA) was used to measure the fluorescence of solutions in the 384-well plate at excitation wavelength of 355 nm and emission wavelength of 460 nm. A Flexdrop IV Precision reagent dispenser (Perkin Elmer, Waltham, MA) could be used to facilitate the addition of reagents to the 96- and 384-well plate. The Origin Pro software (OriginLab, Northampton, MA) was used to perform linear and nonlinear regression analyses.

L-Cystine Crystallization Inhibition Assay Procedure

A. Preparation of test compounds for assay (Timing: 1–1.5 h)

  1. Prepare 10 mM stock solutions of test compounds and positive controls in Millipore deionized water

  2. Prepare serial dilutions of test compounds and positive controls in Millipore deionized water to various 100x concentrations
    1. For inhibitor screening experiments, three concentrations are used: 10 μM, 1 μM, and 100 nM
    2. For dose-response experiments, 11 concentrations, chosen based on inhibitor screening experiments, are recommended to ensure adequate number of data points to define the lower and upper limits of the sigmoidal curve
    3. A blank sample containing Millipore deionized water in place of inhibitor is included in the screening and dose-response experiments

B. Preparation of supersaturated L-cystine aqueous solution (Timing: 2–2.5 h)

  1. Add 70 mg of L-cystine (MW = 240.30) to a clean and oven-dried 500-mL single-neck round-bottom flask with a Teflon-coated egg-shaped magnetic stir bar and dissolve in 100 mL Millipore deionized water (2.9 mM) [8]

  2. Fit the round-bottom flask with a reflux condenser

  3. Heat solution under reflux at 120 °C on an oil bath for 60 min with stirring to ensure complete dissolution of L-cystine

  4. Cool supersaturated solution slowly (~1 °C per minute) with stirring until oil bath temperature reaches 40–45 °C

C. Set-up for L-cystine crystallization inhibition assay (Timing: 1–1.5 h + 72 h incubation)

  1. Vortex serially diluted test compound solutions prepared in step A2

  2. Pipette 5 μL of serially diluted test compound solutions to pre-labeled 1.5-mL microcentrifuge tubes placed in a microtube rack

  3. Pipette gently 495 μL of freshly prepared supersaturated L-cystine solution from step B4 to microcentrifuge tubes in step C2 to dilute 100x inhibitor solutions to 1x concentration

  4. Incubate mixtures undisturbed for 72 hours within an incubator set at 20°C to maintain consistent temperature during the entire incubation period

D. Preparation of L-cystine standard solutions (Timing: 1 h)

  1. Add 1.0 mg L-cystine to a 40-mL reaction vial with a pressure relief cap containing a Teflon-coated magnetic stir bar and dissolve in 10.4 mL Millipore deionized water

  2. Heat and stir solution at 100 °C on dry heat bath for 30 minutes to ensure complete dissolution to yield a 0.4 mM L-cystine standard solution

  3. Allow the 0.4 mM L-cystine standard solution to cool to room temperature

  4. Perform 2-fold serial dilutions starting from the 0.4 mM L-cystine standard solution to prepare the 0.2, 0.1, and 0.05 mM L-cystine standard solutions

E. Preparation of samples for L-cystine concentration measurement (Timing: 1–1.5 h)

  1. After the 72-hour incubation, centrifuge mixtures at 16,110 × g or 14,000 rpm (maximum centrifuge speed) for 10 minutes at room temperature to pack the crystallized L-cystine to the bottom of the microcentrifuge tubes

  2. Carefully withdraw 50 μL of the supernatant from each microcentrifuge tube without disturbing the pellet and dilute it 10-fold (450 μL) with Millipore deionized water. Diluted L-cystine solutions may be stored at r.t. for at most 24 h or at 2–8 °C for at most 72 h, if necessary

F. Measurement of the L-cystine concentration

Note: In the following section of the procedure, users may choose either method to measure the L-cystine concentrations in the test compound samples (from step E2) using the solutions prepared in section D for the standard curves.

  1. Cyanide-nitroprusside colorimetric assay method (Timing: 1.5–2.5 h)
    1. Pipette 40 μL of the L-cystine standard solution prepared in section D, diluted test compound samples prepared in step E2, and blank (Millipore deionized water) in triplicates into a 96-well plate (Corning 3631 black, clear bottom; Fig. 4A)
      1. Only two inhibitors (11 concentrations each and a blank) and a set of standard solutions can be plated on a single 96-well plate
      2. Each plate must include a set of standard solutions because the red-orange color produced by the cysteine-nitroprusside complex fades over time
    2. Add 80 μL of aqueous solution of potassium cyanide (30% w/v) in PBS (pH 7.4) (1:7) to each well
    3. Shake the plate at 550 rpm for 25 minutes
    4. Add 10 μL of aqueous solution of sodium nitroprusside (20% w/v) to each well using automated dispenser
    5. Centrifuge the plate immediately at 1,500 rpm for 20 seconds
    6. Immediately measure the absorbance of the samples at 530 nm
  2. Fluorometric assay method (Timing: 2.5–3.5 h)
    1. Mix 10 μL of each L-cystine standard solution prepared in section D, diluted test compound solution prepared in step E2, and blank (Millipore deionized water) with 100 μL of DTT solution (12.5 mM) in 0.1 M dibasic sodium phosphate solution (1:9) in a 0.65-mL microcentrifuge tube; allow to react at room temperature for 10 minutes
    2. Add 10 μL of iodoacetic acid (100 mM) to each mixture in the previous step and allow to react at room temperature for an additional 15 minutes
    3. Add 20 μL of OPA (100 mM in methanol)/NBC (100 mM in methanol) (1:1) to each mixture in the previous step and allow derivatization to proceed at room temperature for 3 minutes
    4. Pipette 40 μL of each mixture prepared in the previous step in triplicates into a 384-well plate (Corning 3575 black, opaque bottom; Fig. 4B)
      1. A total of ten inhibitors (11 concentrations each and a blank) and one or two sets of standard solutions can be plated on a single 384-well plate
      2. Standard curve is repeated for each set of experiments and used to calculate the concentration of L-cystine in each sample
    5. Centrifuge the plate at 1,500 rpm for 1 minute
    6. Measure the fluorescence of samples at excitation wavelength of 355 nm and emission wavelength of 460 nm
Fig. 4.

Fig. 4

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Typical arrangement of samples on an assay plate for dose-response experiments using the cyanide-nitroprusside colorimetric method (A) and the fluorometric method (B)

Data Analysis

A. Generation of standard curve

  1. Calculate the average of the triplicate absorbance or fluorescence emission readings for each standard solution

  2. Graph the average of the absorbance (Fig. 2A) or fluorescence emission (Fig. 2B) readings for the standard solutions against the L-cystine concentration (0.4, 0.2, 0.1, and 0.05 mM) in each standard solution to obtain a standard curve

  3. Determine the line of best fit for the data set and use the equation for the line to derive the L-cystine concentration in each test compound solution and blank
    1. A dilution factor of 10 must be applied to L-cystine concentrations obtained from the standard curve equation, which accounts for the 10-fold dilution of the supersaturated solutions in Procedure Step E2

B. Generation of dose-response curves and calculation of inhibitor potency

  1. Calculate the average and standard error for the L-cystine concentration (triplicates) for each test compound solution and blank

  2. Plot the averages of the L-cystine concentration for each test compound solution against the inhibitor concentration in each test compound solution as a scatter plot

  3. Use graphing software (e.g., OriginPro) to perform nonlinear regression analysis of the data and to determine either the EC2x or EC50 of the inhibitors (Fig. 3)

Acknowledgements:

Longqin Hu 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. Yanhui Yang developed and optimized the fluorometric assay. Haifa Albanyan applied the colorimetric assay in evaluating inhibitor potency. Jeffrey Yang and Yiling Wang were responsible for further optimization of the assay protocol to ensure assay reproducibility. Haifa Albanyan, Yanhui Yang, Jeffrey Yang, and Yiling Wang performed the statistical analyses. Jeffrey Yang wrote the initial manuscript draft. Jeffrey Yang and Longqin Hu 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].

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

Footnotes

Conflict of Interest:

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

References

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