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

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).

Design, synthesis, and evaluation of l-cystine diamides as l-cystine crystallization inhibitors for cystinuria.

To overcome the chemical and metabolic stability issues of l-cystine dimethyl ester (CDME) and l-cystine methyl ester (CME), a series of l-cystine diamides with or without Nα-methylation was designed, synthesized, and evaluated for their inhibitory activity of l-cystine crystallization. l-Cystine diamides 2a-i without Nα-methylation were found to be potent inhibitors of l-cystine crystallization while Nα-methylation of l-cystine diamides resulted in derivatives 3b-i devoid of any inhibitory activity of l-cystine crystallization. Computational modeling indicates that Nα-methylation leads to significant decrease in binding of the l-cystine diamides to l-cystine crystal surface. Among the l-cystine diamides 2a-i, l-cystine bismorpholide (CDMOR, LH707, 2g) and l-cystine bis(N'-methylpiperazide) (CDNMP, LH708, 2h) are the most potent inhibitors of l-cystine crystallization.

l-Cystine Diamides as l-Cystine Crystallization Inhibitors for Cystinuria.

l-Cystine bismorpholide (1a) and l-cystine bis(N'-methylpiperazide) (1b) were seven and twenty-four times more effective than l-cystine dimethyl ester (CDME) in increasing the metastable supersaturation range of l-cystine, respectively, effectively inhibiting l-cystine crystallization. This behavior can be attributed to inhibition of crystal growth at microscopic length scale, as revealed by atomic force microscopy. Both 1a and 1b are more stable than CDME, and 1b was effective in vivo in a knockout mouse model of cystinuria.

Discovery of direct inhibitors of Keap1-Nrf2 protein-protein interaction as potential therapeutic and preventive agents.

The Keap1-Nrf2-ARE pathway is an important antioxidant defense mechanism that protects cells from oxidative stress and the Keap1-Nrf2 protein-protein interaction (PPI) has become an important drug target to upregulate the expression of ARE-controlled cytoprotective oxidative stress response enzymes in the development of therapeutic and preventive agents for a number of diseases and conditions. However, most known Nrf2 activators/ARE inducers are indirect inhibitors of Keap1-Nrf2 PPI and they are electrophilic species that act by modifying the sulfhydryl groups of Keap1׳s cysteine residues. The electrophilicity of these indirect inhibitors may cause "off-target" side effects by reacting with cysteine residues of other important cellular proteins. Efforts have recently been focused on the development of direct inhibitors of Keap1-Nrf2 PPI. This article reviews these recent research efforts including the development of high throughput screening assays, the discovery of peptide and small molecule direct inhibitors, and the biophysical characterization of the binding of these inhibitors to the target Keap1 Kelch domain protein. These non-covalent direct inhibitors of Keap1-Nrf2 PPI could potentially be developed into effective therapeutic or preventive agents for a variety of diseases and conditions.

Current practices for labeling medications in hospitals in Riyadh, Saudi Arabia.

BACKGROUND: Good medication labeling practices are imperative to ensure safe medication use. Non-adherence to labeling protocols is reported as one major source of medication errors. OBJECTIVE: This study was intended to evaluate and compare adherence to labeling guidelines for dispensed medications among the hospitals of the five different health sectors in the city of Riyadh, Saudi Arabia. METHODS: A descriptive, cross-sectional analysis was conducted among 14 public hospitals in the city of Riyadh, Saudi Arabia. Labeling guidelines issued by the Institute for Safe Medication Practices were used as a standard assessment tool. A total of 218 medication labels were collected and evaluated for labeling adequacy. Descriptive statistics were used to elaborate the study findings. All analyses were performed with Microsoft Access. RESULTS: The study showed a substantial rate of adherence to the labeling guidelines. In terms of the established criteria, community and mail orders were reported to adhere strongly (90.5%), whereas injectables adhered least to the labeling guidelines. The labeling format, contents of the label, instructions on the labels, abbreviations used on the labels and drug names were also consistent with the guidelines (80.0%, 84.0%, 88.0%, 97.7% and 85.5%, respectively). Organizations belonging to the public sector reported a higher level of adherence (⩾80.0%) than the level found for private hospitals (70.0%). CONCLUSION: In Riyadh hospitals, medication labeling following the guidelines issued by the Institute for Safe Medication Practices, is well accepted and rationally practiced. However, a nationwide study is recommended to evaluate if the guidelines are followed throughout Saudi Arabia.