Novel Multiplexed High Throughput Screening of Selective Inhibitors for Drug-Metabolizing Enzymes Using Human Hepatocytes.

Selective chemical inhibitors are critical for reaction phenotyping to identify drug-metabolizing enzymes that are involved in the elimination of drug candidates. Although relatively selective inhibitors are available for the major cytochrome P450 enzymes (CYP), they are quite limited for the less common CYPs and non-CYPs. To address this gap, we developed a multiplexed high throughput screening (HTS) assay using 20 substrate reactions of multiple enzymes to simultaneously monitor the inhibition of enzymes in a 384-well format. Four 384-well assay plates can be run at the same time to maximize throughput. This is the first multiplexed HTS assay for drug-metabolizing enzymes reported. The HTS assay is technologically enabled with state-of-the-art robotic systems and highly sensitive modern LC-MS/MS instrumentation. Virtual screening is utilized to identify inhibitors for HTS based on known inhibitors and enzyme structures. Screening of ~4600 compounds generated many hits for many drug-metabolizing enzymes including the two time-dependent and selective aldehyde oxidase inhibitors, erlotinib and dibenzothiophene. The hit rate is much higher than that for the traditional HTS for biological targets due to the promiscuous nature of the drug-metabolizing enzymes and the biased compound selection process. Future efforts will focus on using this method to identify selective inhibitors for enzymes that do not currently have quality hits and thoroughly characterizing the newly identified selective inhibitors from our screen. We encourage colleagues from other organizations to explore their proprietary libraries using a similar approach to identify better inhibitors that can be used across the industry.

Characterization of CYP3A5 Selective Inhibitors for Reaction Phenotyping of Drug Candidates.

CYP3A is one of the most important classes of enzymes and is involved in the metabolism of over 70% drugs. While several selective CYP3A4 inhibitors have been identified, the search for a selective CYP3A5 inhibitor has turned out to be rather challenging. Recently, several selective CYP3A5 inhibitors have been identified through high-throughput screening of ~ 11,000 compounds and hit expansion using human recombinant enzymes. We set forth to characterize the three most selective CYP3A5 inhibitors in a more physiologically relevant system of human liver microsomes to understand if these inhibitors can be used for reaction phenotyping studies in drug discovery settings. Gomisin A and T-5 were used as selective substrate reactions for CYP3A4 and CYP3A5 to determine IC50 values of the two enzymes. The results showed that clobetasol propionate and loteprednol etabonate were potent and selective CYP3A5 reversible inhibitors with selectivity of 24-fold against CYP3A4 and 39-fold or more against the other major CYPs. The selectivity of difluprednate in HLM is much weaker than that in the recombinant enzymes due to hydrolysis of the acetate group in HLM. Based on the selectivity data, loteprednol etabonate can be utilized as an orthogonal approach, when experimental fraction metabolized of CYP3A5 is greater than 0.5, to understand CYP3A5 contribution to drug metabolism and its clinical significance. Future endeavors to identify even more selective CYP3A5 inhibitors are warranted to enable accurate determination of CYP3A5 contribution to metabolism versus CYP3A4.

Design, synthesis, and pharmacological evaluation of azetedine and pyrrolidine derivatives as dual norepinephrine reuptake inhibitors and 5-HT(1A) partial agonists.

Compounds with combined norepinephrine reuptake inhibitor (NRI) and serotonin 1A (5-HT(1A)) partial agonist pharmacology may offer a new therapeutic approach for treating symptoms of neuropsychiatric disorders including ADHD, depression, and anxiety. Herein we describe the design and optimization of novel chemical matter that exhibits favorable dual NRI and 5-HT(1A) partial agonist activity. Lead compounds in this series were found to be devoid of activity at the dopamine transporter and were shown to be brain penetrant with high receptor occupancy.

Identification and SAR around N-{2-[4-(2,3-dihydro-benzo[1,4]dioxin-2-ylmethyl)-[1,4]diazepan-1-yl]-ethyl}-2-phenoxy-nicotinamide, a selective alpha2C adrenergic receptor antagonist.

The discovery of the CNS-penetrant and selective alpha(2C) adrenergic receptor antagonist N-{2-[4-(2,3-dihydro-benzo[1,4]dioxin-2-ylmethyl)-[1,4]diazepan-1-yl]-ethyl}-2-phenoxy-nicotinamide, 13 is described. Structure-activity studies demonstrate the structural requirements for binding affinity, functional activity, and selectivity over other alpha(2)-AR subtypes.