2-Aminopyridine Analogs Inhibit Both Enzymes of the Glyoxylate Shunt in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen responsible for many hospital-acquired infections. P. aeruginosa can thrive in diverse infection scenarios by rewiring its central metabolism. An example of this is the production of biomass from C2 nutrient sources such as acetate via the glyoxylate shunt when glucose is not available. The glyoxylate shunt is comprised of two enzymes, isocitrate lyase (ICL) and malate synthase G (MS), and flux through the shunt is essential for the survival of the organism in mammalian systems. In this study, we characterized the mode of action and cytotoxicity of structural analogs of 2-aminopyridines, which have been identified by earlier work as being inhibitory to both shunt enzymes. Two of these analogs were able to inhibit ICL and MS in vitro and prevented growth of P. aeruginosa on acetate (indicating cell permeability). Moreover, the compounds exerted negligible cytotoxicity against three human cell lines and showed promising in vitro drug metabolism and safety profiles. Isothermal titration calorimetry was used to confirm binding of one of the analogs to ICL and MS, and the mode of enzyme inhibition was determined. Our data suggest that these 2-aminopyridine analogs have potential as anti-pseudomonal agents.

1,2,4-Triazolyl 5-Azaspiro[2.4]heptanes: Lead Identification and Early Lead Optimization of a New Series of Potent and Selective Dopamine D3 Receptor Antagonists.

A novel series of 1,2,4-triazolyl 5-azaspiro[2.4]heptanes with high affinity and selectivity at the dopamine (DA) D3 receptor (D3R) is described. Some of these compounds also have high selectivity over the hERG channel and were characterized with respect to their pharmacokinetic properties both in vitro and in vivo during lead identification and early lead optimization phases. A few derivatives with overall favorable developability characteristics were selected for further late lead optimization studies.

Contribution of rat intestinal metabolism to the xenobiotics clearance.

Michaelis-Menten constants K m and V max values were determined by product formation and substrate depletion at several substrate concentrations of 4-methylumbelliferone using rat intestinal microsomes. K m and V max values determined by measuring product formation were in good agreement with substrate depletion approach. We also investigated hepatic and intestinal in vitro intrinsic clearance (CLint) in the liver and intestinal microsomes and compare with reports in the literature using nine test compounds, atorvastatin, 7-ethoxycoumarin, indomethacin, 4-methylumbelliferone, midazolam, nifedipine, testosterone, terfenadine and verapamil, in rats. CLint was determined from the substrate disappearance rate at 0.1 and 0.5 µM in the rat intestinal and liver microsomes, respectively. These results showed that both the liver and the intestine contributed to the metabolism of these compounds. The intestinal intrinsic clearance values of all these drugs, except for terfenadine in the rat intestinal microsomes, were lower than their hepatic intrinsic clearance per milligram protein, showing that there was an organ difference in metabolism between the liver and intestinal. These results make the evaluation using the intestinal more useful and provide a basis for predicting clearance using intestinal.

A comparative study of the CYP450 inhibition potential of marketed drugs using two fluorescence based assay platforms routinely used in the pharmaceutical industry.

Semi-automated high throughput screening for the inhibition of major human cytochrome P450 enzymes (1A2, 2C9, 2C19, 2D6 and 3A4) expressed in Escherichia Coli (Cypex bactosomes) or human lymphoblastoid cells (Gentest cDNA microsomes) using fluorescent probes has been evaluated using 68 marketed drugs. In general lower IC50 values were obtained with Cypex bactosomes compared with Gentest cDNA microsomes. This could be due to use of higherconcentration of protein and also the lower activity of Gentest cDNA microsomes. Notably, when compared with in vivo clinical drug-drug interactions (cDDIs) gathered from clinical studies reported in the scientific literature Cypex bactosome data was better at predicting in vivo cDDI. Consequently, from the data obtained in this comparative study, a fluorescence based assay using Cypex bactosomes is more suitable as a front-line screen for the prediction of potential downstream CYP450 driven cDDIs.

Evaluation of cytochrome P450 inhibition assays using human liver microsomes by a cassette analysis /LC-MS/MS.

In vitro inhibition assays are used to screen new chemical entities (NCEs) for inhibition studies by using human liver microsomes. High-throughput inhibition assays using pooling methods have been developed to keep pace with screening requirements at the lead ADME stage. This method can determine IC(50) NCEs using microsomes from various organs from any species. A cassette analysis inhibition assay for five of the major CYP enzymes (phenacetin for CYP1A2, diclofenac for CYP2C9, (S)-mephenytoin for CYP2C19, bufurarol for CYP2D6 and midazolam, nifedipine and atorvastatin for CYP3A4) in pooled human liver microsomes using ultraperformance liquid chromatography tandem mass spectrometry (LC/MS/MS) were developed. The major metabolites of seven CYP-specific probe substrates for the five P450 isoforms were monitored and quantified to determine IC(50) values. Human liver microsomal incubation samples at each test compound concentration were combined and analyzed simultaneously by the LC/MS/MS method. The incubation was performed using the selective CYP inhibitors for each isoform: fluvoxamine (CYP1A2), sulphaphenazole (CYP2C9), ticlopidine (CYP2C19), quinidine (CYP2D6), and Ketoconazole (CYP3A4). Similar IC(50) results were obtained using the cassette analysis and discrete analysis method. The IC(50) values determined for typical CYP inhibitors were reproducible and consistent with those reported in the literature. The assay was fully automated in 96 well plate formats using Microlab 4000 series (Hamilton) coupled with two termomixer comfort (Eppendorf). An overall 70% time savings was achieved by pooling four isoforms samples (1A2, 2C9, 2C19 and 2D6) into one sample and also by pooling three CYP 3A4 substrate samples (MDZ, ATR, NIF) into one sample. Cassette analysis minimized the number of injections on LC/MS/MS analysis which results in a decrease in the LC/MS/MS analysis time.

Identifying a higher throughput assay for metabolism dependent inhibition (MDI).

A higher throughput method of screening for the metabolism dependent inhibition of 56 marketed drugs was evaluated and compared data from the PHLM assay (using midazolam as probe) with Cypex assay (using diethoxyfluoresin (DEF) as probe) for CYP3A4 by using 96 well plate. Also 27 marketed drugs were selected to evaluate the reproducibility of Cypex assay using 7-benzyloxyquinoline (7-BQ) as second probe substrate for CYP3A4. Furthermore Cypex CYP2D6 was used to evaluate the reproducibility of this system using 4-methylaminomethyl-7-methoxycoumarin (MMC) as probe substrate with 15 marketed drugs. The fold change was estimated using the fold change obtained from triplicates experiments in same day or different days. All replicates in agreement (i.e. all positive or all negative) for >80% of compounds. The IC50 values for the two assays closely matched only for 13 compounds (23%). Only 5 of the variant 56 compounds had higher IC50 values with the recombinant enzymes, whereas 38 had lower IC50 values with the recombinant cypex CYP3A4 enzyme. The Cypex assay is comparable to PHLM assay in terms of predictivity and reproducibility. The Cypex assay therefore offers a higher throughput, reproducible alternative to PHLM for placement earlier in the lead optimisation process. In conclusion, the results obtained from a fluorescence-based method using Cypex CYP3A4 reflect mostly those obtained from conventional assay using human liver microsomes. This method provides more rapid and reliable detection of MDI inhibitors and may be useful in drug discovery.