Software automation tools for increased throughput metabolic soft-spot identification in early drug discovery.

BACKGROUND: The ability to supplement high-throughput metabolic clearance data with structural information defining the site of metabolism should allow design teams to streamline their synthetic decisions. However, broad application of metabolite identification in early drug discovery has been limited, largely due to the time required for data review and structural assignment. The advent of mass defect filtering and its application toward metabolite scouting paved the way for the development of software automation tools capable of rapidly identifying drug-related material in complex biological matrices. Two semi-automated commercial software applications, MetabolitePilot™ and Mass-MetaSite™, were evaluated to assess the relative speed and accuracy of structural assignments using data generated on a high-resolution MS platform. RESULTS/CONCLUSION: Review of these applications has demonstrated their utility in providing accurate results in a time-efficient manner, leading to acceleration of metabolite identification initiatives while highlighting the continued need for biotransformation expertise in the interpretation of more complex metabolic reactions.

Development of a high-speed, multiplexed sample-delivery instrument for LC-MS/MS bioanalysis.

BACKGROUND: The number of new chemical entities and types of in vitro and in vivo samples that require bioanalysis in drug discovery is large and diverse. In addition, method development time is limited as data turnaround is the highest priority. These circumstances require that a well-defined set of bioanalysis options be available in short timeframes to triage samples for analysis. METHOD: The Apricot Designs Dual Arm (ADDA) instrument is an LC-MS/MS sample delivery system that features a flexible hardware design coupled with software automation to enhance throughput in LC-MS/MS bioanalysis drug discovery. The instrument can perform high-throughput LC-MS/MS (8-10 s/sample) for screening and in vitro bioanalysis, as well as multiplexed LC for traditional gradient or isocratic LC approaches. The instrument control software is designed to integrate with DiscoveryQuant™ software (AB Sciex) and a global database of MS/MS conditions. CONCLUSION: Development of the sample delivery platform and its application in high-throughput and gradient LC will be described.

The effect of breast cancer resistance protein and P-glycoprotein on the brain penetration of flavopiridol, imatinib mesylate (Gleevec), prazosin, and 2-methoxy-3-(4-(2-(5-methyl-2-phenyloxazol-4-yl)ethoxy)phenyl)propanoic acid (PF-407288) in mice.

The role of breast cancer resistance protein (Bcrp) and the combined activities of Bcrp and P-glycoprotein (P-gp, Mdr1a/1b) in limiting the brain penetration of drugs at the blood-brain barrier (BBB) were investigated using wild-type FVB, Mdr1a/1b(-/-), (-/-), Bcrp(-/-), and Mdr1a/1b(-/-), (-/-)Bcrp(-/-) mice. Four drugs, flavopiridol, imatinib mesylate (Gleevec), PF-407288, and prazosin, with different transport specificity for BCRP/Bcrp and MDR1/Mdr1a were selected, and the drug levels in plasma, cerebrospinal fluid, and brain of mice were determined. Flavopiridol and prazosin were identified as substrates for both mouse Bcrp and Mdr1a with greater transport associated with Bcrp. The brain/plasma (B/P) ratios at 0.5 and 2 h in Mdr1a/1b(-/-), (-/-) and Bcrp(-/-) mice were 1- to 2-fold for both compounds, whereas the ratios in Mdr1a/1b(-/-), (-/-)Bcrp(-/-) mice were more than 5-fold of those observed in FVB mice. For imatinib, a better substrate of P-gp than Bcrp, the B/P ratios in Bcrp(-/-) were comparable to those in FVB mice, whereas the B/P ratios in Mdr1a/1b(-/-), (-/-) and Mdr1a/1b(-/-), (-/-)Bcrp(-/-) mice were more than 4- and 28-fold of those in FVB mice at both time points, respectively. Finally, the Bcrp-specific substrate PF-407288 exhibited comparable B/P ratios in Mdr1a/1b(-/-), (-/-) and Bcrp(-/-) mice and slightly but significantly increased B/P ratios in Mdr1a/1b(-/-), (-/-)Bcrp(-/-) mice compared with those in FVB mice. The B/P ratios of compounds in Mdr1a/1b(-/-), (-/-)Bcrp(-/-) mice compared with those in Mdr1a/1b(-/-), (-/-) mice clearly demonstrate that Bcrp impairs the brain penetration of its substrates. Moreover, P-gp and Bcrp at BBB function synergistically to limit the brain penetration of shared substrates.

High throughput ADME screening: practical considerations, impact on the portfolio and enabler of in silico ADME models.

Evaluation and optimization of drug metabolism and pharmacokinetic data plays an important role in drug discovery and development and several reliable in vitro ADME models are available. Recently higher throughput in vitro ADME screening facilities have been established in order to be able to evaluate an appreciable fraction of synthesized compounds. The ADME screening process can be dissected in five distinct steps: (1) plate management of compounds in need of in vitro ADME data, (2) optimization of the MS/MS method for the compounds, (3) in vitro ADME experiments and sample clean up, (4) collection and reduction of the raw LC-MS/MS data and (5) archival of the processed ADME data. All steps will be described in detail and the value of the data on drug discovery projects will be discussed as well. Finally, in vitro ADME screening can generate large quantities of data obtained under identical conditions to allow building of reliable in silico models.

Bradykinin B2 receptor signaling: structural and functional characterization of the C-terminus.

Over the last few years the importance of the intracellular C-terminus in the signaling of G-protein coupled receptors (GPCR) has become increasingly evident. In an effort to provide a structural framework for biological function, we have determined the conformation of the C-terminus of the bradykinin (BK) B2 receptor. Using a uniformly 15N- and 13C-enriched sample of the BKB2 receptor [309-366], NMR results clearly define three alpha-helices lying on the zwitterionic surface of the dodecylphosphocholine. The proximal helix consisting of residues 311-326 was previously predicted based on homology modeling with rhodopsin. This corresponds to what is often called helix-8 of the GPCRs. The two distal helices, residues 333-345 and 348-363, are clearly borne out by the NMR data. The functional importance of these secondary structural elements was probed by determination of the signaling properties (inositol phosphate formation) of mutant BKB2 receptors lacking the domains (deletion mutants) or containing the corresponding region from the related GPCR, angiotensin II AT1a (chimera receptors). We demonstrate that the regions between the helices (residues 327-333 and 346-347) can be exchanged without loss of signaling. In contrast, modification of the three helices, particularly the hydroxyl-containing residues, has drastic effects on the signaling profile of the BKB2 receptor. By coupling of the structural features with the functional data, the molecular mechanisms of signaling by the BKB2 receptor are beginning to be established.