High-throughput cytochrome P450 inhibition screening via cassette probe-dosing strategy. IV. Validation of a direct injection on-line guard cartridge extraction/tandem mass spectrometry method for simultaneous CYP3A4, 2D6 and 2E1 inhibition assessment

A highly efficient direct injection on-line guard cartridge extraction/tandem mass spectrometry (DI-GCE/MS/MS) method has been validated for high-throughput evaluation of cytochrome P450 (CYP) 3A4, 2D6 and 2E1 inhibition potential via cassette dosing of midazolam, dextromethorphan and chlorzoxazone using human hepatic microsomes and 96-well microtiter plates. Microsomal incubations were terminated with formic acid, centrifuged, and the resulting supernatants were injected for analysis by DI-GCE/MS/MS. Due to the novel use of an extremely short C(18) guard cartridge (4 mm in length), this method exhibits several advantages such as no sample preparation, excellent on-line extraction, short run time (2.5 min), and minimized source contamination and performance deterioration. The DI-GCE/MS/MS method demonstrates acceptable accuracy and precision for the simultaneous quantification of 1'-hydroxymidazolam, dextrorphan and 6-hydroxychlorzoxazone in microsomal incubations. The inhibition potential of CYP3A4, 2D6 and 2E1 has been evaluated using their known selective inhibitors. The IC(50) values measured by the cassette dosing approach (high-throughput) are consistent with those observed by an individual dosing regimen (conventional) and are all in good agreement with the literature values. The results suggest that the cassette probe-dosing strategy may provide an in vitro approach to minimize cost while maximizing throughput of CYP inhibition evaluation of new chemical entities in support of drug discovery and development.     

High-throughput cytochrome P450 (CYP) inhibition screening via a cassette probe-dosing strategy. VI. Simultaneous evaluation of inhibition potential of drugs on human hepatic isozymes CYP2A6, 3A4, 2C9, 2D6 and 2E1

The inhibition potential of drugs towards five major human hepatic cytochrome P450 (CYP) isozymes (CYP2A6, 3A4, 2C9, 2D6, and 2E1) was investigated via cassette dosing of the five probe substrates (coumarin, midazolam, tolbutamide, dextromethorphan, and chlorzoxazone) in human liver microsomes using a 96-well plate format. After microsomal incubations had been terminated with formic acid, the five marker metabolites (7-hydroxycoumarin, 1'-hydroxymidazolam, 4-hydroxytolbutamide, dextrorphan, and 6-hydroxychlorzoxazone) were simultaneously quantified using direct injection/online guard cartridge extraction/tandem mass spectrometry (DI-GCE/MS/MS). Several advantages resulted from the use of a short C(18) guard cartridge (4 mm in length) for DI-GCE/MS/MS, including minimal sample preparation, fast online extraction, short analysis time (2.5 min), and minimal source contamination. In addition, this method demonstrated an inter-day accuracy range from -8.7 - 7.4% with a precision less than 8.3% for the quantification of all the marker metabolites. The inhibition assay for the five CYP isozymes was evaluated using their known selective inhibitors via individual and cassette dosing of the probe substrates. The IC(50) values measured via cassette dosing were consistent with those observed via individual dosing, which were all in agreement with the reported values. In addition, the validated assay was used to evaluate the inhibitory potential of 23 generic drugs (randomly selected) towards the five CYP isozymes. The results suggest the integration of the cassette dosing strategy and the DI-GCE/MS/MS method can provide a reliable in vitro approach to screening the inhibitory potential of new chemical entities, with maximal throughput and cost-effectiveness, in support of drug discovery and development.     

Use of engineered unique cysteine residues to facilitate oriented coupling of proteins directly to a gold substrate

A prerequisite for any "lab on a chip" device that utilizes an electrical signal from the sensor protein is the ability to attach the protein in a specific orientation onto a conducting substrate. Here, we demonstrate the covalent attachment to a gold surface of light-harvesting membrane proteins, from Rhodobacter sphaeroides, via cysteine (Cys) residues engineered on either the cytoplasmic or periplasmic face. This simple directed attachment is superior in its ability to retain light-harvesting complex (LHC) function, when compared to a similar attachment procedure utilizing a self-assembled monolayer on gold. LH 1 has previously been observed to have superior photostability over LH 2 (Magis et al. [2010] Biochim. Biophys. Acta, 1798, 637-645); this characteristic is maintained even with the introduction of Cys residues.     

Structure-based secondary structure-independent approach to design protein ligands: Application to the design of Kv1.2 potassium channel blockers

We have developed a structure-based approach to the design of protein ligands. This approach is based on the transfer of a functional binding motif of amino acids, often referred as to the "hot spot", on a host protein able to reproduce the functional topology of these residues. The scaffolds were identified by a systematic in silico search in the Protein Data Bank for proteins possessing a group of residues in a topology similar to that adopted by the functional motif in a reference ligand of known 3D structure. In contrast to previously reported studies, this search is independent of the particular secondary structure supporting the functional motif. To take into account the global properties of the host protein, two additional criteria were taken into account in the selection process: (1) Only those scaffolds sterically compatible with the positioning of the functional motif as observed in a reference complex model were retained. (2) Host proteins displaying electrostatic potentials, in the region of the transferred functional motif, similar to that of the reference ligand were selected. This approach was applied to the development of protein ligands of the Kv1.2 channel using BgK, a small protein isolated from the sea anemone Bunodosoma granulifera, as the reference ligand. Four proteins obtained by this approach were produced for experimental evaluation. The X-ray structure of one of these proteins was determined to check for similarity of the transferred functional motif with the structure it adopts in the reference ligand. Three of these protein ligands bind the Kv1.2 channel with inhibition constants of 0.5, 1.5, and 1.6 microM. Several mutants of these designed protein ligands gave binding results consistent with the presumed binding mode. These results show that protein ligands can be designed by transferring a binding motif on a protein host selected to reproduce the functional topology of this motif, irrespective to the secondary structure supporting the functional motif, if the host protein possesses steric and electrostatic properties compatible with the binding to the target. This result opens the way to the design of protein ligands by taking advantage of the considerable structural repertoire of the Protein Data Bank.     

A novel approach to perform metabolite screening during the quantitative LC-MS/MS analyses of in vitro metabolic stability samples using a hybrid triple-quadrupole linear ion trap mass spectrometer

In vitro metabolic stability experiments using microsomes or other liver preparations are important components in the discovery and lead-optimization stages of compound selection in the pharmaceutical industry. Currently, liquid chromatography-tandem mass spectrometric (LC-MS/MS) support of in vitro metabolic stability studies primarily involves the monitoring of disappearance of parent compounds, using selected reaction monitoring (SRM) on triple-quadrupole instruments. If moderate to high turnover is observed, separate metabolite identification experiments are then conducted to characterize the biotransformation products. In this paper, we present a novel method to simultaneously perform metabolite screening in addition to the quantitative stability measurements, both within the same chromatographic run. This is accomplished by combining SRM and SRM-triggered, information-dependent acquisition (IDA) of MS/MS spectra on a hybrid triple-quadrupole linear ion trap (QqQLIT) mass spectrometer. Microsomal stability experiments using model compounds, bufuralol, propranolol, imipramine, midazolam, verapamil and diclofenac, were used to demonstrate the applicability of our approach. This SRM + SRM-IDA approach generated metabolic stability results similar to those obtained by conventional SRM-only approach. In addition, MS/MS spectra from potential metabolites were obtained with the enhanced product ion (EPI) scan function of LIT during the same injection. These spectra were correlated to the spectra of parent compounds to confirm the postulated structures. The time-concentration profiles of identified metabolites were also estimated from the acquired data. This approach has been successfully used to support discovery programs.     

Enhanced photocurrent generation by photosynthetic bacterial reaction centers through molecular relays, light-harvesting complexes, and direct protein-gold interactions

The utilization of proteins as nanodevices for solar cells, bioelectronics, and sensors generally necessitates the transfer of electrons to or from a conducting material. Here we report on efforts to maximize photocurrent generation by bacterial photosynthetic reaction center pigment-protein complexes (RCs) interfaced with a metal electrode. The possibility of adhering RCs to a bare gold electrode was investigated with a view to minimizing the distance for electron tunneling between the protein-embedded electron-transfer cofactors and the metal surface. Substantial photocurrents were achieved despite the absence of coating layers on the electrode or engineered linkers to achieve the oriented deposition of RCs on the surface. Comparison with SAM-covered gold electrodes indicating enhanced photocurrent densities was achieved because of the absence of an insulating layer between the photoactive pigments and the metal. Utilizing RCs surrounded by light-harvesting 1 complex resulted in higher photocurrents, surprisingly not due to enhanced photoabsorption but likely due to better surface coverage of uniformly oriented RC-LH1 complexes and the presence of a tetraheme cytochrome that could act as a connecting wire. The introduction of cytochrome-c (cyt-c) as a molecular relay also produced increases in current, probably by intercalating between the adhered RCs or RC-LH1 complexes and the electrode to mediate electron transfer. Varying the order in which components were introduced to the electrode indicated that dynamic rearrangements of RCs and cyt-c occurred at the bare metal surface. An upper limit for current generation could not be detected within the range of the illumination power available, with the maximum current density achieved by RC-LH1 complexes being on the order of 25 μA/cm(2). High currents could be generated consecutively for several hours or days under ambient conditions.     

A two-sided Arnoldi algorithm with stopping criterion and MIMO selection procedure

In this paper we introduce a two-sided Arnoldi method for the reduction of high order linear systems and we propose useful extensions, first of all a stopping criterion to find a suitable order for the reduced model and secondly, a selection procedure to significantly improve the performance in the multi-input multi-output (MIMO) case. One application is in micro-electro-mechanical systems (MEMS). We consider a thermo-electric micro thruster model, and a comparison between the commonly used Arnoldi algorithm and the two-sided Arnoldi is performed.