Development of a miniaturized 384-well high throughput screen for the detection of substrates of cytochrome P450 2D6 and 3A4 metabolism.

The identification of a large number of biologically active chemical entities during high throughput screening (HTS) necessitates the incorporation of new strategies to identify compounds with druglike properties early during the lead prioritization and development process. One of the major steps in lead prioritization is the assessment of drug metabolism mediated by the cytochrome P(450) (CYP) enzymes to evaluate the potential drug-drug interactions. CYP2D6 and CYP3A4 comprise the main human CYP enzymes involved in drug metabolism. The recent availability of specific CYP cDNA expression systems and the development of specific fluorescent probes have accelerated the ability to develop robust in vitro assays in HTS format. The aim of this study was to optimize conditions for the CYP2D6 and CYP3A4 HTS assays and subsequently adapt those assays to a miniaturized 384-well format. Assay conversion to a miniaturized format presents certain difficulties, such as robustness of the signal and of compound delivery. Thus the assay optimization involved the comparison of different substrates to identify those most suitable for use in a miniaturized format. Because of current technical limitations in liquid dispensing of nanoliter volumes, assay sensitivity to organic solvents also provides a main concern during assay miniaturization. Therefore, compound activity from redissolved dry films and from DMSO stocks directly delivered into assay buffer was compared. The data indicate that compound activity was comparable in both formats. The data support the conclusion that CYP2D6 and CYP3A4 in vitro metabolism assays can be successfully performed in 384-well plate format and the substrate potencies, as evaluated by the IC(50) values, determined.

Identification of inhibitors of bacterial transcription/translation machinery utilizing a miniaturized 1536-well format screen.

This report presents the miniaturization of a HTS screen to identify inhibitors of prokaryotic transcription-translation in a 1536-well format. The in vitro assay design utilized the bacterial expression machinery to drive expression of a firefly luciferase reporter gene, which was read as an endpoint luminesence measurement. This multicomponent system permits identification of inhibitors at different steps in this pathway. Successful miniaturization required integration of homogeneous assay formats, robust liquid-handling workstations, and second-generation imaging systems. Comparison of data from a triplicate 1536-well screen of a subset of a target library that had been previously validated and followed up for hit confirmation in a 384-well plate format confirmed that triplicate screening yields data of higher confidence and quality, eliminates the time-consuming and potentially error-prone step of cherry-picking, and reduces the number of false positives and negatives. The substantial savings of reagents and reduction of the numbers of plates to process obtained in a 1536-well format as compared to a 384-well format allowed a full triplicate evaluation of the entire library of 183,000 compounds at lower cost and in less time. The triplicate-screen statistics are consistent with a highly reliable data set with a coefficient of variation of 14.8% and Z' and Z values of 0.57 and 0.25, respectively. This screen resulted in the identification of 1,149 hits (0.63% hit rate), representing a compound population at 2.5 standard deviations from the mean cutoff. Furthermore, the data demonstrate good agreement between IC(50) values derived for this assay in a 1536-well format and 384-well format.

Development of a high throughput equilibrium dialysis method.

The identification of large numbers of biologically active chemical entities during high throughput screening (HTS) necessitates the incorporation of new strategies to identify compounds with drug-like properties early during the lead prioritization and development processes. One of the major steps in lead prioritization is an assessment of compound binding to plasma proteins, because it affects both the pharmacokinetics and pharmacodynamics of the compound in vivo. Equilibrium dialysis is the preferred method to determine the free drug fraction, because it is less susceptible to experimental artifacts. However, even low-volume standard equilibrium dialysis is currently not amenable to the HTS format. Those considerations dictate the development of a high throughput equilibrium dialysis device, without compromising the analytical quality of the data. The present paper demonstrates successful development of a 96-well format equilibrium dialysis plate. Plasma protein binding of three drugs, propranolol, paroxetine, and losartan, with low, intermediate, and high binding properties, respectively, were chosen for assay validation. The data indicate that the apparent free fraction obtained by this method correlates with the published values determined by the traditional equilibrium dialysis techniques.

A high-throughput homogeneous assay for reverse transcriptase using generic reagents and time-resolved fluorescence detection.

A homogeneous time-resolved fluorescence (HTRF) assay has been developed for determining the activity of HIV reverse transcriptase (HIV-RT). By using a sequential capping strategy, the assay has been configured to utilize only generic reagents such as biotinylated dUTP, streptavidin-allophycocyanin, and streptavidin-europium. The assay was optimized for a HIV-RT high-throughput screen. Under optimized conditions, a signal-to-background ratio of approximately 10:1 and a Z' factor of 0.8 were obtained. The titration curves of several known HIV-RT inhibitors have been evaluated with this assay format. This HTRF format can be used for high-throughput assays with other nucleotide polymerases.

Confirmation of primary active substances from high throughput screening of chemical and biological populations: a statistical approach and practical considerations.

Many biologically important substances are discovered through screening of relevant chemical or biological libraries. The ability to find the active substances ("hits") from any random collection is largely determined by the quality of the assay and screening conditions. When a large population is screened for a specific characteristic, each member of that population is usually tested only once. Errors in the measurements require additional follow-up tests to confirm that each hit from the primary screen is truly active. In this report, we present a statistical model system that predicts the reliability of hits from a primary test as affected by the error in the assay and the choice of the hit threshold (hit limit). The hit confirmation rate, as well as false positive (representing substances that initially fall above the hit limit but whose true activity are below the hit limit) and false negative (representing substances that initially fall below the hit limit but whose true activity are in fact greater than the hit limit) rates have been analyzed with this model by computational simulation. This model can also be used in screen validation and post-screening data analysis. The statistical analysis presented here has broad implications and is applicable to screening of any large population for any specific characteristic. Obvious applications include drug discovery, gene chip analysis, population biology, directed molecular evolution, biological panning, and combinatorial material sciences.

Analysis of protein-peptide interaction by a miniaturized fluorescence polarization assay using cyclin-dependent kinase 2/cyclin E as a model system.

As a result of the increasing size of chemical libraries, more rapid and highly sensitive strategies are needed to accelerate the process of drug discovery without increasing the cost. One means of accomplishing this is to miniaturize the assays that enter high-throughput screening (HTS). Miniaturization requires an assay design that has few steps, has a large degree of separation between the signal and background, and has a low well to well signal variation. Fluorescence polarization (FP) is an assay type that, in many cases, meets all of the above requirements. FP is a homogenous method that allows interactions between molecules to be measured directly in solution. This article demonstrates the application of FP in a miniaturized HTS format, using 1536-well plates, to measure direct binding between cyclin-dependent kinase 2/cyclin E complex (CDK2/E) and an 8-mer-peptide kinase inhibitor. The data indicate that low variability and high specificity allow rapid and precise identification of antagonist compounds affecting CDK2/E-peptide interactions.

Development of a carbon dioxide-capture assay in microtiter plate for aspartyl-beta-hydroxylase.

CO2-capture methods have been used for assaying many decarboxylating enzymes including hydroxylation-coupled decarboxylation reactions. The traditional CO2-capture method involves performing the reaction in capped tubes and radiometric measurement of trapped 14CO2 by scintillation counting. In this report, a 14CO2-capture method in a 96-well microtiter plate format has been developed and a phosphor imaging system has been employed for sample measurement. The new assay method has been used successfully to assay aspartyl-beta-hydroxylase activity in microtiter plate format. The results obtained here compare favorably with those obtained from the traditional tube method. The method is sensitive, suitable for high throughput, and generally applicable to many CO2-releasing enzyme assays.

Recombination-mediated PCR-directed plasmid construction in vivo in yeast.

We have extended the technique of PCR-directed recombination in Saccharomyces cerevisiae to develop a simple method for plasmid or gene construction in the absence of suitable restriction sites. The DNA to be cloned is PCR-amplified with 30-40 bp of homology to a linearized yeast plasmid. Co-transformation into yeast results in homologous recombination at a position directed by the PCR oligonucleotides.

Conformational studies on analogs of recombinant parathyroid hormone and their interactions with phospholipids.

Through the use of oligonucleotide-directed mutagenesis we have generated variants of a recombinant human parathyroid (PTH) hormone-(1-34)-homoserine (RPTH) in which a positively charged residue (Arg or Lys), a negatively charged residue (Glu), or a neutral residue (Gly) has been substituted at every position throughout the peptide. These 106 PTH analogs have been tested for their ability to stimulate cAMP production in the rat osteosarcoma cell line, UMR106. Analysis of these peptides led to the construction of several analogs containing multiple substitutions at sites of potential structural importance. Several of these analogs were shown to have 3-5-fold enhanced activity and receptor affinity. Circular dichroism (CD) and lipid binding studies were then performed on these analogs. Circular dichroism demonstrates enhanced helical content in the presence of lipid vesicles, particularly anionic lipid. The [Arg15,19,22,Lys29]RPTH (+6RPTH) analog requires higher concentrations of trifluoroethanol to attain enhanced helicity. The intrinsic tryptophan fluorescence of the peptides are blue shifted more in the presence of the anionic lipid dimyristoyl phosphatidylglycerol (DMPG) than with the zwitterionic lipid dimyristoyl phosphatidylcholine (DMPC). Effects of the peptides on the phase transition behavior of DMPC shows that +6RPTH has less effect on the lipid than does RPTH. This difference in lipid interaction is also exhibited with isothermal titration calorimetry, in which RPTH reacts exothermally with DMPG, while +6RPTH shows little or no heat change. The pH dependence of binding of the hydrophobic probe 1,1'-bis(4-anilino)-naphthalene-5,5'-trisulfonic acid, also shows a difference in exposure of hydrophobic sites between RPTH and +6RPTH. The +6RPTH has about a 5-fold greater affinity for receptor binding. We suggest that this enhanced activity is a consequence of the altered lipid interaction of +6RPTH, combined with increased conformational flexibility, particularly in the carboxyl-terminal region of the molecule.

Iontophoretic delivery of oligonucleotides across full thickness hairless mouse skin.

In recent years there has been an increased interest in the use of oligonucleotides as therapeutic agents. Oligonucleotide therapeutics may have significant potential over traditional drugs due to their high degree of specificity and increased affinity. The major drawbacks to the use of oligonucleotide therapeutics are the problems associated with their delivery and their relative instability in serum. The serum instability problem has been partially overcome through the use of oligonucleotides with modified backbones. Transdermal electrotransport may be used to overcome the problems associated with delivery. Here we report the use of transdermal electrotransport in the delivery of oligonucleotides across hairless mouse skin. The effects of pH, salt concentration, current density, and oligonucleotide concentration, structure, and length have been investigated.

A method for the high-level expression of a parathyroid hormone analog in Escherichia coli.

A recombinant parathyroid hormone analog, rPTH(1-34*), was obtained from Escherichia coli using a gene polymerization strategy. The PTH gene polymer contains up to 8 copies of the gene, each separated by a cleavable linker. The polymer was expressed at very high levels and formed inclusion bodies which could be easily isolated by low-speed centrifugation. A polyhistidine leader peptide allows rapid purification via nickel chelation chromatography of the PTH polymer solubilized from the inclusion bodies. Yields of greater than 500 mg/liter have been obtained. After isolating the polymer, monomeric rPTH(1-34*) is released from the polymer by chemical cleavage with cyanogen bromide. Following cyanogen bromide cleavage and high-performance liquid chromatography purification, highly purified, biologically active rPTH(1-34*) is obtained at a yield of approximately 300 mg/liter. This is a general strategy for the high-level production of a variety of peptides and small proteins.

Peptide ligands for a sugar-binding protein isolated from a random peptide library.

Peptide ligands for the carbohydrate-binding protein concanavalin A (Con A) have been identified by screening a large, diverse peptide library expressed on the surface of filamentous phage. A dodecapeptide containing the consensus sequence Tyr-Pro-Tyr was found to bind Con A with an affinity (dissociation constant, Kd) of 46 microM, comparable to that of a known carbohydrate ligand, methyl alpha-D-mannopyranoside (Kd of 89 microM). In addition the peptide inhibited precipitation of the alpha-glucan dextran 1355 by Con A. Given the complexity of oligosaccharide synthesis, the prospect of finding peptides that competitively inhibit carbohydrate-specific receptors may simplify the development of new therapeutic agents.

Invertebrate rhodopsin cleavage by an endogenous calcium activated protease.

Calcium-activated proteases have been purified from a number of vertebrate tissues, including the retina and lens. These proteases exhibit similar characteristics and are believed to be involved in the regulation of cytoskeletal elements. Here we report the partial purification and characterization of a calcium-activated protease from the squid photoreceptor cell which, when activated, specifically removes 10 kDa from the carboxyl-terminal of squid rhodopsin. No other detectable soluble proteins from the invertebrate photoreceptor are susceptible to cleavage and only one non-opsin, integral membrane protein shows evidence of cleavage. The enzyme requires 5 mM calcium for half maximal activation, and is not significantly activated by other divalent ions. The protease has a molecular weight of approximately 350 kDa, as determined by gel filtration, and when partially purified by casein affinity chromatography, it runs as three main bands of 76, 63 and 36 kDa on SDS-PAGE. The crude protease loses as much as 80% of its activity in 24 hr, whereas the partially purified protease is stable up to 4 weeks at 4 degrees C.