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Development of high throughput screening (HTS) assays for evaluation of a compound's toxicity and potential for drug-drug interactions is a critical step towards production of better drug candidates and cost reduction in the drug development process. HTS assays for drug metabolism mediated by cytochrome P450s are now routinely used in compound library characterization and for computer modeling studies. However, development and application of HTS assays involving UDP-glucuronosyltransferases (UGTs) are lagging behind. Here we describe the development of a fluorescence-based HTS assay for UGT1A1 using recombinant enzyme and fluorescent substrate in the presence of an aqueous solution of PreserveX-QML (QBI Life Sciences, Madison, WI) polymeric micelles, acting as a stabilizer and a blocker of nonspecific interactions. The data include assay characteristics in 384-well plate format obtained with robotic liquid handling equipment and structures of hits (assay modifiers) obtained from the screening of a small molecule library at the University of Wisconsin HTS screening facility. The application of the assay for predicting UGT-related drug-drug interactions and building pharmacophore models, as well as the effects of polymeric micelles on the assay performance and compound promiscuity, is discussed.

Purpose: Polymeric micelles have been used for solubilization of insoluble drugs and as carriers for drug delivery applications. Here we evaluated an application of the synthetic polymeric micelles in experiments designed to improve the handling and stability of membrane proteins targets., Methods: Particle sizing by dynamic light scattering was performed in a Zeta Plus Photon Correlation Spectrometer at 532 nm. UGT1A1 activity has been measured in fluorescent assay using scopoletin as a substrate. COX-2 activity has been measured in a fluorescent assay using Amplex Red. Fluorescence Resonance Energy Transfer (FRET) was monitored using either 463 nm excitation wavelength (the emission range 500-600 nm) or 395 nm excitation wavelength (the emission range 500-600 nm)., Results: Incorporation of membrane proteins into PreserveX-QML polymeric micelles resulted in improved homogeneity and stability of the preparation and in reduced light scattering. Stabilization of the biological activity of micelle-incorporated membrane proteins, such as the human UGT1A1 and COX-2 both during extended incubations at room temperature and during multiple freeze/thaw cycles, has been achieved., Conclusion: PreserveX-QML polymeric micelles help to homogenize and disperse membrane proteins preparations and stabilize the biological activity of the proteins making it more suitable for pharmaceutical assays and applications.

Highly miniaturized P450 screening assays designed to enable facile analysis of P450 drug interactions in a 1536-well plate format with the principal human cytochrome P450 enzymes (CYP3A4, 2D6, 2C9, 2C19, and 1A2) and Vivid fluorogenic substrates were developed. The detailed characterization of the assays included stability, homogeneity, and reproducibility of the recombinant P450 enzymes and the kinetic parameters of their reactions with Vivid fluorogenic substrates, with a focus on the specific characteristics of each component that enable screening in a low-volume 1536-well plate assay format. The screening assays were applied for the assessment of individual cytochrome P450 inhibition profiles with a panel of selected assay modifiers, including isozyme-specific substrates and inhibitors. IC(50) values obtained for the modifiers in 96- and 1536-well plate formats were similar and comparable with values obtained in assays with conventional substrates. An overall examination of the 1536-well assay statistics, such as signal-to-background ratio and Z' factor, demonstrated that these assays are a robust, successful, and reliable tool to screen for cytochrome P450 metabolism and inhibition in an ultra-high-throughput screening format.

CYP2C9 is a genetically polymorphic human cytochrome P450 isozyme involved in the oxidative metabolism of many drugs, including nonsteroidal anti-inflammatory compounds. Individuals genotyped heterozygous or homozygous for CYP2C9 allelic variants have demonstrated altered metabolism of some drugs primarily metabolized by CYP2C9. The ability to expand screening of CYP2C9 allelic variants to a larger set of drugs and pharmaceutical agents would contribute to a better understanding of the significance of CYP2C9 polymorphisms in the population and to predictions of possible outcomes. The authors report the development of an in vitro fluorescence-based assay employing recombinant CYP2C9 variants (CYP2C9*1, CYP2C9*2, and CYP2C9*3) and fluorogenic Vivid(R) CYP2C9 substrates to explore the effects of CYP2C9 polymorphisms on drug metabolism, using drugs primarily metabolized by CYP2C9. Several chemically diverse fluorogenic substrates (Vivid(R) CYP2C9 blue, green, and red substrates) were used as prototypic probes to obtain in vitro CYP2C9 metabolic rates and kinetic parameters, such as apparent K(m), V(max), and V(max)/K(m) ratios for each allelic variant. In addition, a diverse panel of drugs was screened as assay modifiers with CYP2C9*1, CYP2C9*2, CYP2C9*3, and the fluorogenic Vivid(R) CYP2C9 substrates. The inhibitory potential of this large group of chemically diverse drugs and compounds has been assessed on the basis of their ability to compete with Vivid(R) CYP2C9 substrates in fluorescent reporter assays, thus providing a sensitive and quick assessment of polymorphism-dependent changes in CYP2C9 metabolism.

CYP2B6 is a highly polymorphic P450 isozyme involved in the metabolism of endo- and xenobiotics with known implications for the activation of many procarcinogens resulting in carcinogenesis. However, lack of validated high-throughput screening (HTS) CYP2B6 assays has limited the current understanding and full characterization of this isozyme's involvement in human drug metabolism. Here, we have developed and characterized a fluorescence-based HTS assay employing recombinant human CYP2B6 and 2 novel fluorogenic substrates (the Vivid CYP2B6 Blue and Cyan Substrates). Assay validation included testing the inhibitory potency of a panel of drugs and compounds known to be metabolized by this isozyme, including CYP2B6 substrates, inhibitors, and known inducers. Compound rankings based on inhibitory potency in the Vivid CYP2B6 Blue and Cyan Assays matched compound rankings based on relative affinity measurements from previously published data (K(i), K(d), or K(m) values) for the CYP2B6 isozyme. In conclusion, these assays are proven to be robust and sensitive, with broad dynamic ranges and kinetic parameters allowing screening in HTS mode of a large panel of compounds for CYP2B6 metabolism and inhibition, and are a valuable new tool for CYP2B6 studies.

Large-scale screening of multiple compound libraries and combinatorial libraries for pharmacological activity is one of the novel approaches of the modern drug discovery process. The application of isozyme-specific high-throughput screening (HTS) assays for characterizing the interactions of potential drug candidates with major human drug-metabolizing cytochrome p450 enzymes (p450s) is newly becoming an essential part of this process. Fluorescence-based HTS assays have been successfully employed for in vitro assessment of drug-drug interactions and enzyme inhibition with several p450 isoforms, including CYP3A4, CYP2D6, CYP2C9, and CYP2C19. Here we describe a fluorescence-based HTS assay for detecting drug metabolism and inhibition with human CYP2E1. CYP2E1 plays an important role in the metabolism of several drugs, many solvents, and toxins and therefore has been repeatedly linked to numerous pathologies, including cancer, liver and kidney toxicity, diabetes, and alcoholism. The assay is based on the ability of a drug to compete with the fluorogenic Vivid CYP2E1 Blue Substrate for CYP2E1 metabolism and thus enables rapid screening of lead molecules for their inhibitory potential. We have used this assay to screen a panel of drugs and compounds for their effects on CYP2E1 metabolism and inhibition. Our results demonstrate the assay's usefulness in identifying CYP2E1 substrates and inhibitors and in enabling in-depth characterization of their interactions with the CYP2E1 isozyme. We also present detailed characteristics of the assay, including its dynamic range and Z'-factor values, which indicate that this robust assay is well suited for kinetic and inhibition studies in HTS formats.

Beta-glucuronidase cleavage of 4-methylumbelliferyl beta-D-glucuronide generates the highly fluorescent compound, 4-methylumbelliferone. We show that other beta-D-glucuronide compounds act as competitors in this assay. The 4-methylumbelliferyl beta-D-glucuronide cleavage assay can easily be adapted to high throughput formats to detect the presence of beta-D glucuronides generated using recombinant glycosyl transferase preparations.

Dunkin Hartley guinea pigs develop spontaneous, age-related osteoarthritis (OA) of the knee and other joints. Histologic changes are observed beginning at 3 months of age. Disease severity increases with age, and at 18 months moderate to severe OA is observed. A study was undertaken to assess the morphologic and biochemical changes of 22-month-old animals, and to compare them with values in 2-month-old guinea pigs. Biochemical indices characteristic of OA, from tibial cartilage, indicated an increase in proteoglycan content from 233 +/- 2 micrograms/mg (mean +/- SEM) at 2 months of age to 365 +/- 6 micrograms/mg at 22 months. Collagen concentration in cartilage decreased from 364 +/- 2 micrograms/mg at 2 months to 223 +/- 3 micrograms/mg at 22 months. Proteoglycan fragments found in synovial fluid measured 4.6 +/- 1 micrograms/ml at 2 months and increased to 37 +/- 2 micrograms/ml at 22 months. Radiographic changes observed at 22 months included marginal osteophytes of the tibia and femur, sclerosis of the subchondral bone of the tibial plateau, femoral condyle cysts, and calcification of the collateral ligaments. Histologic evaluation revealed severe OA, with a Mankin score of 10.7 +/- 0.5 in 22-month-old animals. In contrast, 2-month-old animals had no histologic or radiographically detectable lesions. The results of the study reported here indicate that the lesions observed in this model are similar to those of human OA. Spontaneous development of OA in guinea pigs is amenable to the study of the pathogenesis of OA and to the evaluation of potential disease-modifying agents.

Lysyl oxidase is secreted from fibrogenic cells as a 50-kDa proenzyme that is proteolytically processed to the mature enzyme in the extracellular space. To characterize the secreted proteinase activity, a truncated, recombinant form of lysyl oxidase was prepared as a proteinase substrate containing the sequence of the propeptide cleavage region. The processing proteinase activity secreted by cultured fibrogenic cells resists inhibitors of serine or aspartyl proteinases as well as tissue inhibitor of matrix metalloproteinases-2 (MMP-2) but is completely inhibited by metal ion chelators. Known metalloproteinases were tested for their activity toward this substrate. Carboxyl-terminal procollagen proteinase (C-proteinase), MMP-2, and conditioned fibrogenic cell culture medium cleave the lysyl oxidase substrate to the size of the mature enzyme. The NH2-terminal sequence generated by arterial smooth muscle conditioned medium and the C-proteinase but not by MMP-2, i.e. Asp-Asp-Pro-Tyr, was identical to that previously identified in mature lysyl oxidase isolated from connective tissue. The C-proteinase activity against the model substrate was inhibited by a synthetic oligopeptide mimic of the cleavage sequence (Ac-Met-Val-Gly-Asp-Asp-Pro-Tyr-Asn-amide), whereas this peptide also inhibited the generation of lysyl oxidase activity in the medium of fetal rat lung fibroblasts in culture. In toto, these results identify a secreted metalloproteinase activity participating in the activation of prolysyl oxidase, identify inhibitors of the processing activity, and implicate procollagen C-proteinase in this role.