Screening MT1-MMP Activity and Inhibition in Three-Dimensional Tumor Spheroids.

Membrane type 1 matrix metalloproteinase (MT1-MMP) has been shown to be crucial for tumor angiogenesis, invasion, and metastasis, and thus MT1-MMP is a high priority target for potential cancer therapies. To properly evaluate MT1-MMP inhibitors, a screening protocol is desired by which enzyme activity can be quantified in a tumor microenvironment-like model system. In the present study, we applied a fluorogenic, collagen model triple-helical substrate to quantify MT1-MMP activity for tumor spheroids embedded in a collagen hydrogel. The substrate was designed to be MT1-MMP selective and to possess fluorescent properties compatible with cell-based assays. The proteolysis of the substrate correlated to glioma spheroid invasion. In turn, the application of either small molecule or protein-based MMP inhibitors reduced proteolytic activity and glioma spheroid invasion. The presence of MT1-MMP in glioma spheroids was confirmed by western blotting. Thus, spheroid invasion was dependent on MT1-MMP activity, and inhibitors of MT1-MMP and invasion could be conveniently screened in a high-throughput format. The combination of the fluorogenic, triple-helical substrate, the three-dimensional tumor spheroids embedded in collagen, and Hit-Pick software resulted in an easily adaptable in vivo-like tumor microenvironment for rapidly processing inhibitor potential for anti-cancer use.

Kinetic operational models of agonism for G-protein-coupled receptors.

The application of kinetics to research and therapeutic development of G-protein-coupled receptors has become increasingly valuable. Pharmacological models provide the foundation of pharmacology, providing concepts and measurable parameters such as efficacy and potency that have underlain decades of successful drug discovery. Currently there are few pharmacological models that incorporate kinetic activity in such a way as to yield experimentally-accessible drug parameters. In this study, a kinetic model of pharmacological response was developed that provides a kinetic descriptor of efficacy (the transduction rate constant, kτ) and allows measurement of receptor-ligand binding kinetics from functional data. The model assumes: (1) receptor interacts with a precursor of the response ("Transduction potential") and converts it to the response. (2) The response can decay. Familiar response vs time plots emerge, depending on whether transduction potential is depleted and/or response decays. These are the straight line, the "association" exponential curve, and the rise-and-fall curve. Convenient, familiar methods are described for measuring the model parameters and files are provided for the curve-fitting program Prism (GraphPad Software) that can be used as a guide. The efficacy parameter kτ is straightforward to measure and accounts for receptor reserve; all that is required is measurement of response over time at a maximally-stimulating concentration of agonist. The modular nature of the model framework allows it to be extended. Here this is done to incorporate antagonist-receptor binding kinetics and slow agonist-receptor equilibration. In principle, the modular framework can incorporate other cellular processes, such as receptor desensitization. The kinetic response model described here can be applied to measure kinetic pharmacological parameters than can be used to advance the understanding of GPCR pharmacology and optimize new and improved therapeutics.

Retrospective evaluation of the Duracon periapatite-coated tibial tray: midterm results and factors affecting success.

This study reports the retrospective radiographic outcome of a series of 63 consecutive total knee arthroplasties using an uncemented hydroxyapatite-coated Duracon cruciate-retaining tibial baseplate (Stryker Howmedica Osteonics Corp, Mahwah, NJ). Sixty-three knees were assessed at a mean follow-up of 65 months. The knees were primarily diagnosed with osteoarthritis with a mean age of 61 years. Radiographic analysis showed 6 knees with tibial baseplate radiolucencies, with all of these resolving or improving over the course of the study. All tibial baseplates were implanted with the concomitant use of autologous bone slurry. There were no reoperations for aseptic loosening, fracture, or patellofemoral problems. This intermediate study demonstrates excellent radiographic outcomes for uncemented hydroxyapatite-coated Duracon cruciate-retaining tibial baseplates and evaluates other factors felt to be important in the success of a cementless implant.

Automation of cell-based drug absorption assays in 96-well format using permeable support systems.

Cell-based drug absorption assays, such as Caco-2 and MDCK-MDR1, are an essential component of lead compound ADME/Tox testing. The permeability and transport data they provide can determine whether a compound continues in the drug discovery process. Current methods typically incorporate 24-well microplates and are performed manually. Yet the need to generate absorption data earlier in the drug discovery process, on an increasing number of compounds, is driving the use of higher density plates. A simple, more efficient process that incorporates 96-well permeable supports and proper instrumentation in an automated process provides more reproducible data compared to manual methods. Here we demonstrate the ability to perform drug permeability and transport assays using Caco-2 or MDCKII-MDR1 cells. The assay procedure was automated in a 96-well format, including cell seeding, media and buffer exchanges, compound dispense, and sample removal using simple robotic instrumentation. Cell monolayer integrity was confirmed via transepithelial electrical resistance and Lucifer yellow measurements. Proper cell function was validated by analyzing apical-to-basolateral and basolateral-to-apical movement of rhodamine 123, a known P-glycoprotein substrate. Apparent permeability and efflux data demonstrate how the automated procedure provides a less variable method than manual processing, and delivers a more accurate assessment of a compound's absorption characteristics.

Automated triplexed hepatocyte-based viability and CYP1A and -3A induction assays.

Cytochrome P450 (CYP) enzymes are key players in drug metabolism. Therefore, it is essential to understand how these enzymes can be affected by xenobiotics with regards to induction and toxicity to avoid potential drug-drug interactions. Typically, information has been gathered by combining data from multiple experiments, which is time-consuming and labor intensive, and interassay variability may lead to misinterpretation. Monitoring CYP induction and cytotoxicity by xenobiotics using an automated, multiplexed format can decrease workload and increase data confidence. Here the authors demonstrate the ability to monitor CYP1A and CYP3A4 induction, combined with a cytotoxicity measurement, from a single microplate well using cryopreserved human hepatocytes. The assay procedure was automated in a 384-well format, including cell manipulations, compound titration and transfer, and reagent dispensing, using simple robotic instrumentation. EC(50) and E(max) values were derived for multiple known CYP1A and -3A4 inducers. Induction and toxicological responses in the triplex system were validated based on literature values from conventional single-parameter assays. Validation and pharmacology data confirm that multiplexed cell-based CYP assays can simplify workload, save time and effort, and generate biologically relevant data.

Automation and miniaturization of the bioluminescent UGT-Glo assay for screening of UDP-glucuronosyltransferase inhibition by various compounds.

The uridine 5'-diphospho-glucuronosyltransferase (UGT) family of enzymes is involved in the metabolism of various compounds. These enzymes transfer a hydrophilic glucuronic acid moiety to their substrates, rendering them more water soluble and amenable to excretion. The UGTs act on various endogenous substrates, such as bilirubin, 17ß-estradiol, and testosterone, and drugs and other xenobiotics. The function of these enzymes is essential for the clearance of drugs and toxicants, and alteration of UGT activity is a potential cause of adverse drug-drug interactions in vivo. This has stimulated an increased interest in the study of UGT function and inhibition, and the desire to profile new drug entities against UGT enzymes, similar to CYP450 profiling. However, certain factors have hindered the development of a robust method for UGT profiling. Current methods for assessing UGT enzyme activity are laborious and involve protein precipitation and/or chromatographic separation steps, which are not amenable to rapid screening applications for UGT inhibitors or substrates. The approach presented here is a bioluminescent assay for measuring UGT enzyme activity and inhibition in vitro. Using flexible, robust instrumentation in a 384-well microplate format, this study highlights the quick and easy assay implementation for estimation of inhibition kinetics with a variety of known and suspected UGT substrates and inhibitors.

Automated luminescence-based cytochrome P450 profiling using a simple, elegant robotic platform.

The determination of inhibitory effects that lead compounds have on cytochrome P450 (CYP) enzymes is an important part of today's drug discovery process. Assays can be performed early in the discovery process to predict adverse drug-drug interactions caused by CYP inhibition and to minimize the costs associated with terminating candidates in late stage development or worse, removing a drug from the market after launch. For early discovery work, testing substantial numbers of compounds is desirable, thus automated "mix and read" assays are beneficial. Here, we demonstrate the automation of the CYP profiling process using a simple, yet robust robotic platform. Compound titration, as well as transfer of compounds and assay components was performed by the same automated pipetting system. IC(50)s of small molecule drugs were determined using recombinant CYP enzymes, CYP3A4, -2C9, and -2D6 and luminogenic substrates specific to each. Compounds were profiled against all three enzymes on the same 384-well assay plate.

A kinematic comparison of spring-loaded and traditional crutches.

CONTEXT: A novel spring-loaded-crutch design may provide patients additional forward velocity, relative to traditional axillary crutches; however, this idea has not yet been evaluated. OBJECTIVE: To quantify elastic potential energy stored by spring-loaded crutches during crutch-ground contact and determine whether this energy increases forward velocity for patients during crutch ambulation. Because elastic potential energy is likely stored by the spring-loaded crutch during ambulation, the authors hypothesized that subjects would exhibit greater peak instantaneous forward velocity during crutch-ground contact and increased preferred ambulation speed during spring-loaded-crutch ambulation, relative to traditional-crutch ambulation. DESIGN: Within-subject. SETTING: Biomechanics laboratory. PARTICIPANTS: 10 healthy men and 10 healthy women. INTERVENTIONS: The independent variable was crutch type: Subjects used spring-loaded and traditional axillary crutches to ambulate at standardized and preferred speeds. MAIN OUTCOME MEASURES: The primary dependent variables were peak instantaneous forward velocity and preferred ambulation speed; these variables were quantified using high-speed videography and an optoelectronic timing device, respectively. Between-crutches differences for the dependent variables were evaluated using paired t tests (α = .05). Elastic potential energy stored by the spring-loaded crutches during crutch-ground contact was also quantified via videography. RESULTS: Peak forward velocity during crutch-ground contact was 5% greater (P < .001) for spring-loaded-crutch ambulation than for traditional-crutch ambulation. Preferred ambulation speed, however, did not significantly differ (P = .538) between crutch types. The spring-loaded crutches stored an average of 2.50 ± 1.96 J of elastic potential energy during crutch-ground contact. CONCLUSIONS: The spring-loaded crutches appear to have provided subjects with additional peak instantaneous forward velocity. This increased velocity, however, was relatively small and did not increase preferred ambulation speed.

The utility of semi-automating multiplexed assays for ADME/Tox applications.

ADME-Tox testing examines the effects of an organism, tissue or cell on a compound, as well as the effects that the compound has on an organism, tissue or cell, and has gained in importance in the overall drug discovery process over the past twenty years. This is due to the rising percentage of drug candidate attrition in the 1990s and early 2000s due to adverse ADME/Tox profiles. The increased importance placed upon ADME/Tox testing has brought about new types of in-vitro assay technologies utilizing microplates to deliver the most pharmacologically relevant data. These tests, however, have typically been performed sequentially, where multiple assays over multiple microplates are used. This typically leads to increased time and cost required to generate the required information, and can sacrifice data quality. Multiplexed assays, however, where more than one piece of data can be attained from a single well or a single microplate, performed using appropriate liquid handling and detection instrumentation, can improve data quality and reduce the time and expense required to attain this information.

Characterization and optimization of a red-shifted fluorescence polarization ADP detection assay.

ATP depletion and ADP formation are generic detection methods used for the identification of kinase and other ATP-utilizing enzyme inhibitors in high-throughput screening campaigns. However, the most widely used nucleotide detection approaches require high ATP consumption rates or involve the use of coupling enzymes, which can complicate the selection of lead compounds. As an alternative, we have developed the Transcreener (BellBrook Labs, Madison, WI) platform, which relies on the direct immunodetection of nucleotides. Here we describe the development of antibodies with >100-fold selectivity for ADP versus ATP, which enable robust detection of initial velocity rates (Z' > 0.7 at 10% substrate consumption) at ATP concentrations ranging from 0.1 microM to 1,000 microM in a competitive fluorescence polarization (FP) immunoassay. Competitive binding experiments indicate similar affinities for other nucleotide diphosphates, including 2' -deoxy ADP, GDP, and UDP. The antibody-tracer complex and the red-shifted, ratiometric FP signal are stable for at least 24 h at room temperature, providing suitable conditions for high-throughput screening. A method for calculating a kinase ATP Km with this FP immunoassay is also presented. The Transcreener ADP assay provides a simple, generic assay platform for inhibitor screening and selectivity profiling that can be used for any ADP-generating enzyme.

A Simple and robust automated kinase profiling platform using luminescent ADP accumulation technology.

Kinases continue to be one of the most important targets in today's drug discovery efforts. Following the identification of lead compounds through screening efforts, it is important to profile these leads against other kinases within that family, as well as from other families, to ascertain potential off-target effects. Because many kinase assays require the use of different substrates, optimization time and costs during profiling can be prohibitive. Here we demonstrate the versatility of a luminescent ADP accumulation assay, where one set of reagents can be used for a wide variety of kinases with differing K(m app) for ATP and substrates. Assay sensitivity allows for the use of low enzyme concentrations and small percent ATP conversion levels while still maintaining high signal:background ratios. We have used a simple, inexpensive automated pipetting system to automate the entire process from enzyme optimization through generation of compound IC(50) values. Agreement with literature values proves this combination of chemistry and instrumentation provides a simple, yet robust solution for automated kinase profiling.

Fluoroscopically-assisted hamstring ACL reconstruction.

The fluoroscope can be a valuable tool during hamstring ACL reconstruction when appropriate measures are taken to minimize radiation exposure risk to patients and operating room personnel.

Clinical stage EGFR inhibitors irreversibly alkylate Bmx kinase.

Irreversible HER/erbB inhibitors selectively inhibit HER-family kinases by targeting a unique cysteine residue located within the ATP-binding pocket. Sequence alignment reveals that this rare cysteine is also present in ten other protein kinases including all five Tec-family members. We demonstrate that the Tec-family kinase Bmx is potently inhibited by irreversible modification at Cys496 by clinical stage EGFR inhibitors such as CI-1033. This cross-reactivity may have significant clinical implications.

Bicycle seat designs and their effect on pelvic angle, trunk angle, and comfort.

PURPOSE: To examine whether bicycle seats with anterior-medial cutouts influence pelvic angle, trunk angle, and comfort in female subjects during cycling. METHODS: Twenty female cyclists pedaled a stationary bicycle with their hands on the tops and drops of the handlebars under three different saddle conditions (standard, partial, and complete cutout designs). Pelvic angle was measured using an inclinometer attached to a caliper whereas trunk angle was quantified from digitization of video images. Comfort level was assessed subjectively by having participants rank the saddles from most to least comfortable. RESULTS: Anterior pelvic tilt angles for the partial and complete cutout saddles were 8% and 16% greater, respectively, than values for the standard saddle condition ( P < 0.05). Trunk flexion angles were greater for the complete versus standard and partial cutout designs ( P< 0.05). Participants displayed a 77% greater anterior pelvic tilt angle and an 11% greater trunk flexion angle in the drop versus top handlebar positions ( P < 0.05). A total of 55% of the subjects ranked the partial cutout saddle as the most comfortable, and 30% ranked the standard saddle as the most comfortable. CONCLUSIONS: These data indicate that partial and complete cutout saddle designs may increase anterior pelvic tilt, and saddles with a complete cutout design may increase trunk flexion angles under select cycling conditions. A saddle with a partial cutout design may be more comfortable than a standard or complete cutout saddle design.