featuring the Echo Acoustic Technology
TITLES and AUTHORS
Cytochrome P450 (CYP) and monoamine oxidases (MAO) are two groups of enzymes that metabolize drugs. The interactions of these enzymes and compounds are studied carefully in early drug discovery to facilitate greater success in clinical trials. Luminescent metabolism assays consist of the P450-Glo™ Screening Systems (Promega, Madison, WI) for CYP 1A2, 2C9, 3A4, 2C19 and 2D6, as well as the MAO-Glo™ Assay System (Promega) for monoamine oxidase A. A collection of drug-like compounds was profiled by IC50 analyses against a panel of seven (7) metabolism assays in 1536-well format using acoustic droplet ejection (ADE) coupled with low-volume reagent dispensers. ADE was performed with the Labcyte® Echo™ 550 acoustic liquid handler and low-volume reagent fills were made with a Deerac Fluidics Equator HTS reagent dispenser. ADE and low-volume reagent dispensing provided total enzyme assay volumes of 5 μL. Final analysis volumes were 10 μL.
This article describes metabolic profiling applications in which compounds are assayed against several different drug metabolizing enzymes on the same assay plate. We obtain potency data for cytochrome P450 (CYP450) and monoamine oxidase A (MAO) using this parallel approach and testing compounds in a dose-response format using both 384-well and 1536-well plate formats.
Dose-response experiments, such as IC50 analyses, are time- and labor-intensive. They usually require multiple serial dilutions and large amounts of sample. Aqueous serial dilutions of concentrated stock solutions can precipitate or otherwise become biologically unavailable generating false negatives. Serial dilutions may also accumulate significant error. Cross-contamination of wells is a constant possibility. Finally, even low levels of DMSO in the final assay can dramatically affect the apparent activity of the compound especially in the case of cell-based assays. A system incorporating acoustic ejection of nanoliter droplets of active compounds dissolved in DMSO improves IC50 analyses by eliminating accumulated error (CV%<10% over the entire sample concentration range), eliminating compound precipitation in intermediate dilutions, reducing consumables (plastics and solvents), reducing DMSO concentrations in the final assay to significantly less than 1% and reducing the amount of compound used in the analysis to nanomoles.
IC50 analyses are typically time- and labor-intensive, requiring multiple dilution steps and significant amounts of sample compound. Aqueous dilutions of concentrated stock solutions can lead to sample precipitation and the generation of false negatives. Multiple serial dilutions result in significant accumulated error. Some compounds may stick to pipette tips, reducing the concentration of the analyte in the dilution and also increasing the possibility of contaminating other dilutions. DMSO levels in the final assay solution can affect the apparent activity of the compound especially in the case of cell-based assays.A system incorporating acoustic ejection of nanoliter droplets of active compounds dissolved in DMSO improves IC50 analyses by eliminating accumulated error (CV%<10% over the entire sample concentration range), eliminating compound precipitation in intermediate dilutions, reducing consumables (plastics and solvents), reducing DMSO concentrations in the final assay to significantly less than 1% and reducing the amount of compound used in the analysis to nanomoles. Quantitative improvements in each of these areas will be shown.
- Control of hydration level of DMSO in library stocks
- Control of the concentration of the compound in library stocks
- Use of acoustics for determination of hydration and volume
-Experiment: hydration, dehydration and restoration of DMSO hydration level and compound concentration
The stage has been set for low-microliter compound storage systems dedicated to closed-loop screening. Reliable low-nanoliter dispensing, robust low-microliter assays and accurate high-density microplate readers were necessary, yet insufficient, precursors to closed-loop screening. Hundreds of assays, each using 5 nL of compound solution extracted from the same well of a 1536-well microplate can address compound availability. The next challenge is to extend compound lifetime. Reduction of solvent hydration and evaporation is essential to ensure compound integrity. We show methods for preserving compound in 1536-well microplates. In particular, a novel room-temperature storage method will be introduced that maintains a dry, dehydrating and DMSO-rich microclimate for all the wells within a 1536-well storage plate. We compare the results from microplates stored with conventional seals or lids to novel techniques. Preservation of compound for use in a closed-loop screening environment where plates are accessed hundreds of times per year will be explored.
May of the HTS assays at Merck are performed in the 3456-well format to enable rapid screening of the sample collection and to reduce the cost of an HTS campaign. Our laboratory has successfully miniaturized many HTS assays to the 3456 format using Aurora Discovery's Flying Reagent Dispenser™ (FRD) and the tcPR™ detector. However, we did not have the capability to transfer 5nL of compound solutions to the 3456 assay plate. The Echo 550 was chosen to perform this task. We will show reproducibility, accuracy and precision data of the 5nL acoustic droplet ejection into the 3456-well plates using fluorescent dyes. Results from a panel of compounds tested in a 3456-well enzymatic assay will also be presented and compared to the original 384-well format.
Even a cursory look at the laboratory automation and robotics field will reveal that key recent accomplishments revolve largely around the ability to handle tiny volumes efficiently and accurately and on innovative technologies that render liquid handling systems far more capable. "In the last few years the key advancement in laboratory automation has really come in the miniaturization process, that is, driving down from 384 well plates to 1536 well plates and beyond," says Jonathan O'Connell, PhD, Group Leader in Lead Discovery, Bristol Myers Squibb (BMS).
The continued miniaturisation of assay technologies in high throughput screening, compound management and protein crystallisation has created a pressing need for improved nanolitre liquid handling systems.A recent industry market survey suggests that although the total liquid handing market in pharma drug discovery is contracting, the nanolitre dispensing segment is expected to grow to $56 million US dollars in 2004 with a 45% annual growth rate. Growth over the next two years is predicted to be especially strong for devices based on acoustic transducers and solenoid sensors.This review takes a look at some of the latest offerings in this increasingly competitive market place and discusses some recent industry trends. Customer expectations have now shifted from lower volumes to improved performance and reliability, added value and maximising the quality to price ratio.>
Bristol-Myers Squibb has implemented a flexible, fully automated UHTS process. Key to the process is compound reformatting with the Echo 550 nanoliter dispensing technology. They plan to implement a flexible closed loop screening process.
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