Introduction to Enzyme-Drug Assays
Instructor: Roberta King, PhD, University of Rhode Island
Location: URI
Session 1: June 3 – June 5 ( Lab: Room 305 / Lecture Room: Avedisian Rm 207)
Session 2: June 10 – June 12 ( Lab: Room 305 / Lecture Room: Avedisian Rm 207)
Course Overview
This WDT Module (Drug Interactions by CYP450 Enzymes) is designed to instruct students in an important biochemical assay that is used in the pharmaceutical industry during pre-clinical drug discovery and development. Instruction will include both theoretical and hands-on practical training.
Participants will learn to conduct the enzyme assay used to detect drug-drug interactions through inhibition of cytochrome P450 (CYP) drug metabolizing enzymes using the widely used Vivid® CYP450 Screening Kit.
Students will review basic skills such as micropipetting and making solutions of known concentrations while gaining more advanced skills in fluorescence spectroscopy to measure concentration, enzyme inhibition kinetics, and drug metabolism-based drug-drug interactions.
The module will be completed over 2.5 days and participants who complete the module will receive an RI-INBRE Certificate of completion
Learning Outcomes
- Appreciate the role of CYP450 inhibition assays in pre-clinical drug development
- Conduct CYP450 enzyme assays under appropriate conditions for product formation and inhibition at microliter volume scale
- Appreciate how chemical structure influences fluorescence spectroscopy regarding excitation and emission wavelengths, and how intensity is related to concentration and volume
- Learn how to calculate inhibition constants (IC50 values) from experimental data
- Calculate and perform with accuracy and precision serial dilutions of test compounds, microliter transfers of enzyme reagents, and timed measurements of enzyme activity
Lab Report
- Students will be expected to maintain detailed laboratory notes to include:
- Calculations need for each step, showing main equation and details for that step.
- Volumes and concentrations of reagents used in each pipetting step.
- Diagram of the placement of solutions in student’s 96-well plate (i.e., what is in each well).
- Recording of data obtained from the instruments.
- Graphical analysis of data using Excel graphing features.
- Conclusions draws from each step and plans for next step.
- Sources of reagents and all equipment used.
Additional Information:
It is recommended that students bring a tablet, iPad, or laptop to make entries in their electronic lab notebook. Lecture slides and laboratory handouts will be provided to students.
Timeline
| Day 1 | Day 2 | Day 3 | |
|---|---|---|---|
| 9:00 AM-10:00 AM | Overview (Classroom) | Discuss results from previous day (classroom) | CYP assay set #5 |
| 10:00 AM-11:00 AM | CYP assay #1: Detect known concentrations of your metabolite (dilution series and fluorescence measurement) (Hands-on) | CYP assay set #3 | CYP assay set #5 continued |
| 11:00 AM-12:00 PM | Graph data, discuss results, prepare for afternoon. (Hands-On) | Graph data, discuss results, prepare for afternoon. (Lecture and Hands-On) | Graph data, Discussion of results, Wrap up. |
| 12:00 - 1:00 PM | Break | Break | WDT Survey and Certificate Distribution |
| 1:00 PM-2:00 PM | CYP assay set #2 (Hands-On) | CYP assay set #4 | |
| 2:00 PM-3:00 PM | CYP assay set #2 continued | CYP assay set #4 continued | |
| 3:00 PM-4:00 PM | Prepare for CYP assay #3, make own inhibitor dilution series. Graph data, Discussion of results | Graph data, Discussion of results |