EDA – The Eserine Experiment

1. EDA – The Eserine Experiment • Setting the Scene • We work in the Technical Services Dept of a pharmaceutical company • They are interested in new compounds that –ve ACh-esterase • They want to measure the half-life of their putative inhibitors • We are tasked with seeing if an existing assay for ACh-esterase can be used to measure the half-life of an existing –ve, Eserine

2. EDA – The Eserine Experiment • Why the interest in Acetylcholinesterase Inhibitors? • Acetylcholine (ACh) is a NT used extensively by central and peripheral nervous system • eg the neuromuscular junction (NMJ) that mediates the control of skeletal muscle contraction by α-motoneurones • Alzheimer’s Disease (AD) is characterised by an ACh deficit • AChE is the enzyme responsible for breaking down ACh, so -ve the enzyme will potentiate the effects of ACh • At NMJ  uncontrolled contraction  paralysis • Fly spray    nerve gasses • Medium potency AChE –ve used to treat myasthenia gravis • Centrally, could help redress memory loss in AD

3. EDA – The Eserine Experiment • What Acetylcholinesterase Inhibitor will we use? • Fly spray  • Nerve gas  • Eserine (physostigmine)  • Medium potency (ie toxic!), used in MG • Binds to active site – covalently when cleaved • Effects persist

4. EDA – The Eserine Experiment • How are we going to use Eserine? • We are trying to measure the half-life of AChE inhibitors, using Eserine as a test-case • So, we need… • a way of measuring the ‘biological’ activity of Eserine samples • ie an assay for its target enzyme, AChE, to which we can add Eserine • a way of simulating Eserine degradation at a known rate • so we are dealing with a ‘known’ half-life • A successful assay could then be applied to new compounds synthesised by the company

5. EDA – The Eserine Experiment • AChE Assay

6. EDA – The Eserine Experiment • AChE Assay with Eserine

7. EDA – The Eserine Experiment • AChE Assay

8. EDA – The Eserine Experiment • AChE Assay with Eserine

9. EDA – The Eserine Experiment • Simulating the degradation of Eserine • By definition, a half-life is the time taken for something to reduce by a factor of 2 • Don has serially diluted Eserine by a factor of 2, and then labelled the solutions 0 min, 30, min , 60 min, etc… • so, the simulated half-life is 30 mins • If we take those samples through the AChE assay, then we can check to see if the ‘measured’ half-life is accurate

10. EDA – The Eserine Experiment • Learning the assay (30 mins) • Can you show that AChE produces a measurable colour change? • Is there any colour change without AChE (ie spontaneous breakdown)? • Does the ‘0 min’ Eserine (0.075 uM) give about 75% inhibition?

11. EDA – The Eserine Experiment • Doing the Experiment • Use the ‘standard’ assay protocol twice to establish baseline rates for uninhibited enzyme (ie no Eserine) • Use the ‘eserine’ assay protocol twice for each of the serine samples (0, 30, 60, 90 and 120 mins) • If time is tight, you may wish to do them singly first, then repeat • Measure the rate of change in absorbance for each of the above • Calculate the percent inhibition of AChE for each of the above

12. EDA – The Eserine Experiment

13. EDA – The Eserine Experiment • Tips • Calibrate/blank the spectrophotometer against DTNB • Eserine is toxic! Gloves and goggles, please. Empty cuvettes down sink, wash and put in tray • You have available 0-20, 0-200 and 0-1000 µl Gilsons. Use the correct ones, and use them correctly! • You have limited ATC and AChE. Pipette with care! • 020 means different things on different Gilsons! • You have limited time. Stagger the Eserine samples to save time.