1. Principles of Drug Discovery & Development Bioassay development B19FE Semester 2 8 Lectures Dr Colin Rickman (c.rickman@hw.ac.uk)

2. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 2 Enzymes as pharmaceutical targets Enzymes catalyse the conversion of a substrate in to a product. This is essential for almost all physiological processes and may be the causative agent of a pathological state. Enzymes are also excellent targets for treatment of infections. The enzymes present in the pathogen may not be present in the host. If present their amino acid sequence may have diverged sufficiently during evolution to provide a therapeutic window. 3. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 3 Enzyme catalysis - Michaelis-Menten For reactions which can be described by the simple steps above, the rate is given by the Michaelis-Menten equation. Under steady state conditions the rate of formation and breakdown of the enzyme-substrate complex is equal. The efficiency of enzymes can vary from very low to limited only by diffusion (catalytically perfect). 4. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 4 Enzyme catalysis Measurement of the initial rate To measure enzymatic rates requires the accurate measurement of an enzyme catalysed reaction under conditions where [ES] is constant. In a time course of reaction this occurs early on, before substrate is consumed sufficiently to alter the rate of ES formation. Depending on the catalytic rate of the enzyme this can be very fast. To measure this value accurately requires rapid reagent mixing and detection. 5. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 5 Enzyme catalysis Measurement of the initial rate Suitable detection of reaction products. Simultaneous recoding of reaction products during experiment. Ability to stop reaction and measure products offline. Rapid initiation of the reaction. Rapid mixing of enzyme and reagents. Ability to pre-mix enzyme and reagents before triggering start of reaction. 6. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 6 Methods for detection of reaction products - Spectrophotometry Many substances absorb light in the ultraviolet or visible regions of the spectrum. By shining a light of known wavelength through a solution the concentration can be measured. This is calculated using the Beer-Lambert law: 7. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 7 Methods for detection of reaction products - Spectrophotometry This can be measured in a cuvette-based or plate-based spectrophotometer. Choice depends on the method used and the volume of information required. 8. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 8 Methods for detection of reaction products - Spectrophotometry Almost all spectrophotometers work using a monochromator. This splits white light in to a spatially separated spectrum of UV or visible light. By moving the slit position and width a small range of wavelengths can be sampled. 9. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 9 Methods for detection of reaction products - Spectrophotometry This approach can be used wherever the substrate and product differ substantially in their absorption at a specific wavelength. An example of this is alcohol dehydrogenase. The NADH product absorbs light at 340 nm in comparison to NAD+. The sensitivity is limited by the path length (normally 1cm or less for a cuvette) and the extinction coefficient. 10. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 10 Methods for detection of reaction products - Spectrophotometry The advantage of spectrophotometry is it can be monitored over time without altering the reaction conditions. It can also be adapted to high throughput screening. However, a small extinction coefficient will severely limit the sensitivity of this approach. A disadvantage is that other components of the reaction may also have significant absorbance at the required wavelength. This decreases the overall sensitivity of the assay. 11. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 11 Methods for detection of reaction products - Spectrofluorimetry 12. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 12 Methods for detection of reaction products - Spectrofluorimetry Fluorescence is defined as the Emission of radiation, generally light, from a material during illumination by radiation of a higher frequency. The difference in wavelength between the absorbed and emitted photon is referred to as the Stokes shift. 13. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 13 Methods for detection of reaction products - Spectrofluorimetry This shift in wavelength is utilised in a spectrofluorimeter. By blocking the excitation wavelength with a filter the emission is detected against a very low level of background signal. c.f. spectrophotometry where the aim is to detect a decrease in intensity of a bright light. This makes spectrofluorimetry a highly sensitive technique (theoretically ~100 times more sensitive than spectrophotometry). 14. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 14 Methods for detection of reaction products - Spectrofluorimetry Can be used whenever the substrate or product differ in their fluorescent properties. An example of a highly fluorescent drug is quinine used to treat malaria. Quinine blocks the biocrystallisation of heme in to hemozoin inside the parasite. This results in build-up of toxic heme leading to their death. It is also the flavouring using in tonic water. Quinine 15. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 15 Methods for detection of reaction products - Spectrofluorimetry 16. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 16 Methods for detection of reaction products - Spectrofluorimetry The advantage of spectrofluorimetry is it can be monitored over time without altering the reaction conditions. It can also be adapted to high throughput screening. It is also theoretically more sensitive than spectrophotometry. However, it is only useful if either the substrate or product are fluorescent Although it may be possible to engineer them to be. 17. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 17 Methods for detection of reaction products Coupled assays - Chemiluminescence If the reaction substrate or products are not naturally chromophores/fluorophores (and then cant be engineered to be so) a coupled reaction can be used. This feeds the reaction products from the test enzyme in to a subsequent reaction generating a detectable output. A simple example of this is the use of the luciferase enzyme from fire flies. 18. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 18 Methods for detection of reaction products Coupled assays - Chemiluminescence Luciferase catalyses the reaction of luciferin with oxygen to form oxyluciferin and light. This process requires ATP. If the enzyme reaction to be assayed produces ATP then it can be coupled to the luciferase reaction and light output measured. Alternative coupling assays can make use of NADH production and assayed using spectrophotometry. 19. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 19 Methods for detection of reaction products - Radioactivity Radioactive detection of products is the most sensitive approach available (~106 times more sensitive than spectrophotometry). Radioactive 3H or 14C can be incorporated in to the substrate during synthesis. However it cannot be detected simultaneously during the reaction. The reaction must be stopped for detection. The product and substrate must be separated to allow specific detection. Normally both product and substrate are radioactive. 20. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 20 Methods for detection of reaction products - Radioactivity Separation can be achieved by thin layer chromatography or electrophoresis followed by scintillation counting. Alternatively, separation by HPLC can be combined with simultaneous detection of radioactive components. If the product and substrate are sufficiently different in their chemical properties they may be simply separated using a filter pad. Isotope Half-life Max energy of emission (MeV) 14C 5730 yr 0.156 3H 12.35 yr 0.0186 32P 14.3 days 1.709 21. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 21 Enzyme catalysis Measurement of the initial rate Suitable detection of reaction products. Simultaneous recoding of reaction products during experiment. Ability to stop reaction and measure products offline. Rapid initiation of the reaction. Rapid mixing of enzyme and reagents. Ability to pre-mix enzyme and reagents before triggering start of reaction. 22. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 22 Rapid initiation of the reaction Continuous flow Two syringes (containing enzyme and substrate) are compressed at a constant rate. They mix thoroughly and pass down the flow tube. The flow rate must be sufficiently high to ensure a turbulent flow. For a 1 mm diameter tube a flow rate in excess of 2 m s-1 is required. The advantage is a dead time as low as 10 s, however, large amounts of substrate and enzyme are consumed. 23. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 23 Rapid initiation of the reaction Stopped flow 24. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 24 Rapid initiation of the reaction Stopped flow The reaction mix in stopped flow fills the stopping syringe which once filled to the required level sends a trigger to the detector and stops the flow. This allows recording with a dead time of 0.5 ms and observations over several minutes. In comparison to the continuous flow approach, stopped flow requires only 100 400 l. Both continuous flow and stopped flow are ideal for spectrometric recording. 25. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 25 Rapid initiation of the reaction Quenched flow The quenched flow technique is an adpatation of the continuous flow approach which does not require simultaneous detection during the experiment. The quencher can contain an acid or denaturant to stop the reaction after a period of time determined by the flow rate and l. The minimum dead time is approximately 5 ms with a maximum recording time of around 150 ms. The quenched reaction can then be analysed (often radioactivity). 26. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 26 Rapid initiation of the reaction Flash photolysis Flash photolysis requires the use of caged compounds which may be the main substrate or a required co-factor (more common). A pulsed laser source (340 nm) provides the energy to break the caged compound releasing the active molecule (in this case ATP). This allows pre-mixing of enzyme and substrate minimising dead time. However, this approach is limited by the availability of caged compounds. 27. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 27 Enzymes as pharmaceutical targets - Summary Enzymes are a common therapeutic target permitting the regulation of a biochemical process. Michaelis-Menten kinetics provides a standardised means of measuring enzyme kinetics and the influence of targets on this reaction. Spectrophotometry, spectrofluorimetry, radioactivity and coupled reactions are the principle means of detection. Stopped flow is the most popular means for the rapid mixing and measurement of initial rates and velocities. Every assay needs to be specifically designed for the enzyme reaction to be assayed.