Bioassay development part 1

• 1. Principles of Drug Discovery & Development Bioassay development B19FE Semester 2 8 Lectures Dr Colin Rickman ([email protected])

2. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 2 The process of drug development A multi-step process initiated by advances made in basic biomedical research. At any stage retrograde steps may be made in the process as a result of failure. Throughout this process the cost incurred increases steeply. 3. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 3 Bioassays Any assay involving a biological sample. They typically are either quantal or graded. Quantal assays ideally provide a yes or no answer. Graded assays measures a property under multiple conditions. They can be used to test: Drug/Target interactions either presence or a measure of a kinetic property. Influence of a drug on target functions such as disruption of protein- protein interactions or enzyme kinetics. Physiological outcome of drug on disease state. Graded assays can be adapted to give a quantal result through the use of defined thresholds. 4. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 4 Bioassays Bioassays fall in to two broad categories. High throughput bioassays are used in high throughput screens (HTS). They provide small amounts of information from millions of small scale bioassays. Ideally provide a QUANTAL yes or no answer which is 100% accurate. High content bioassays are used in lead identification and optimisation. They provide very large amounts of information from a small number of bioassays. Need to provide accurate GRADED information with a high degree of sensitivity. 5. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 5 Bioassays High Throughput Screening 6. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 6 Bioassays High Content Screening 7. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 7 Bioassay considerations What property is to be measured? Binding of compound to target. Influence of compound on target interactions with other molecules. Effect of compound on an enzymatic process. Result of compound on a downstream cellular process. How is this performed and what is the readout? High throughput versus high content. Monitored over time or an endpoint. Measured using light, heat, radiation. What biological system is going to be used? Using purified targets in solution (in vitro). Cell based assays (in vivo). Animal based assays (in vivo). 8. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 8 Bioassay development lecture plan Measurement of binding. Recap of mathematical principles (KD, KON, KOFF). Radio-ligand binding. Isothermal titration calorimetry. Surface plasmon resonance. Measurement of kinetics. Recap of mathematics on enzyme kinetics (KM, VMAX). Rapid mixing and sampling techniques. Flash photolysis. Cell-based assays. Gene expression reporter constructs. Frster resonance energy transfer 9. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 9 Measurement of binding Binding is the process of a ligand (A) binding to a target (B) resulting in a complex. The target can be DNA, protein or lipids and the ligand can be endogenous or exogenous (a drug). Most drugs in development today target proteins due to their wide variation in structure and properties (allowing specificity) and their key role in cell physiology and pathology. Example: Influenza neuraminidase bound to oseltamivir (Tamiflu). 10. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 10 Measurement of binding Measurement of the affinity of a ligand for its target is important to distinguish between similar compounds in the lead optimisation stage. Optimisation of affinity allows lower concentrations of a drug to be used therapeutically. This minimises side effects which typically have lower affinities for the drug than the target. Also minimises costs if the drug is expensive to synthesise. So how is the affinity of a ligand for a target measured? 11. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 11 Measurement of binding To calculate the affinity we need to make some assumptions: All receptors are equally accessible to ligands. All receptors are either free or bound to ligand. The model ignores any states of partial binding. Neither ligand nor receptor are altered by binding. Binding is reversible. For binding analysis theory and the influence of inhibitors see lectures given in B19FD (on Vision with notes for these lectures). 12. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 12 Measurement of binding Radioligand binding assays This is the general process to perform a radioligand binding assay. 13. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 13 Measurement of binding Radioligand binding assays The target can be either a purified protein or cells expressing surface receptors. Purified proteins can be fused to an affinity tag (e.g.. GST, myc, biotin) to allow immobilisation on beads. The radiolabelled ligand can either be synthesised de novo incorporating 3H. Expensive for each molecule but highly specific and no change to properties. Or labelled post synthesis on an aromatic hydroxyl group (tyrosine) by 125I. Can degrade and may change properties of compound. 14. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 14 Measurement of binding Radioligand binding assays It is important that the reaction reaches equilibrium for all of the different concentrations of ligand used in the assay. The lower the concentration the longer it takes to achieve equilibrium. This is achieved more quickly at higher temperatures. However, purified proteins may be unstable above 4oC and cell viability will be hindered above/below 37oC. 15. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 15 Measurement of binding Radioligand binding assays Unbound ligand is typically removed by filtration or centrifugation of the sample. The sample is normally washed to remove as far as possible unbound ligand. Calculation of affinity is performed by non-linear regression. The ionic composition of solutions and the temperature the experiment is performed at all influence the measured affinity and must be standardised. By setting a threshold of detected radiation above which binding is deemed to have occurred this can be applied to HTS for a small number of concentration points. 16. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 16 Measurement of binding Isothermal titration calorimetry Isothermal titration calorimetry (ITC) measures the enthalpy change which occurs upon binding. Ligand is added stepwise to a chamber containing the binding target. With each injection the concentration of ligand increases in the chamber allowing a binding curve to be built up. 17. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 17 Measurement of binding Isothermal titration calorimetry In general two cells are used one containing the sample reaction and another a reference cell. The heat energy injected by the machine in to the sample cell or reference cell to maintain the temperature between the two cells is used to measure the enthalpy change of each ligand injection. 18. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 18 Measurement of binding Isothermal titration calorimetry 19. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 19 Measurement of binding Isothermal titration calorimetry The top graph shows the energy input to counteract the binding of ligand to target. This can be converted in to a binding curve from which the enthalpy change of binding and the stoichiometry can be calculated. The gradient of the slope around the point of inflexion is the KA which is 1/KD. Disadvantage of this approach is it needs large amounts of target and ligand. 20. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 20 Measurement of binding Surface plasmon resonance (Biacore) Surface plasmon resonance (SPR) has been adapted to measure binding kinetics and affinities in solution. In comparison to ITC which measures binding as a change in energy, biacore measures interactions using a special property of light. 21. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 21 Measurement of binding Surface plasmon resonance (Biacore) Total Internal Reflection (TIR) 22. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 22 Measurement of binding Surface plasmon resonance (Biacore) Total Internal Reflection (TIR) When light hits an interface between two materials with different refractive indices the light bends. If the light hits this interface with a sufficiently shallow angle the light will reflect off the interface in a process called total internal reflection. 23. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 23 Measurement of binding Surface plasmon resonance (Biacore) Total Internal Reflection (TIR) When light hits an interface between two materials with different refractive indices the light bends. If the light hits this interface with a sufficiently shallow angle the light will reflect off the interface in a process called total internal reflection. Perpendicular to this surface an evanescent electromagnetic wave is set up which can interact with a gold layer and generate a SPR. 24. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 24 Measurement of binding Surface plasmon resonance (Biacore) The SPR is an electronic oscillation at the boundary between the gold layer and the medium. As such is it is very sensitive to local changes to the surface of the gold (such as a target binding a ligand). This results in a slight change in the angle of the reflected light which is detected by the biacore machine and used to report interaction. 25. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 25 Measurement of binding Surface plasmon resonance (Biacore) Target is immobilised on to the gold surface. Light undergoes total internal reflection through a prism generating SPR. As ligand is added to the sample through a microfluidic channel binding occurs. This alters the SPR causing a slight deflection in the angle of the reflected light. This shift in angle is recorded in real time as the binding occurs. 26. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 26 Measurement of binding Surface plasmon resonance (Biacore) 27. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 27 Measurement of binding Surface plasmon resonance (Biacore) From this you can measure: The concentration of immobilised target (excess ligand in solution) or ligand concentration (if immobilised target is in excess). The rate of association and dissociation KON/KOFF. The affinity KD. The target surface can also be regenerated for the next experiment. 28. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 28 Measurement of binding Surface plasmon resonance (Biacore) Edin Univ. Centre for translational and chemical biology 29. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 29 Measurement of binding Microscale thermophoresis 30. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 30 Measurement of binding Microscale thermophoresis 31. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 31 Measurement of binding Microscale thermophoresis By examining the flow of molecules through the temperature gradient at different ligand concentrations you can determine binding affinities. This requires the constant molecule to be fluorescently labelled (or using intrinsic tryptophan fluorescence. This process uses very small amounts of material. Also compatible with crude lysates. 32. B19FE (Semester 2) Principles of Drug Discovery & Development Bioassay Development 32 Measurement of binding - Summary Detection of binding can be important both in hit identification (although a physiological outcome of the ligand is probably more important to establish) and lead identification/optimisation. Radioligand and microscale thermophoresis binding is applicable to both in vitro and in vivo analysis of interactions but does require labelling. ITC and Biacore both allow label free detection of binding and calculation of binding/kinetic rate constants. The measurement of binding affinities is an essential step for the selection and optimisation of leads, iterative increase in specificity and ultimately the decrease of dosage and off-target effects.