Isothermal Titration Calorimetry in Drug Discovery. Geoff Holdgate Structure & Biophysics, Discovery Sciences, AstraZeneca October 2017

Transcription

1 Isothermal Titration Calorimetry in Drug Discovery Geoff Holdgate Structure & Biophysics, Discovery Sciences, AstraZeneca October 217

2 Introduction Introduction to ITC Strengths / weaknesses & what is required for ITC experiments ITC in the drug discovery process ITC in reagent / assay QC ITC in Hit confirmation & Mechanistic studies Using the thermodynamic data? Spotting some issues with ITC data Summary

3 ITC - Power compensation As chemical reactions occur in the sample cell, heat is generated or absorbed The temperature difference between the sample and reference cells ( T) is kept at a constant value, close to zero, (i.e. baseline) by the addition or removal of heat to the sample cell, by applying more or less power as required adiabatic jacket sample cell reference cell feedback heaters The integral of the power applied to maintain T = constant, and close to zero over time is a measure of total heat resulting from the process studied Τ

4 ITC - Operational procedure

5 Systems most amenable to study by ITC Binding: A + B AB Protein ligand binding DNA ligand binding Protein protein interactions Protein DNA interactions Catalysed reactions A B Enzyme catalysed reactions Association / Dissociation: xa B x Oligomer dissociation Micellisation / de-micellisation

6 ITC - Strengths and limitations Strengths Label-free, no limit to molecular species Broad applicability in the entire drug discovery process Fast set-up times for new projects Comprehensive data set ( H, K d, n, C p ) Method is easy to implement High impact on quality control of proteins by stoichiometry readout Mechanistic studies on drug interactions Limitations Throughput is restricted to about 7 cpds / day Requires high protein amounts (around 25 µg/expt) Suitable protein targets/constructs (stability, quantity, quality) For LMW compounds the concentration window is practically limited by solubility Data has to be carefully evaluated, overinterpretation easily possible as data is a sum of multiple simultaneous effects Thermodynamics accessible Cross-validation and contribution to all molecular interaction methods

7 Protein ligand binding requirements Typical requirements: - Protein ( 1 2 µm) in cell - Ligand ( 12 3 µm) in syringe - Binding partners in the same buffer Considerations - Concentrations of each reactant should be known accurately - Presence of linked equilibria should be evaluated Eg protonation events causing additional heat effect due to buffer ionisation - Control experiments C value (c = [Protein] tot.n / K d ) 1 1 feasible when titrating to low molar ratios Must titrate to high multiples of K d, when working at low c values

8 Biophysics and use of ITC in Drug Discovery Deliverables & impact on projects Feasibility assessment, construct design & LG planning Protein QC Tool ligand evaluation Assay QC Target Ligandability Structures of HTS hits Fragment hits & design Validated hits post HTS/ELT screens Hit binding modes Binding affinity determination outside biochemical assay regime Structure based DMTA Mechanistic characterisation Full kinetic, thermodynamic & structural profile

9 ITC as a tool for quality control Titration of compounds in 5mM Tris/Cl, 15mM NaCl,1% Glycerol, 1mM TCEP, 1% DMSO ph 7.5 at 25 o C Inactive Pr Activated Pr3 kcal/mole of injectant kcal/mole of injectant Molar Ratio Molar Ratio

10 Protein QC continued Protein supplied by PT for enzyme assay found to contain cofactor at concentration 2 orders of magnitude above K d Prevented monitoring of competitive ligand binding Issues around immobilisation Protein supplied by Specialist Team for biophysics has no cofactor Suitable for monitoring cofactor / ligand binding fluorescence, ITC, SPR Suitable for immobilisation Cofactor affinity ~5 µm (close agreement in ITC, fluorescence, SPR) KCal/Mole of Injectant Data: Data2_NDH Model: OneSites Chi^2/DoF = 3.124E4 N 1.7 ±.268 Sites K 1.49E5 ±1.31E4 M -1 H -1.45E4 ±511.6 cal/mol S -25. cal/mol/deg Molar Ratio [NADH] um Thermal stability measurements: 1 C stabilisation for cofactor DMSO effects: 5% causes ~5 fold shift in affinity, suggests DMSO Kd ~ 175 mm RFU Resp. Diff. RU Time RUs [NADH] um s

11 Evaluation of Tool compounds KCal/Mole of Injectant Thermodynamics Data: PARG - ADP-HPD Model: OneSites Chi^2/DoF = 8.515E4 N 1.4 ±.663 Sites K 1.4E7 ±3.47E6 M -1 H ±14.9 cal/mol S 1.91 cal/mol/deg Resp. Diff. RU Kinetics [ADP-HPD] nm Time s Signal (RUs) HT Enzyme assay used alphascreen SPR and ITC used to evaluate affinity of standard compound relative to potency Molar Ratio

12 Assay QC In vitro assay used GST-SH2 fusion protein (p85 C-terminal SH2 domain of PI3K) immobilised on 96-well plates Assay measured compound ability to perturb binding of an 11 residue biotinylated phosphopeptide (Bio-P-pep 11 ), derived from PDGFR Representative from quinoxaline lead series found to have IC 5 = 7 nm

13 Assay QC kcal/mole of injectant Molar Ratio kcal/mole of injectant Molar Ratio kcal/mole of injectant Molar Ratio Quinoxaline titrated into isolated SH2 (no heat change observed at 2 temperatures), lack of binding confirmed by NMR Also no shift in K d for P-pep 5 titrated into mixture of Quinoxaline and SH2 P-pep 5 titrated into isolated SH2, confirms that isolated protein capable of supporting binding Urea denatured and refolded GST-SH2 suitable and functional Quinoxaline shows no binding Bio-P-pep 11 shows protein capable of supporting binding Also no shift in K d for P-peps binding in presence of saturating Quinoxaline

14 Mechanistic Characterisation Mechanistic characterisation is vital for drug discovery Allows evaluation of the effect of other ligands on the binding of the test compound Can be vital to understand SAR and extrapolate to effects in cells Can be achieved using enzyme kinetics Facilitated by ITC

15 Reductase Inhibitor MoA Cofactor Kd None n/a 1mM NAD+ 9.3 µm.1mm NADH 9.6 nm kcal mol -1 of injectant Data: Data1_NDH Model: OneSites Chi^2/DoF = 3.36E4 N.858 ±.263 Sites K 1.4E8 ±5.E7 M -1 H ±68.45 cal/mol S 5.37 cal/mol/deg Molar Ratio

16 Binding to Kinases Effect of ATP kcal/mole of injectant Model: OneSites Chi^2/DoF = 3.573E4 N 1.11 ±.85 K 5.38E6 ±3.7E5 H E4 ±185 S Molar Ratio kcal/mole of injectant Model: OneSites Chi^2/DoF = 2.28E4 N 1.17 ±.11 K 6.16E6 ±5.5E5 H -1.67E4 ±132 S Molar Ratio For this compound, ATP has no effect on K d for compound binding showing Non-competitive binding

17 Binding to Kinases Effect of ATP kcal/mole of injectant Model: OneSites Chi^2/DoF = 1.834E4 N 1.27 ±.63 K 6.36E6 ±3.3E5 H E4 ±94.7 S kcal/mole of injectant Model: OneSites Chi^2/DoF = 2.686E4 N 1.41 ±.4 K 1.76E8 ±2.5E7 H E4 ±63.2 S Molar Ratio Molar Ratio For this compound, there is a 3 fold increase in binding affinity in the presence of ATP - showing Mixed tending towards Uncompetitive binding

18 MoA studies: identification of two binding events Binding of compound to Kringle domains of plasminogen. Results suggest two binding events O HN O NH KCal/Mole of Injectant Data: Data1_NDH Model: TwoSites Chi^2 = 713 N1.377 ±.411 Sites K1 6.36E6 ±4.63E6 M -1 H ±332 cal/mol S cal/mol/deg N2.985 ±.43 Sites K2 3.33E5 ±6.85E4 M -1 H ±187 cal/mol S cal/mol/deg Molar Ratio ITC study was performed in 5mM Na-P i, 1mM NaCl, 2% DMSO, ph 7.6 at 37 o C Protein concentration was 35-5uM and ligand concentration 1-2mM

19 Ligase Mechanism Complex K d (µm) n H (kcal/mol) Enz Enz + S Enz + S1 + S Enz + P Enz + S1 analogue Test compound designed as S2 analogue was shown to bind to multiple enzyme forms. ITC shows that following inhibitor SAR in enzyme assays may be difficult if the dominant binding mode changes with assay conditions or compound modification

20 The driving forces for binding interface desolvation H-bonds, vdw, ionic bonds conformational change G = H - T S = -R T ln(1/k D ) = H int + H solv -T S T+R - T S conf - T S solv Enthalpy Contributions from forces within the complex (H-bonding, v d Waals, electrostatic) Penalty from desolvation processes (polar surfaces >>unpolar surfaces) Entropy Contribution from surface desolvation = increase of disorder (number of microstates) Penalty from formation of rigid structures = loss of degree of freedom 2

21 Thermodynamic information l o /m J k -2 H T S kj/mol Increasing affinity means Making G more negative This can be achieved by: 1.Making H alone more negative 2.Making -T S alone more negative 3.A combination of changes in H and -T S together being negative

22 But caution E-E compensation Enthalpy kj/mol -2-4 Enthalpy kj/mol -2 Enthalpy kj/mol Entropy kj/mol/k.5.1 Entropy kj/mol/k Entropy kj/mol/k Figure 1. Enthalpy-Entropy for compounds binding at domains in a single protein, a bromodomain: Bromodomain 1 (left), Bromodomain 2 (middle), Bromodomains 1&2 (right) Enthalpy kj/mol Enthalpy kj/mol Enthalpy kj/mol Entropy kj/mol/k Entropy kj/mol/k Entropy kj/mol/k Figure 2. Enthalpy-Entropy for compounds binding to different proteins: synthase (left), protease (middle), kinase (right)

23 ITC is a dual probe technique T S or G (kcal/mol) Open symbols: T S Filled symbols: G Circles: Triazines Squares: Coumarins Slope for T S vs H is.95 ±.2 ITC may help identify compounds with altered binding mode, not identified by K d, but due to the larger than expected H H (kcal/mol)

24 Spotting & resolving issues with ITC data Injection spacing too short need to allow signal to return to baseline Increase injection spacing, change the feedback mode (low or no feedback requires longer injection spacing (longer instrument response time), high feedback has faster response time and so can operate with shorter injection times

25 Spotting & resolving issues with ITC data No saturation is observed during the titration, may be due to buffer mismatch, or due to weak binding Dialyse or buffer exchange the protein, use dialysate to dissolve / dilute ligand. Check concentration of ligand. Use higher concentration of ligand / lower protein concentration. Possible competition expt. Check affinity with another technique

26 Summary ITC is a valuable part of the biophysics toolbox It can play a vital role at several stages of the drug discovery process As a dual probe technique can identify differences that affinity only methods may miss Always useful to combine biophysical methods for developing greater mechanistic understanding of binding interactions

27 Acknowledgements A large number of people across Discovery Sciences and the IMED Biotech units at AstraZeneca