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Proprietary and Confidential © AstraZeneca 2008 NMR to characterise protein-ligand interaction Applications in drug discovery Rational drug design – the concept • Traditional drug design … Observation (1928) • Drug (1942) Target (1965) Rational drug design …. N N N N N O N N Identify potential attack point target drug What makes a good drug? siRNA strategies (protein) target antibodies Small molecule inhibition Ideally a good (oral) small molecule drug is “rule of 5” compliant …. … MW < 500 … ClogP < 5 larger molecules diffuse more slowly water/octanol partition coefficient small logP means: soluble in octanol AND water … < 5 H bond donors … < 10 H bond acceptors … < 5 rotatable bonds restricts degrees of conformational freedom Drug discovery • Bringing a new medicine to market is a long, complex, expensive, risky and difficult process • It takes on average over 10 years and costs around $1-5bn to bring a new medicine to market NMR > 10 years • Most projects fail: Of every 100 drug research projects started, only 1 will make it as a drug What options do we have? Protein observe methods define binding sites Ligand observe methods find ligands in mixtures not-binder binder 116 117 118 STD 119 120 121 1D 122 123 124 10.4 10.0 9.6 9.2 • identifies binding sites • needs larger amounts of protein • protein size limitation • requires isotopic labelling (15N or 13C) 9.0 8.8 8.6 8.4 8.2 8.0 • no labelling • minimal amounts of protein • no protein size limitation 7.8 7.6 ppm Ligand binding equilibrium + N N E+L Association constant Dissociation constant NB KA= KD= EL [EL] [E] [L] [E] [L] [EL] (hardly ever used) pKD=-log KD KD mM µM nM pM pKD 3 6 9 12 Enzymatic tests often use IC50 (50% inhibition) Conversion through Cheng-Prusoff eqn: K i = IC50 1+ K[ S ] M Affinity ranges KD mM pKD 3 µM 6 nM 9 pM 12 Typical biochemical interactions Marketed drugs Fragments Practical detection limit Detection range ligand-observe NMR Detection range protein-observe NMR How can we describe the KD? … thermodynamically .. by the mass law [E] [L] KD= [EL] ∆GD=∆HD+∆SD=-RTlnKD KD … kinetically Off rate KD= On rate The thermodynamic description ∆GD=∆HD+T∆SD=-RTlnKD dissociation 1 mM 1 µM 18 kJ/mol @ 37C 36 kJ/mol 1 nM 53 kJ/mol binding 40 kJ/mol Loss of translational entropy 40 kJ/mol Loss of rotational entropy 6 kJ/mol per uncharged H bond 19 kJ/mol per charged H bond 110 J/mol Å2 Hydrophobic surface Concept of fragments …. A weakly binding fragment is actually doing a pretty good job … nM µM mM … a few additional interactions might turn it into a proper drug! nM µM mM Pocket properties must match … … not too big, not too small … shape complementary to small molecules (not too shallow - “convexity”) … availability of H bond donors/acceptors Concept of druggability 1. binding of a small molecule requires certain properties of the binding pocket 2. if such a pocket is available, we have a high chance to find fragments indeed many fragments are found to bind to “natural” binding sites 3. if we find no fragments, there is probably no such pocket 4. Without a suitable “small molecule” pocket this target might not bind to small molecules From: Hajduk et al., J. Med. Chem. (2005) 48, 2518 NMR fragment screening hitrate is predictive for success in small molecule approaches! The mass law description [E] [L] KD= [EL] [ EL] fb = [ E ]tot Relevant for protein observe NMR Fraction bound [ EL] fb = [ L]tot Relevant for ligand observe NMR ([ E ]tot + [ L]tot + K D ) 2 − 4[ E ]tot [ L]tot [ E ]tot + [ L]tot + K D − [ EL] = 2 2 • fraction bound, KD and concentrations are linked • optimal experimental concentration ranges depend on • which KD range? • ligand observe or protein observe? • achievable concentrations for ligand (solubility? DMSO conc?) Protein (supply? Stability? Solubility?) Fraction bound (protein) depending on [L] and KD 100.00% 80.00% 100 µM protein (typical concentrations) problematic …. … with 100 mM stocks it means 5% DMSO in solution … many ligands are not that soluble 60.00% 40.00% 20.00% KD=0.05 mM KD=0.5 mM 0.00% 0.01 0.05 mM mM 0.1 mM 0.5 mM [L] Practical detection limit for a binder ~20% KD=5 mM 1 mM 5 mM More realistic maximal ligand concentration Millimolar binders will be difficult to detect and characterise Difficult to achieve full titration curve (and get KD) 100.00% 90.00% 80.00% 70.00% 60.00% 50.00% 40.00% 30.00% 20.00% 10.00% 0.00% 0.01 0.05 mM mM [L] KD=0.05 mM KD=0.5 mM 0.1 mM 0.5 mM KD=5 mM 1 mM 5 mM A KD=5 mM ligand will only just be visible @ 1 mM ligand/0.1 mM protein Tight binders will quickly be saturated Quickly saturated …. 100.00% 80.00% 60.00% 40.00% 20.00% KD=0.05 mM 0.00% 0.01 0.05 mM mM [L] KD=0.5 mM 0.1 mM 0.5 mM KD=5 mM 1 mM 5 mM Very tight binders follow a step function Increasing saturation only monitors [L] 100.00% 90.00% 80.00% 70.00% 60.00% 50.00% 40.00% 30.00% 20.00% 10.00% 0.00% 0.01 0.05 mM mM [L] KD=5 nM KD=0.5 mM 0.1 mM 0.5 mM KD=5 mM 1 mM 5 mM Typical concentrations in practice Protein observe - screening • [E] > 50 µM Protein observe - titration • • Practical detection limit for reasonable throughput • Solubility? Availability? • [L] = 1 mM • Titration might have to be repeated with a more suitable [E] • Lowest [L] should be ½ [E] • Concentrations should give good coverage at minimum no. of points – ‘rule of 3’ • Safely detects mM binders Ligand observe • [L] > 100 µM • Practical detection limit for reasonable throughput • [E] ~ 1 …. 5 µM • Typically [L]: [E]=20 ..50:1 • Depends on size and method used [E] ~ KD • e.g. 50 – 150 – 450 – 1350 µM • Protein should reach >80% occupancy at highest [L] • CHECK THE CONCENTRATIONS (i.e. run a 1D NMR – many compounds are not very soluble!) The kinetic description Off rate KD= On rate In many practical cases (at least for fragments …) the on rate will be diffusion controlled For molecules of similar structure and binding mode: KD ∝ off rate Always the slower step (otherwise there would not be binding) Slow exchange (often observed for tight binders) Fast exchange (often observed for weaker binders) Kinetics of ligand binding: protein observe NMR Fast exchange In true fast exchange ∆δ is a measure of fraction bound 200 µM protein +100 µM ligand (1:2) +300 µM ligand (3:2) +900 µM ligand (9:2) +2700 µM ligand (27:2) Fit uses parameters KD and δ(complex) Kinetics of ligand binding: protein observe NMR Slow exchange Slow exchange ∆δ>koff Rel. occupancy determined by fB Classification as “fast”, “intermediate” or “slow” is ‘per resonance’, not ‘per system’! Intermediate exchange ∆δ~koff 200 µM protein +100 µM ligand (1:2) +300 µM ligand (3:2) +900 µM ligand (9:2) Complex lineshape Kinetics of ligand binding: ligand-observe NMR In fast exchange all observed parameters are weighted averages: δav = fB δbound + (1-fB)δfree T1 av= fB Τ1 bound + (1-fB)Τ1 free T2 av= fB Τ2 bound + (1-fB)Τ2 free Much shorter than T2 free Influences lineshape even for small fB’s (<5%) Effect can be experimentally increased through T1ρ filter [Protein] Ligand fB Kinetics of ligand binding: ligand-observe NMR In fast exchange all observed parameters are weighted averages: Transferred NOE δav = fB δbound + (1-fB)δfree T1 av= fB Τ1 bound + (1-fB)Τ1 free T2 av= fB Τ2 bound + (1-fB)Τ2 free NOE av= fB ΝΟΕ bound + (1-fB)NOE free large and -ive -prot +prot WaterLOGSY +prot small and +ive -prot Logistics 4 1 Compound library on 384-well plates • stock solutions in DMSO-d6 • the more concentrated the better – but there is a limit! • QC control of screening libraries? 2 protein • need to plan protein supply before screening • how much do I need? (100s of mgs for protein observe screening) • stability? Can it be recycled? • basic project management skills are useful 3 Robotic sample preparation 5 Automatic acquisition Summary • Rational drug design usually means optimising an interaction between a small molecule and a protein target • NMR can support rational design by characterising this interaction • In order to do so we need to understand the mass law and its implications • Just like in any other biophysical technique! • Choose the right concentrations depending on KD • Choose the right method depending on the exchange regime • Don’t underestimate the logistical aspect! • While NMR is capable of delivering structures, crystallography is usually the preferred method • Localising binding sites is easy though! • We might get binding modes from more sophisticated experiments Structural information from NMR • protein-ligand structures yield binding modes - very valuable to understand ligand SAR (structure-activity relationship) • crystallography is the preferred option • a soakable system can yield structures in high throughput (and even be used for screening) Develop crystal system Apo structure develop soakable system complex structure • NMR structures take long time ….. (apo and even more so as a complex) Structural information from NMR CSPs can map binding sites onto a crystal structure …. O N S NH N ME F2 a SB203580 pe p ti de F assigned unassigned CSP This information comes “for free” with all protein observe methods … but - how does the ligand bind? Kinetics of ligand binding: ligand-observe NMR • No need to label protein • Can use protein from natural source or insect cell expression • Minimal protein requirements • At least one order of magnitude less than protein observe • ligand observe methods rely on fast exchange • Cannot find tight or covalent binders!! What if I want to characterise these – but cannot label the target? Ligand observe reporter screen Reporter ligand (known affinity) Test ligand (affinity to be determined) Ligand observe reporter screen Dalvit et al JACS 2002, 124, 7702-7709 Reporter ligand (known affinity) Test ligand (affinity to be determined) [Protein] Ligand fB “calibration” Competition measurement KI=47 µM (KD=41 µM)