BIOINF 4371 Drug Design 1 Oliver Kohlbacher & Jens Krüger

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1 BIIF 4371 Drug Design 1 liver Kohlbacher & Jens Krüger Winter 2013/ Receptor- Ligand InteracAons

2 verview Receptor structure Structure elucida0on of proteins Structural change upon binding Receptor- ligand interac0ons Types of interac0ons Rela0ve strength of interac0ons Structure- based drug design Virtual screening, docking, de novo design 1

3 Structure- Based Drug Design Given 3D structure of a target Find Ligands binding to the target Binding strength of these ligands HS H? HS H H HS H H HS CH 3 H CH 3 H H CH 3 SH H 2

4 Structure- Based Drug Design Prerequisites Geometric and chemical complementarity of ligand and receptor High- quality target structure (high resolu0on) HS H? HS H H HS H H HS CH 3 H CH 3 H H CH 3 SH H 3

5 Structure- Based Drug Design Protein Candidate Structures 3D Structure Candidates Active Ligands Structure Elucidation Molecular Modeling Syn- thesis In vitro Testing Preclinical Tests Structure Elucidation (complex) HS H HS H H H H CH 3 SH H CH 3 H HS CH 3 H HS H SH H 4

6 verview of Methods Key Problems Which ligands will bind? (op0miza0on of libraries for HTS) ) Virtual Screening (in silico HTS) RelaAve binding strength of some ligands? (lead op0miza0on) ) Docking Which new ligands could be made? (no known ligands, avoid IP issues) ) de novo Design 5

7 Virtual Screening Test many(!) ligand structures quickly for one target ApplicaAons Improve enrichment in HTS, find new scaffolds Speed is more important than accuracy Large number of ligands have to be processed (millions) Precise predic0on of binding affinity not essen0al CH CH 3 HS CH 3 H H 3 C HS H H H CH 3 H H H CH 3 H HS H H CH 3 H? H H RH SH H H H CH 3 H S H CH H 2 CH 3 CH 3 Cl H H H H 2 S H F 3 C H CH 3 CH 3 CH 3 CH 3 HS H H H H HS CH 3 H RH S CH 3 CH 3 CH H 6

8 Docking Predict binding conforma0on (pose) and affinity (ΔG) of a small number of ligands for one target ApplicaAons Predic0on/understanding of binding mode, lead op0miza0on Accuracy is more important than speed Small number of compounds (hundreds?) Precise ordering of compounds is essen0al for lead op0miza0on H 2 -C CH 3 H 2 H C-? ΔG =... 7

9 De ovo Design Construct a novel compound fixng into the (known) binding pocket of a target ApplicaAons Try to come up with new leads/scaffolds if HTS did not come up with any viable hits, escape patented structure space Structures are constructed from scratch in a way that ensures op0mal interac0on with the target Problem: synthesizability of the structures H H H H HS H 2 HS H 2 8

10 Prerequisites Docking/VS/de novo design require Accurate receptor geometries Conforma0on genera0on that is complementary to the receptor Predic0on of binding free energy for the ligand Requirements Protein structure elucidaaon Conforma0onal analysis Modeling of receptor- ligand interacaons 9

11 Protein Structure Since about 1960: determina0on of protein structures using X- ray crystallography (Kendrew, Perutz) Later also nuclear magneac resonance spectroscopy (MR) Receptor structure is the basis for structure- based drug design 10

12 Protein Structure Structure reveals shape (e.g., binding pockets) Structure also reveals atomic details (interac0ons) 11

13 X- Ray Crystallography X-ray source Protein Crystal Detector Analysis 12

14 X- Rays 13

15 Protein Crystals Proteins are difficult to crystallize Irregular shape large holes in the crystal Rather large crystals required ( mm) Large amounts of protein necessary Protein needs to be very pure Crystal growth is very slow (weeks to months) Some proteins do not crystallize at all (membrane proteins!) Branden, Tooze, p

16 CrystallizaAon Hanging Drop ölting, p. 70, Branden/Tooze, p

17 DiffracAon Bragg s Law 2d sin θ = λ ConstrucAve interference occurs under angles corresponding to reflec0on at the laxce planes of the crystal d: distance between laxce planes θ: angle λ: wavelength θ d 16

18 DiffracAon Pa_ern of a Protein 17

19 verview X- Ray Crystallography 18

20 Electron Density Map 19

21 Electron Density Map 20

22 ResoluAon Resolu0on of a structure determines informa0on content Determined by quality of the crystal: Purity Inclusions Water content Stability under irradia0on Resolu0on can be es0mated from diffrac0on paiern 21

23 ResoluAon Resolu0on determines which atomic details are recognizable Poor resolu0on (large value) blurs the details of the structure Resolu0on is measured in Å Resolu0on of 2 Å does not mean, that the error for the atom coordinates is about 2 Å! Error in the atom coordinates would be about 0,3 Å in that case 22

24 uclear MagneAc Resonance 1 H nuclei possess nuclear magneac moment In an external magne0c field B 0, every nucleus assumes one of two possible states (spins) : α or β The two states differ in energy, spin state α (parallel to B 0 ) is energe0cally more favorable β B 0 α ΔE 23

25 uclear MagneAc Resonance 1 H nuclei possess nuclear magneac moment In an external magne0c field B 0, every nucleus assumes one of two possible states (spins) : α or β The two states differ in energy, spin state α (parallel to B 0 ) is energe0cally more favorable Addi0on of energy can invert the spin state β h ν α ΔE 24

26 uclear MagneAc Resonance 1 H nuclei possess nuclear magneac moment In an external magne0c field B 0, every nucleus assumes one of two possible states (spins) : α or β The two states differ in energy, spin state α (parallel to B 0 ) is energe0cally more favorable Addi0on of energy can invert the spin state β α ΔE 25

27 MR Hardware 26

28 MR Hardware 27

29 1 H- MR Spectrum of a Protein 28

30 2D- MR Spectrum Peaks on the diagonal correspond to the shins in 1D spectrum Cross peaks (off- diagonal) are caused by transfer of magne0za0on between two nuclei, i.e., interac0on between these nuclei δ 1 δ A δ B It usual implies closeness of these nuclei δ B δ A δ 2 29

31 (H,H)- CSY 30

32 Result: Structure Family MR data is compa0ble with a whole range of structures (proteins in solu0on are moving!) The result is thus a structure family Data from 1SVQ 31

33 Flexibility Different regions of the proteins show different flexibility Flexibility can be measured, for example, as RMSD between individual structure Figure on the right displays backbone flexibility as backbone thickness Termini usually highly flexible 32

34 Comparison XRD MR XRD MR Also for large proteins Requires crystals Hydrogen atoms invisible Unlabeled protein Higher spa0al resolu0on < 30 kda From solu0on Hydrogen atoms are essen0al Isotope- labeled protein required Informa0on on flexibility 33

35 Databases PDB PDB (Protein Data Bank) h_p:// Database for biomolecular structures Maintained by the RCSB (Research Collaboratory for Structural Bioinforma=cs) Deposi0on of structures in the PDB is prerequisite for the publica0on of the structure in a journal Each structure is given a unique iden0fier (PDB ID) 4 characters 1st character version 2nd 4th character structure ID Example: 2PTI, 3PTI, 4PTI are different structures of protein BPTI 2PTI: 1973, 3PTI: 1976, 4PTI:

36 PDB Contents & Growth yearly total Data from: 0

37 Receptor Structures Very good structures are required to analyze and model receptor- ligand interac0ons (resolu0on beier than 2.5 Å) Receptor structure onen reveals binding pockets and interacaons with the ligand Difficult: integral membrane proteins (ion channels, GPCRs,...) Cannot be crystallized in most cases Too large for MR, insoluble However, we onen need only the structure of an individual domain to understand receptor- ligand interac0on, not the whole receptor XRD/MR of individual (globular) domains onen sufficient and feasible 36

38 Bound Structures Details of the binding mode can be obtained from the complex structure (receptor with bound ligand) Interac0ons can be iden0fied through contacts in the structure (e.g., spa0al proximity of acceptor/donor) Good resolu0on of the structure is essen0al to iden0fy interac0ons reliably nen the receptor structure differs (slightly) between the bound and unbound state The same is true for the ligand: the bound conforma0on is not necessarily the lowest- energy conforma0on 37

39 Structural Flexibility Comparison of the unbound receptor (blue) and bound complex (red) reveals structural changes upon binding (here: MTX/DHFR, PDB: 1DF7/1DG8) 38

40 Receptor- Ligand InteracAons Two possible modes of interac0on for ligands: Irreversible covalent binding of the ligand to the receptor ) difficult to model Reversible receptor and ligand are in equilibrium with the receptor- ligand complex 39

41 Reversible/Irreversible InteracAons Irreversible (e.g., CX- 1 and aspirin) Reversible (e.g., DHFR and methotrexate) 40

42 Receptor- Ligand InteracAons Binding determined by binding free energy ΔG Entropic and enthalpic contribuaons ΔG = ΔH TΔS ΔH: binding enthalpy ΔS: binding entropy T: absolute temperature The smaller (more nega0ve) ΔG, the stronger the interac0on, the 0ghter the binding ΔH and ΔS are sums of several enthalpic/entropic contribuaons arising from different physical interacaons 41

43 Hydrogen Bonds Hydrogen bound to strongly electronega0ve atoms are polar (e.g., bound to,, F) Polar H atom at a donor atom D interacts with electron pair (lone pair) of an hydrogen bond acceptor A D H A Energe0cally, H- bonds are somewhere between covalent bonds and most other intermolecular interac0ons H 42

44 Hydrogen Bonds Can be described accurately using quantum mechanics 4- electron 3- center bond: 2 e - from lone pair of A 2 e - from D H bond Direc0onality Depends on orienta0on of acceptor lone pair Depends on direc0on of D H bond Typical distances well below the sum of van der Waals radii of paracipaang atoms \Å(X A H) H \Å(D H A) d(d A) \Å(D H A) \Å(X A H) d(d A) Å 43

45 Hydrogen Bonds EnergeAcs Hydrogen bonds possess opamal angle and length The greater the devia0on from ideal geometry, the smaller the hydrogen bonding energy Free energy contributed by a single H- bond typically 2-20 kj/mol Important acceptor groups C= H C - with lone pair with lone pair Important donor groups H H 44

46 Salt Bridges Ionic interac0on Electrosta0c interac0on between posi0vely and nega0vely charged groups - + H 3 Very strong interacaon: large charges (+/- 1 e 0 ) at a small distance ( Å) Can also involve metal ions (e.g., Zn 2+ ) Counterparts in proteins: charged side chains (Arg, Lys, Asp, Glu, His) H - + H H H 45

47 ComplexaAon of Metal Ions Many proteins contain metal ions as cofactors (Zn, Fe, Mg,...) Apart from the electrosta0c interac0on, metal ions can also form coordinaaon bonds to ligands and receptor (complexaaon) Thiols (- SH) Hydroxyamate (- CHH) - heterocycles with a lone pair Zn complexa0on by a ligand (SC 12155) in LF (lethal factor) from Bacillus anthracis Panchal et al., ature Struct. Biol., 2004, 11, 67 46

48 Hydrophobic InteracAon Favorable interac0on between apolar side chains of the receptor and lipophilic groups in the ligand ot really an interac0on o real pairwise interac0on between atoms (only vdw interac0ons and those are rather weak) Hydrophobic groups cannot form polar interac0ons Thus no polar interac0on with water possible hydrophobic groups disturb the hydrogen bond network of surrounding water Energe0c contribu0on of hydrophobic interac0on is based on reduced disturbance of the surrounding water (entropic effect) 47

49 Hydrophobic Effect Entropy comes from the solvent Apolar groups/side chains cannot form H bonds with water Water is forced to assume cage- like structures Loss of disorder in the solvent ) ΔS < 0 Free energy contribu0on approximately proporaonal to hydrophobic surface area 48

50 ther Solvent Effects Receptor and ligand are both surrounded by water and interact with water Water modulates interac0on Forms H bonds with acceptors/donors in receptor/ligand Shields electrosta0c interac0ons (dielectric constant of water: 78!) Ligand binding requires Breaking the solvent shell surrounding receptor and ligand Releasing bound waters in the receptor- ligand interface 49

51 Explizites Wasser 50

52 Solvent Effects Water forms H bonds with acceptors/donors of receptor and ligand For each H bond formed between receptor and ligand, an H bond to water has to be broken ) relaave strength of H bond is important, not their number! H H H H H + + H H H H H 51

53 Water- Mediated Hydrogen Bonds Fixed water molecules are part of many receptor- ligand complexes Example: methotrexate forms an H bond with a water molecule that is kept in place by two addi0onal H bonds with the receptor (DHFR) 52

54 Entropic ContribuAons Loss/gain of degrees of freedom (DFs) corresponds to entropic contribu0on InteracAons reduce degrees of freedom of receptor and ligand Ligand frozen in place Loses transla0onal and rota0onal degrees of freedom Loses internal degrees of freedom (flexibility) Loss of entropy energeacally unfavorable ΔG = ΔH TΔS ΔS < 0 ) TΔS > 0 53

55 Entropic ContribuAons Ligand freezing Loss of rota0onal/transla0onal DFs Loss of internal DFs Displacement of water from binding site Solvent hull of ligand and receptor stripped Water molecules displaced from binding site are released into the bulk solvent and gain rota0onal/ transla0on DFs Klebe, p

56 Strength of InteracAons Typical binding free energies of receptor- ligand complexes are between - 40 and - 10 kj/mol Each complex has its unique mix of interac0ons contribu0ng Depending on receptor and ligand, different interac0ons can dominate the binding Some complexes are dominated by enthalpic contribu0ons, some by entropic contribu0ons However: only sum of ALL contribuaons is decisive ) We need to model ALL interacaons accurately! 55

57 Example: Thermolysine Inhibitors Binding constants were determined for 80 different ligands of thermolysine Binding strength is shown as a func0on of the number of H bonds formed nly weakly correlated Reasons Desolva0on effects Strength of H bonds varies Klebe, p

58 Example: Avidin and BioAn Complex of avidin and biotin is one of the strongest interactions known: ΔG = -78 kj/mol Two dominant interactions: Seven hydrogen bonds Hydrophobic interaction BKK, p.?? 57

59 Example: Avidin and BioAn Complex of avidin and biotin is one of the strongest interactions known: ΔG = -78 kj/mol Two dominant interactions: Seven hydrogen bonds Hydrophobic interaction PDB: 2AVI 58

60 Example: CyAdine Deaminase Cy0dine deaminase (CD) catalyzes conversion of cy0dine to uridine Reac0on involves a tetrahedral transiaon state TransiAon state analogs are good inhibitors for CD Cy0dine H 2 H 2 H H Uridine H H H Ribose K i = 1.2 pmol/l H H H Ribose Ribose Tetrahedral transiaon state H Ribose Ribose K i = 30 µmol/l BKK, p

61 Example: CyAdine Deaminase Extreme difference between the two inhibitors can be explained by their ability to displace a water molecule Hydroxyl group can take the place of a water in the binding site and form favorable interac0ons with the receptor CH 2 group cannot displace the water BKK, p

62 Summary Structure- based drug design tries to iden0fy drug candidates based on the 3D structure of the receptor Structure of these receptors is elucidated using XRD or MR High- resolu0on structures reveal detailed interac0ons between receptor and ligands Key interac0ons: hydrogen bonds, hydrophobic interac0ons, electrosta0c interac0ons Ligand displaces water molecules upon binding Water plays a key role and its influence can dominate the binding free energy Ligand binds in a fixed conforma0on and thus loses internal degrees of freedom upon binding 61

63 References Books [Klebe] Klebe, Wirkstoffentwurf, 2 nd ed., Spektrum, 2009 [BKK] Böhm, Klebe, Kubinyi: Wirkstoffdesign, Spektrum 2002 [HSRF] H.- D. Höltje, W. Sippl, D. Rognan, G. Folkers: Molecular Modeling Basic Principles and Applica0ons, 2nd ed., Wiley, 2003 More on XRD: Gale Rhodes: Crystallography made crystal clear, Academic Press,