The importance of residence and recognition time of drug-target interactions in understanding efficacy and SAR

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1 GE ealthcare Life Sciences The importance of residence and recognition time of drug-target interactions in understanding efficacy and SAR Markku ämäläinen, PhD Senior scientist in chemometrics Protein Analysis, R&D GE ealthcare Bio-Sciences AB Uppsala, Sweden 1 GE ealthcare Life Sciences Without on you are off! With slow off you might still be on even when the drug is gone! Markku ämäläinen, PhD Senior scientist in chemometrics Protein Analysis, R&D GE ealthcare Bio-Sciences AB Uppsala, Sweden 1/

2 utline Short overview on the use of SPR in drug discovery Why binding kinetics is closely connected with PK/PD and therefore also efficacy The value of residence/recognition time for Structure Kinetic Relations (SKR) Q&A session 3 SPR, ITC & DSC fits into the interface between different DD-disciplines Medicinal Chemistry SAR and QSAR based on on/off/kd-maps & enthalpy Direct binding gives pure interaction info Fragment and focused libraries gives new scaffolds Do not misuse your time with frequent hitters Screening Improved quality on hit identification by elimination of promiscuous binders and false positive/negatives Selectivity screening using panels of proteins Binding site ranking using LF-competition assays Intell. stepwise screening Screening of fragment, target focused and diversity libraries Protein & Structural Chemistry Primary screen of fragments before MR/X-ray Identify binding pockets with competition DSC for QC Validate mutations Label-free Interaction Analysis Biology Interaction proteomics Protein panels for the understanding of binding/biological effect mechanism Selectivity Validate binding difference in animal disease models Biophysical Chemistry Adds kinetic and thermodynamic resolution and throughput Transition state thermodynamics Computational Chemistry Validate your virtual binding site model with pure binding data 3D structure gives not on/only off, i.e. not equal to function/affinity/efficacy LF-screening can feed insilico models ADME & Bioanalytical Chemistry SA/AGP-maps for ranking of plasma protein binding Fa% from interaction analysis with liposomes Compare binding with the serum albumin from disease model animals 4 /

3 Label-free biophysical screening in Drug Discovery Up to ~1 compounds: Fragment libraries Selected diversities its from TS Directed libraries Content: Intent: Technologies: Primary Screen Active to it it To Lead Lead ptimization 1 1-5% 1 ew compounds 5-x.5% scaffolds ~ 1 ~all scaffolds -3 scaffolds Low Medium Medium/igh igh Binding: Yes/o Promiscuous? Covalent? False positive? Ranking: or IC 5 1:1 binding Binding site ID SAR: Full kinetic profile (k on, k off, ) Target selectivity Early ADME SPR and ITC X-ray/MR In-silico SAR: Full kinetic selectivity profile Thermodynamics CD Selection 5 What do Biacore instruments measure? Immobilized target buffer sample Compound buffer Response (RU) association Lead Binding response Fragment Sensorgram X Binding late report point Time dissociation Baseline 3/

4 Kinetic analysis of lmw drug-target interactions 5 4 Fc=1 Spot=1-r corr Estrogen receptor Fc= Spot=1-r corr Thrombin CD8 Adenosine receptor Aa Response (RU) Carbonic anhydrase IV protease Time (s) RU DA sequence CDK Kinase s Albumin s Increasing the number of targets!! 7 Binding kinetics data to understand drug efficacy 4/

5 Affinity Kinetics - Thermodynamics Lead series Affinity 1pM 1nM 1nM 1nM 1 M 1 M 1 M 1 mm Kinetics log (k a ) SPR/Biacore = k off /k on 1 pm 1 nm 1 nm log (k d ) 1 µm 1 µm 1 µm 1 mm 1*1 7 1*1 k a 1*1 5 Vary temperature k a (M -1 s -1 ) 1* Temp (K).1 k d.1 Transition state thermodynamics k d s -1 Equilibrium ow strong? Rates of complex formation and dissociation ow fast? Temp (K) Why that fast? All affinities and rate constants are apparent K i vs. shows good correlation Large differences at high affinities Buffer differences K i =PBS, p 5.5, 1M acl. = BS p 7.4,.15M acl K i (nm) e5 (nm) 1 Clinically used drugs 5/

6 Affinity Kinetics - Thermodynamics Lead series SPR/Biacore ITC/MicroCal Affinity 1 pm 1 nm Kinetics 1 nm 1 M 1 M 1 M 1 mm Equilibrium ow strong? (M -1 s -1 ) log k on = k off /k on 1 pm 1 nm 1 nm log 1 µm 1 µm 1 µm 1 mm Rates of complex formation and dissociation ow fast? Thermodynamics A,B,C kcal/mole G =RTln G = - T S A B C G - Gibbs free energy - Enthalpy T S - Entropy Why that strong? Complementary information Kinetics Thermodynamics n-off rate map log 7 Ethoxzolamide Entropy-Enthalpy map T S (kcal mol -1 ) log (kcal mol -1 ) Furosemide /

7 1 times higher affinity but lower efficacy Both drugs with identical and rapid clearance (short plasma half-life) Concentration 1 % occupied 9 % occupied Residence 1 M M 87 % occupied time - t ½ K 1 - = minutes D = 1 pm k 1-3 = 19 minutes on = 1 7 (M -1 s -1 ) k 1-4 = hours off = 1-3 (s -1 ) % occupied 1-5 = 19 hours 1 Time (h) 1 - = 8 days igher affinity is not always better! = 1 nm k on = 1 3 (M -1 s -1 ) k off = 1-5 (s -1 ) 13 Binding site occupancy (%) at different on/off/ -values at 1/ after 1h of interaction and 1h of dissociation Rapid off-rate limited efficacy A B C 95 D Slow off-rate protected efficacy 14 7/

8 Summary the 4 areas Ancient medicinal chemistry knowledge! Affinity limited efficacy igh affinity do not help if clearance is rapid! Rapid off-rate limited efficacy / 3/3 99/ 1/1 Slow on-rate limited efficacy Without on you are off! Slow off-rate enhanced efficacy With slow off you might still be on even when the drug is gone! 15 When is short residence time better? Short in relation to the effect wanted: e.g. Sleeping pills with very long residence time is very bad business For pharmacological mechanism requiring the endogenous ligand to perform routine fysiological functions a fast binding exogenous ligand displaying short lived interventions might be advisable. 1 Ion-channel antagonism: Ketamine and MK-81 gives mechanism based adverse effect probably due to strong and slowly dissociation binding to MDA Dopamin receptor antagonism Cyclooxygenase inhibitors: naproxen and ibuprofen sometimes better due to rapid reversible contra aspirin which binds covalently and gives side effects 1) Sara úňez et al. DDT (1) 17:1/:pp1- ) -methyl-d-aspartate receptor 1 8/

9 Lead optimization based on Residence and recognition time to understand SAR verview of the spread in on/off-rates IV-1 Protease inhibitors DPP-IV inhibitors Log (k on ) -4 Log (k off ) Ago Anta Carbonic anhydrase inhibitors Estrogen receptor inhibitors CD8/CD8 inhibitors E DC B A Scaffolds cluster in on/off/ - space Leads with slowest offrates all with scaffold E had highest biological potency Range in k on -5 orders of magnitude Range in k off -5 orders in magnitude 18 Also shown in poster 9/

10 The rate of association of the inhibitor to thrombin is of crucial importance for its in vivo effect The slope in the dose response curves from the rat arterial thrombosis model is related with the k on slope AT rate 4 efegatran C 191 melagatran PPACK inogatran argatroban kon (/µm.s) hirulog from: Elg M, Gustafsson D, Deinum J: The importance of enzyme inhibition kinetics for the effect of thrombin inhibitors in a rat model of arterial thrombosis, Thrombosis and aemostasis 78 (1997) Thrombin is the key enzyme in the coagulation process and converts fibrinogen into clottable fibrin. An intensive effort in several pharmaceutical companies has lead to the development of many potent and selective thrombin inhibitors with distinct advantages over heparin and warfarin. The choice of thrombin inhibitors as antithrombotic drugs depends a.o. on their affinity for the target and on their rate of binding. 19 Structure kinetic relations (SKR) of P38 map kinase inhibitors R R1 BIRB 79 1.) J. Regan, C.A. Pargellis, et. al. (3) Bioorg. Med.. Chem. Letters 13, Former BI drug candidate for inflammatoric and auto-immune diseases The t-bu induce rearrangement of activation loop and fills the formed lipofilic pocket 1 pm 1 pm 1 pm 1 nm 1 nm M 1 M 1 M ATP 1 /

11 SKR of P38 inhibitors R1 BIRB 79 Some of the first tool compounds show rapidon/rapid-off type of binding SB 358 R ) J. Regan, C.A. Pargellis, et. al. (3) Bioorg. Med.. Chem. Letters 13, pm 1 pm 1 pm 1 nm 1 nm M 1 M 1 M ATP SKR of P38 inhibitors R1 BIRB 79 The formation and filling of the pocket increase the complex stability longer residence time R pm 1 pm 1 pm 1 nm 1 nm R= R1= -tbu -ipr M 1 M 1 M 1.) J. Regan, C.A. Pargellis, et. al. (3) Bioorg. Med.. Chem. Letters 13, /

12 R SKR of P38 inhibitors R1 BIRB 79 R= Me R1=tBu -Me 1 pm 1 pm 1 pm 1 nm 1 nm Phenyl and tolyl identical affinity but tolyl better efficacy due to longer residence time R = Phenyl k off = 1.5*1-5 s -1 ; t 1/ = 13h R = Tolyl k off = 8.3*1 - s -1 1 M 1 M 1 M ; t 1/ = 3h SKR of P38 inhibitors Polar R gives slower onrates R1 BIRB 79 R1=tBu R R= Me Me Me Me Me C -Me Me 1 pm 1 pm 1 pm 1 nm 1 nm M 1 M 1 M ) J. Regan, C.A. Pargellis, et. al. (3) Bioorg. Med.. Chem. Letters 13, /

13 IV1-protease inhibitors: on-off rate map Ritonavir pm Ind Saq elf B48 B44 B49 B49 B41 A37 B439 A38 U75875 Rit Amp B8 B435 Drugs Cyclic urea Cyclic sulfonamides B8 analogues P1/P1 - modified P/P - modified ther B77 1 pm 1 nm 1 nm B39 B388 B3 B355 B45 A1 B95 A3 A45 A4 A18 B35 A47 A3 A15 B347 A1 XM3 A8 B nm 7 nm 7 nm A17 B / Markku ämäläinen, rion Finland Sep 7 / 1 M 1 M 1 M 1 mm P B8 Cyclic urea AA-8 S Cyclic sulfonamides AA-1 Symmetric B8 TS-analoges P1 P1 P IV1-protease inhibitors SKR pm Saq elf B48 B44 B49 B49 B41 U75875 B39 Rit B388 Amp Ind B3 A37 B439 A38 B8 1 pm 1 nm 1 nm B435 B45 A1 B35 XM3 A8 B nm 7 nm 7 nm B49 B347 1 M Di- Mono-hydroxy 1 M End amid ester 1 M Chirality of dihydroxy S,S R,R 1 mm B8 P1 P 1 3 B / Markku ämäläinen, rion Finland Sep 7 / P1/P1 Bz i-pr igher rotational freedom - ~1 times slower on-rate 13 /

14 IV1 protease inhibitors: CMFA-QSAR 38 Inhibitors ( in the training set, 1 in the test set) from 5 different scaffolds and the clinically used drugs. The structures were aligned and 3D-CoMFA was used for obtaining a QSAR model describing on-rates and off-rates. Predicted pk off Predictive CMFA-QSAR model only for offrate - not for on-rate nor affinity 7 / Markku ämäläinen, rion Finland Sep 7 / bserved pk off 8 / Markku ämäläinen, rion Finland Sep 7 / 14 /

15 Qualitative chemodynamics: IV-p 1 pm 1 nm 1 nm A = p 7.4 B = p 5.1 C = p 4.1 k on Indinavir C B A B C B Scaffold 1 A Scaffold A 1 M 1 5 C B peptide A k off The unknown binding site/binder situation Remedy - label-free/in-silico combination it identification and validation 1. Label free selectivity screening 1-5k compounds 4. LF-competition screen b.sites. Target selective hits Scaffolds identification 3. Structure feeds in-silico selection 5. Binding site selective hits Binding site classification. B-site classes feeds in-silico Lead identification and optimization Co-cryst. Xray Kinetic charact. Thermodyn. charact. Kinetic screening Lead identification & optimization. Early ADME CADD SAR/QSAR Cell based assays Animal models Candidate Drug 3 15 /

16 Conclusions 1. Binding kinetics resolve affinity into new more close to efficacy type of data. Residence time/off-rate of slowly dissociating compounds often dominates the binding site occupancy over time and cannot be neglected when judging the quality of a lead 3. Compounds with slow off-rates need only a peak in bioavailable concentration to have a broader therapeutic window 4. You cannot properly judge pharmacokinetic data without knowing the binding kinetics of the interaction 5. igh residence time selectivity is important to minimize side effects. The slowness of the off-rate should be judged in relation to the time constant of the biological system and the effect wanted 7. Rapid on-rate is important if the bioavailability is low or if a rapid effect is needed 8. There is also a wealth of other information in the Biacore /MicroCal measurements which can be used for identification of good/bad binders: (on/off-rates, enthalpy/entropy, stoichiometry, selectivity, shape, affinity, specificity) 9. Label-free screening, binding kinetics and thermodynamics are now on the way of being fully integrated into the DD-process of most pharma/biotec companies 31 GE, imagination at work and GE monogram are trademarks of General Electric Company. Biacore and MicroCal are trademarks of GE ealthcare companies. All third party trademarks are the property of their respective owners. All goods and services are sold subject to the terms and conditions of sale of the company within GE ealthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE ealthcare representative for the most current information. 13 General Electric Company All rights reserved. GE ealthcare Bio-Sciences AB, a General Electric Company. GE ealthcare Bio-Sciences AB, Björkgatan 3, SE Uppsala, Sweden. 1 /