Fragment Screening in Drug Discovery Marc Martinell SEQT, Sitges, 19th-20th October 2006 Crystax Pharmaceuticals SL Barcelona Science Park Josep Samitier 1-5, E-08028 Barcelona Tel: +34 93 403 4703 Fax : +34 93 403 4788 www.crystax.com Overview The Company Fragment Screening Fragment Library Detection of Fragment Binding Structures of fragment-protein complexes Hit Selection and optimization Summary
The Company CrystaX is a Structure-based Drug Discovery company. Founded in 2002 by recognised scientists J. Aymamí and M. Coll. Current team of 22, among them 12 PhD with international experience, additional technical staff. Advisory agreements with experts in complementary areas (Computational Chemistry, Organic Synthesis, etc.) Our main strength is the combination of structural biology and chemistry into a team that can address any issue in the leadfinding process. Barcelona Science Park Access to top technology equipment and labs X-ray Crystallography NMR for Biomolecules Fine Chemistry Combinatorial Chemistry Microcalorimetry Surface Plasmon Resonance Genomics and Transcriptomics Proteomics Business Model Fee for Service Co-Development of new drugs with pharma partners using our platform technology Development of own pipeline Crystax s approach to business Fee-for-service business and R&D collaborations Deliver value to clients Develop relationships for licensing opportunities Product pipeline from own drug discovery Short term: in collaboration with other companies Mid term: own licensing opportunities B2D2 Common technology platform
Collaborations Fragment Screening
Fragment Screening There is a massive amount of drug like molecules with a suitable molecular weight for drug discovery HTS and traditional discovery techniques often start with relatively large and complex molecules. The main disadvantage of traditional approaches is that finding one right molecule amongst such a vast number is quite difficult and, moreover, it is indeed hard to, once found, jump from one chemical branch to another. 10 60 10 180 Drug like molecules with Mw < 800 Fragment Screening Fragment screening allows to start with a smaller molecule and then add as much complexity as needed. The difficulty is that you start at a pre-hit stage, where functional activity is difficult/impossible to measure.
Fragment Screening Drug-like mm µm nm pm K D Probability 0 2 4 6 8 10 Ligand complexity Probability of detection Probability of binding Adapted from: M.H. Hann et al, J. Chem. Inf. Comput. Sci. 2001, 41, 856-864 Fragment Screening Fragmentlike Fragmentlike Drug-like mm µm nm pm K D Techniques able to detect and develop low affinity binders are needed Probability 0 2 4 6 8 10 Ligand complexity NMR X-Ray Crystallography Biophysical techniques Probability of detection Probability of binding Adapted from: M.H. Hann et al, J. Chem. Inf. Comput. Sci. 2001, 41, 856-864
Fragment Screening SAR-by-NMR (S.B. Shuker et al. Science 1996, 274, 1531-1534) R. Carr and H. Jhoti, DDT, 2002, 7, 522-527 Fragment Screening - Examples Hit Lead N p38 kinase N O OH IC 50 = 1.1mM F N N O OH IC 50 = 200nM J. Fejzo et al, Chem.Biol, 1999, 6, 755-769 N Urokinase N OH Ki = 56µM N N NH Ki = 370nM V.L. Nienaber et al, Nat. Biotech., 2000, 18, 1105-1108 NH 2 NH2 CO 2 H Thymidylate Synthase O O S N CO 2 H Ki = 1000µM O O S N O HN O CO 2 H Ki = 33nM D. A. Erlanson et al, PNAS, 2000, 97, 9367-9372 CO 2 H
Fragment Library Fragments are organic molecules with a low degree of complexity and non-reactive CrystaX s Fragment Library commercial compounds Selection process based on the newest criteria for fragment libraries. The balance between chemical space exploration and efficiency of the hit to lead process is optimized. CrystaX s Fragment Library 1000 3,000.000
CrystaX s Fragment Library commercial compounds Molecular properties (fragment-like) Unwanted reactivity Clustering and selection CrystaX s Fragment Library 80.000 1000 280.000 3,000.000 CrystaX s Fragment Library Quality Control Quality Control of individual compounds Solubility, identity, purity and stability Quality Control of mixtures of 7-9 compounds Designed to obtain the minimum signal overlap among compounds Solubility, identity, purity and stability 615 compounds ready for Fragment Screening in 71 mixtures Constant monitoring of false positive and/or promiscuous binders
Detection of fragment binding Due to their low degree of complexity, fragments are low affinity binders Fragment Screening by NMR Target protein NMR screening STD Relaxation edited spectra WaterLOGSY TrNOE 19 F-NMR Chemical Shift Mapping (CSM) Continuous development Positive Fragments
Fragment Screening by NMR - STD I 0 Each molecule has characteristic signals on a 1D 1 H spectra B. Meyer and T. Peters, Angew. Chem. Int. Ed., 2003, 42, 864-890 Fragment Screening by NMR - STD I 0 In a solution of a protein with a large excess of these molecules, their spectra is almost not affected B. Meyer and T. Peters, Angew. Chem. Int. Ed., 2003, 42, 864-890
Fragment Screening by NMR - STD I 0 I SAT When the protein is saturated with a selective irradiation, this saturation is transferred to the binding molecules. This saturation produces an attenuation of its NMR signal. B. Meyer and T. Peters, Angew. Chem. Int. Ed., 2003, 42, 864-890 Fragment Screening by NMR - STD I 0 I SAT By subtracting both spectra, an NMR difference spectrum is obtained in where ligand molecules that bind to the target can be identified I STD = I 0 -I SAT δ (ppm) B. Meyer and T. Peters, Angew. Chem. Int. Ed., 2003, 42, 864-890
Fragment Screening by NMR The complete library is screened by NMR 1D 1 H mixture Relaxation Edited Experiment The signals of fragment binders disappear Saturation Transfer Difference (STD) The signals of fragment binders appear Direct deconvolution from mixtures of fragments Fragment Screening by NMR Examples 1D 1 H spectrum STD spectrum
Fragment Screening by NMR Examples Direct Deconvolution CXL-8 CXL-7 CXL-9 CXL-11 CXL-12 CXL-18 CXL-20 CXL-23 CXL-24 CXL-28 STD Fragment Screening by NMR Examples Direct Deconvolution Positives fragments: CXL-20 and CXL-23 CXL-20 CXL-23 STD 13
Fragment Screening by NMR Examples STD STD upon addition of known active-site ligand Fragment Screening by NMR Examples CXL-23 CXL-23 Only compound CXL-23 interacts with the active site STD STD upon addition of known active-site ligand
Fragment Screening by NMR Examples 1D 1H mixture Relaxation Edited Experiment STD The signals of fragment binders disappear The signals of fragment binders appear 1D 1H compound CXL-212 Compound CXL-212 interacts with the protein Fragment Screening by NMR Examples 1D 1H mixture + Competitor Relaxation Edited Experiment addition of known active-site ligand STD 1D 1H compound CXL-212 15
Fragment Screening by NMR Examples Using competition studies by NMR ligands for specific binding sites can be identified 1D 1 H mixture Relaxation Edited Experiment STD Compound CXL-212 interacts with the active site of the protein target 1D 1 H compound CXL-212 Projects at CrystaX Project 1 2 3 4 5 6 Field Inflammation Oncology Oncology Auto-immune Oncology - Hit rate 10% 5% 5% 4% 3% 1%
Structures of fragment-protein complexes Due to their low affinity and small size fragments are more difficult to study by Xray crystallography Fragment Screening by Xray Positive Fragments Crystallization (co-crystallization or soaking) Structure determination Fragment Hits
High-Throughput Crystallography Sparse matrix screening for initial crystallization conditions > 1000 conditions Optimization of conditions for crystal growth Reproduction of known crystallization conditions Characterization of crystals Collection of apo -datasets Ab-initio screening for crystallization conditions in the presence of inhibitors Large scale production of crystals Soaking of crystals with ligands Co-crystallization under analogous conditions Collection of diffraction data in the presence of inhibitor Automatic Data Processing & Analysis of Results Data are processed automatically using commercial software and a modular suite of proprietary scripts. Resulting electron densities are inspected individually, analyzed and classified. Models of the protein-ligand complex are partially or completely refined, depending on the needs of the individual project.
Structures of fragment-protein complexes Fragment Screening renders a high amount of structural data, thus increasing the efficiency of the hit to lead process Projects at CrystaX Project Field Hit rate (NMR) Hit confirmation (Xray) 1 Inflammation 10% 40% 2 Oncology 5% 45% 3 Oncology 5% ongoing
Alternative approaches Primary screening method NMR (ligand-based detection) NMR (protein-based detection) SPR Biochemical assays Virtual Screening Xray Hit confirmation Xray Xray Xray Xray Xray - The combination of ligand-based NMR methods and Xray crystallography renders the most general approach with the minimum consumption of protein sample Hit Selection and Optimization Several prioritization criteria are needed in order to select the most interesting hits
Hit selection Fragment Hits Preliminar SAR Selected HITS Hit validation and selection Validation of Binding-mode New chemical structures Evaluation of other molecules within its cluster and/or molecules that contain the same binding motif Hit Optimization Fragment Hits Biophysical methods (B2D2 TM ) SPR K on and K off Microcalorimetry Hº, Sº Selected HITS Synthesis Activity Assays Optimization Molecular Modeling LEADS Fragment Screening!!
Biophysics-Based Drug Discovery (B2D2 TM ) pm Activity Assays Lead nm? µm Fragment Screening Hit mm Biophysics-Based Drug Discovery (B2D2 TM ) pm Activity Assays Lead Fragment Screening nm µm mm Hit ADME Toxicity Selectivity Patentability B2D2 TM Biophysical characterization renders high quality data and increases the efficiency of Hit2Lead process
Biophysics-Based Drug Discovery (B2D2 TM ) Biophysics-Based Drug Discovery B2D2 TM NMR X-Ray crystallography Biophysics-Based Drug Discovery (B2D2 TM ) Biophysics-Based Drug Discovery B2D2 TM NMR X-Ray crystallography Calorimetry Unique technique for thermodynamic data (K D, Hº, Sº) Low throughput Label free High protein consumption G H -T S G H -T S Same K D but different thermodynamics
Use of ITC in Fragment Evolution ITC requirements 10 < nk A [M] T < 100 Fragment Hits: mm - µm K A ~ 10 4 10 8 K D ~ 100µM 10nM Soluble compounds Lead compounds: nm - pm High amounts of sample Use of ITC in Fragment Evolution
Biophysics-Based Drug Discovery (B2D2 TM ) Biophysics-Based Drug Discovery B2D2 TM NMR X-Ray crystallography RU 500 Calorimetry Biacore (SPR) Kinetic data (K A, k on, k off ) Medium-high throughput Immobilization needed Low protein consumption 0 0 120 240 360 480 Time (seconds) Same K D but different kinetics Biophysics-Based Drug Discovery (B2D2 TM ) Biophysics-Based Drug Discovery B2D2 TM NMR X-Ray crystallography Calorimetry Biacore (SPR) Fluorescence spectroscopy Affinity constant (K A ) Medium-high throughput Fluorescent-label needed Low protein consumption
Summary Renders. Novel Structures Fragment Screening B2D2 but also. Summary New binding modes Novel Structures New binding sites Fragment Screening B2D2 Lead Optimization Structural waters Protein Hot-spots Biophysical characterization Protein flexibility
Acknowledgements Joan Aymamí Miquel Coll Maria Kontoyianni Ingo Korndoerfer Montse Soler Xavier Barril Isabel Navarro Franck Chevalier Teresa Luque Irena Bonin Unitat RMN (SCT-UB) Unitat Químic Fina (SCT-UB) Unitat Citometria (SCT-UB) Unitat Química Combinatòria (PCB) Plataforma Raigs-X (PCB) A. Llebaria (RUBAM, IIQAB-CSIC) R. Gutierrez (IMIM-UPF) F. J. Luque (UB) Carolina Moral Marta Masip Sarah Sotil Verónica Toledo Laura Quintana Sonia Soriano Anja Leimpek Marian Domínguez Marta Martín Thank you for your attention