Fragment based drug design O OH O NH2 NH2 N Fragment based drug design ● Nový směr zaváděný v posledních 13 letech v předních farmaceutických společnostech nebo specializovaných high-tech. Firmách ● Využívá nejnovějších vědeckých poznatků a technologií z oblastí genetiky, molekulární biologie, proteinové krystalografie a/nebo NMR spektroskopie, bio-informatiky a počítačové chemie (modelování, docking) Traditional design ● Traditional recipes and random discoveries ● Combinatorial chemistry and natural drugs ● Screening (ultra high-throuput screening, HTS), "hit" → "lead compound" ● Compounds library Typical drug properties ● Molecular mass 350-450 ● Protein affinity, inhibition IC50 50 nM ● Five - rule ● Defined number of bond rotations ● Optimisation usually by increasing molecular mass Five - rule ● Ch.A.Lipinski, 1997. Oral drugs ● Not more than 5 hydrogen donors (OH, NH) ● Not more than 10 (2x5) HB acceptors (N a O atomy) ● Molecular mass up to 500 ● ClogP less than 5 High-Throughput Screening, HTS Up to one million compounds has to be scanned to find activity Limity HTS ● Automated, costly method ● Less productive - high death ratio of hits ● Big libraries of chemical compounds – small drug-like space ● Did not reduce cost NCE/(milion $) ● For 5000 compounds only 5 clinical trials ● Only 5 - 20 % on the market ● Cost $800 mil. Targets Targets ● Week binary complexes leads to stable multiprotein complexes ● Many targets can be found between multiprotein complexes for cell regulation and signalisation Genomics ● „Human genome project“ human DNA. ● Leads to more protein targets ● Structural genomics - gene function determined from structure Genomics Target identification ● Functional genomics - huge number of potential protein targets ● Structural genomics searches relationship between sequence structure and biological function ● PDB is growing Structural biology and drug discovery Fragment based drug design Fragment definition ● Men.islou.enina (Pravidlo 3. Men. ne. 300 Da. Typical 150-250. ClogP 3, not more than 3 HB donors and acceptors ● Medium affinity (100ĘM-1mM)ƒ ● HTS. Smaller library (Graffinity library - 20 000 fragments). ● Biophysical screening: NMR, X-ray, MS ● Binding sites FBDD - advantages ● Smaller library covers bigger chemical space ● 100 fragmens 1 000 000 combinations ● Fragments can be identified by protein crystallography even if it has no biological activity ● Fragments can be found in silico - docking Screening ● Screening fragments against protein targets ● Following techniques are used ● rtg. crystallography ● NMR spektroskopy (Water LOGSY) ● Isothermal titration calorimetry ● Surface plasmon resonance ● Non covalent mass spectrometry NMR Screening ● NMR most productive ● SAR using NMR, (SAR = structure activity relationship) ● Subjective interpretation ● Not automated ● Abbott laboratories, Novartis, Vertex Pharmaceuticals, Hoffmann-La Roche, Triad Therapeutics NMR screening cocktail of three fragments 0.5 mM + enzyme 20 mM Only fragment 1 + enzyme (diference) X-ray screening ● Best for protein - ligand interaction study ● Highly automated ● Fragment visualisation - possible in silico optimization ● Rejection of non specific affinity ● Synchrotron is required ● Astex Therapeutics (Technology), Abbott Laboratories, Structural GenomiX (SD CA) Astex facilities Strukturní screening fragmentů Structure based fragment screening ● 100R1x100R2x100R3 = 1,000,000 cmpds ● Higher hit rate ● Detect unique structures (mM) ● Precise structural data (validate/prioritise the hits) ● Rapid structure based optimisation ● Room to optimise ● High re-use -protein family specific scaffolds X-ray screening X-ray screening X-ray screening ● 4-10 compounds 25-50mM concentration Crystal is submerged and it chose best fragment Identification in electron density maps X-ray process definition ● 10-100 mg purified protein proteinových, 100 ● Every crystal soaksz 5-10 fragments. ● Crystal is mounted and measured (flash cooling, cryo-crystallography) X-ray process definition ● Automated high throughput screening on synchrotron (in Argonne National Lab. X-ray screen 1000 compounds 24-48 h) ● Reasonable speed also on new diffractometersFragment i (54 crystals/80h on Rigaku FR-E Superbright). ● Fragment is shown on difference map Crystallization protocol (PixSys) Astex facilities – X-ray analysis Fragment connection and optimization ● Two fragments in two places and connecting molecule (spacer) ● Self-assembly – template method, Protein catalyses synthesis its own inhibitor ● Binding sites mapping SAR (probe analogy GOLD). ● Connecting fragments increase entropy ● The potency is increased by 3-5 orders of magnitude Fragment→lead. Fragment connection and optimization Fragment connection and optimization Optimalizace ● Znalost způsobu, jak se fragment váže na proteinový cíl umožňuje fragment zvětšovat. ● Používají se počítačové metody ● Docking ● „Experimentální“ metody Isostar, Superstar, Gold suite Gold - docking ● Protein-ligand docking ● GlaxoSmithKline, U.Sheffield, CCDC ● Complete flexibility of the ligand, the partial flexibility protein ● Energy-based functions Isostar ● Discrimination between active and inactive compounds ● Goldmine – decriptors Gold - docking Gold - docking Advantages and disadvantages ● The small fragment has a greater chance that better incorporated into the binding site of the target protein than the whole molecule. ● The fragments have a higher binding energy per unit weight. ● Screening of small fragments leads to a greater number of "hits". The number of fragments is at 100 - 1000 (as compared to a million in HTS). Advantages and disadvantages ● "Leads" from fragments have lower mortality rates. 70% of hits from HTS fails, 80% FSDD hits are successful. ● Allows you to discover the "lead" where HTS failed ● It is possible to obtain a lead outside the standard database and thereby obtain a patentable compound