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