Beruflich Dokumente
Kultur Dokumente
Bernd Wendt
EMBL, Heidelberg
Large and small molecules
• Large molecule
• X-ray structure
• Protein
• Target
• “Lock”
• COX-1
• Small molecule
• 3D model
• Ligand
This is not • “Key”
the keyhole! • Aspirin
• surface representation
Large and small molecules
• Look inside the protein
• Display of the protein fold
• Active site in green dots
• Aspirin
• Heme-group
This is the
keyhole!
• ribbon representation
From serendipity to rational drug design: Aspirin
• 400 BC: Hippocrates: extracts from willow tree (containing salicylic acid) for pain
relieve
• 1890’s: synthesis of acetylsalicylic acid (Aspirin)– improvement on side effects
(irritation of stomach)
• 1970’s: Aspirin target identified as cyclo-oxygenase (COX)
• 1980’s: Two forms of COX identified:
• COX-1, constitutive
• COX-2, induced at sites of inflammation
• Mid 1990’s: X-ray structures of COX-1 and COX-2 available
• Rational design of selective COX-2 inhibitors
• 1998/99: Celebrex and Vioxx approved by FDA for osteoarthritis and rheumatoid
arthritis
• Mid 2000’s: Randomized placebo-controlled trial showed an increased risk of heart
attack and stroke
• Vioxx withdrawn from market
• Celebrex with warning label
A detailed look at Aspirin and its target
Develop-
Biology Chemistry
ment
targets: 16%
Transporters
Cytochrom P450
16%
• 3,051 NHR
Proof-of-
Docking
Concept:
X-ray structure
of bound ligand
13 compounds
Rational Approaches: Fragment-based discovery
• Chemical Microarrays
(Graffinity, Heidelberg)
• ~100,000 fragments linked onto
chip
• parallel detection of weak,
specific interactions
• Confirmation of hits by functional
testing and X-ray
• Thrombin-example
• Novel fragment identified that
binds to S1-pocket (usually
occupied by benzamidine-group)
S1
• Confirmed by X-ray
Rational Approaches: Comparative Molecular Field
Analysis (CoMFA)
Contour
Maps
Bio PLS
QSAR
equation
QSAR Table = SYBYL MSS
Rational Approaches: Comparative Molecular Field
Analysis (CoMFA)
• Example Steroids:
• Dataset of 31 ligands
• Measured activity data
(CBG-Affinity)
• CoMFA-steps
• Build molecules in 3D
• Align all molecules on top of
each other
• Calculate fields
• Run statistical analysis
• Display results in from of
contour maps
• Use regression equation for
prediction of untested
compounds
Rational Drug Design at EMBL: Aurora A
Aurora-A: ATP binding site with bound ATP Aurora-A: ATP-binding site blocked by inhibitor
Aims for Aurora A Project
18
Aurora A: Characterisation of Compounds
19
Aurora A:
Comparison of ATP versus allosteric inhibitor
ATP competitive non-competitive
123671 103444
Inhibition (%) Inhibition (%)
110
80
80
60
40 50
20 20
0
-10
1 10 100 1 10 100
Compound Concentration [µM] Compound Concentration [µM]
ATP [µM] IC50 [µM] r² Hill-Slope ATP [µM] IC50 [µM] r² Hill-Slope
20 6.68 0.997 1.163 20 1.7 0.996 1.537
200 49.8 0.997 1.06 200 1.9 0.993 1.759
1000 >100 0.975 0.42 1000 0.84 0.978 2.165
20
Allosteric inhibitors of Aurora A:
TPX2 competition
IC50 Aurora A
103421:02
IC50 = 4.8 µM; Slope = 1.33; r2 = 0.995 Of ~100 compounds tested
90
Inhibition (%)
60
80
50
20
-10
1 10 100
Concentration (µM)
with TPX2
21
Aurora A Project: Summary
• Rational design of allosteric
inhibitors
• Examine H-bond interactions
• Hydrophobic interactions
• Water molecules
• Fit active compounds into
binding pockets
• Improve hypothesis
• Docking and scoring of
untested compounds
• Help guiding synthesis and
medicinal chemistry efforts One of the TPX2-binding sites
Rational Drug Design:
Unsolved problems and limitations
• Experimental data in early stages is inaccurate
• X-ray structures give static picture
• Conformational changes upon binding
• Role of water molecules and protonation states of functional
groups in binding pocket often unknown
• Limited experimental data in later stages of development
• Complexity of biological systems
• Too many targets (whole proteome)
• Proteome variability
• Short-term versus long-term effects
• Elena Conti
• Friedrich Reinhard (Chemical Biology Core Facility)
• Joe Lewis (Chemical Biology Core Facility)