1 Combinatorial Synthesis and Drug Discovery Random Search e.g., buying paintbrush, mop, hammer,...
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1 Combinatorial Synthesis and Drug Discovery • Random Search e.g., buying paintbrush, mop, hammer, gluestick, nail, etc. etc. for hanging a picture frame on the wall • Rational Design e.g., buying a nail for hanging the frame • Combinatorial Search e.g., buying a box of nails of different sizes for hanging the frame
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Transcript of 1 Combinatorial Synthesis and Drug Discovery Random Search e.g., buying paintbrush, mop, hammer,...
1
Combinatorial Synthesis and Drug Discovery
• Random Searche.g., buying paintbrush, mop, hammer, gluestick, nail, etc. etc.for hanging a picture frame on the wall
• Rational Designe.g., buying a nail for hanging the frame
• Combinatorial Searche.g., buying a box of nails of different sizes for hanging the frame
2
~ 5 IND
~ 250 in vivo
~ 500 in vitro
> 10,000 compounds
> 15 years ~ 1 NDA > $500 million
Time Cost
Why Combinatorial Synthesis?
3
X X X Y Y Y+
……….. 9 products
MIXTURE SYNTHESIS
Traditional and Combinatorial Synthesis
X
X
X
Y
Y
Y
= CH3COOH
= CH3CH2COOH
= (CH3)2CHCOOH
= PhNH2
= p-CH3PhNH2
= p-NO2PhNH2
For example
4Ser Ala Tyr Arg
SPATIALLY ADDRESSABLE SYNTHESIS
X Y+I)
X Y+II)
X Y+III)
5
SPLIT AND MIX STRATEGY
X X X
Y
Y YY
MIX
SPLIT
6
SPLIT AND MIX STRATEGY
MIX and SPLIT
7
DECONVOLUTION BY TAGGING WITH GC-ANALYZABLE MOLECULES
MIX
SPLIT
Cl
Cl
Cl
Cl
Cl
=
=
=
….and many more.
8
DECONVOLUTION BY TAGGING (continued)
MIX and SPLIT
9
DECONVOLUTION BY TAGGING - Real Example
10
DECONVOLUTION BY ENCODED DNA
MIX and SPLIT
SPLIT
A1
N1
A2
N2
A3
N3
A2A2A1A2
N1N2 N2N2
A3A2
N3N2
A2A1A1A1
N1N1 N2N1
A3A1
N3N1
A2A3A1A3
N1N3 N2N3
A3A3
N3N3
A2 A3 A1
11
Parallel Synthesis and Screening of a Solid Phase Carbohydrate Library
Rui Liang, Lin Yan, Jennifer Loebach, Min Ge, Yasuhiro Uozumi, Klara Sekanina, Nina Horan, Jeff Gildersleeve, Chris Thompson, Andri Smith,
Kaustav Biswas, W. Clark Still, Daniel Kahne*
A solid phase carbohydrate library was synthesized and screened against Bauhinia purpurea lectin. The library, which contains
approximately 1300 di- and trisaccharides, was synthesized with
chemical encoding on TentaGel resin so that each bead contained a single carbohydrate. Two ligands that bind more tightly to the lectin than Gal--1,3-GalNAc (the known ligand) have been identified. The strategy outlined can be used to identify carbohydrate-based ligands for any receptor; however, because the derivatized beads mimic the polyvalent presentation of cell surface carbohydrates, the screen may prove especially valuable for discovering new compounds that bind to proteins participating in cell adhesion.
Science (1996) 274; 1520-1522
An Example of Combinatorial Approach in the Discovery of a Potent Ligand
12
Synthesis of the Library
13
High Throughput Screening of the Library
Derivatized TentaGel beads were washed three times with 1 ml of PBST buffer and then suspended in 1 ml of PBST containing 3% bovine serum albumin. The beads were incubated at room temperature for 3 hours on a rotary shaker in 1 ml of a biotin-labeled lectin solution and then washed three times with 1 ml of TBST buffer containing 1% BSA. The beads were incubated on a rotary shaker for 20 min at room temperature in 1 ml of alkaline phosphatase-coupled streptavidin. The beads were washed three times with 1 ml of alkaline phosphatase buffer and kept in the alkaline phosphatase buffer prior to staining. A portion of the beads was transferred to a petri dish and the alkaline phosphatase buffer was replaced with 200 µl of a solution containing 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT). Color development was observed under a low-power microscope. The staining was terminated by washing the beads twice with 200 µl of sodium EDTA solution. The colored beads were picked out manually under the microscope for decoding.
14
High Throughput Screening of the Library
A portion of the beads in the library after 5 min of staining. The dark bead in the center was identified as a hit. This level of contrast shown in the photograph was representative [60X].
15
Major Disadvantages of Combinatorial Synthesis
• Limited structural diversity of synthetic library
• Wastage of much of raw material
• Wastage of assay material
• No de novo drug designed as yet based solely on combinatorial synthesis
• How about combinatorial virtual library screening?
16
Combinatorial Virtual Hexasaccharide Library
_ _
_
_
O
O
C H 2 O S O 3
N H S O 3
O S O 3
O
O H
O H
C O O
O
G l c A G l c N S 3 S 6 S
_ _
_
_
O
O
C H 2 O S O 3
N H S O 3
O S O 3
O
O H
O H
C O O
O
_ _
_
_
O
O
C H 2 O S O 3
N H S O 3
O S O 3
O
O H
O H
C O O
O
G l c A G l c N S 3 S 6 S
_
_
_
O
O
C H 2 O H
N H S O 3
O S O 3
O
O X
O H
C O O
O
G l c A ± 2 S G l c N S 3 S
_
_
_
O
O
C H 2 O H
N H S O 3
O S O 3
O
O X
O H
C O O
O
_
_
_
O
O
C H 2 O H
N H S O 3
O S O 3
O
O X
O H
C O O
O
G l c A ± 2 S G l c N S 3 S
_ O
O
C H 2 O H
N H C O C H 3
O H
O
O
O X
O HC O O
I d o A ± 2 S G l c N A c
_ O
O
C H 2 O H
N H C O C H 3
O H
O
O
O X
O HC O O
_ O
O
C H 2 O H
N H C O C H 3
O H
O
O
O X
O HC O O
I d o A ± 2 S G l c N A c
I d o A ± 2 S G l c N A c 6 S
_ O
O
C H 2 O S O 3
N H C O C H 3
O H
O
O
O X
O HC O O
_
I d o A ± 2 S G l c N A c 6 S
_ O
O
C H 2 O S O 3
N H C O C H 3
O H
O
O
O X
O HC O O
_
_ O
O
C H 2 O S O 3
N H C O C H 3
O H
O
O
O X
O HC O O
_ _
_
_
O
O
C H 2 O S O 3
N H S O 3
O H
O
O
O X
O HC O O
I d o A ± 2 S G l c N S 6 S
_
_
_
O
O
C H 2 O S O 3
N H S O 3
O H
O
O
O X
O HC O O
_
_
_
O
O
C H 2 O S O 3
N H S O 3
O H
O
O
O X
O HC O O
I d o A ± 2 S G l c N S 6 S
_
_
O
O
C H 2 O H
N H S O 3
O H
O
O
O X
O HC O O
I d o A ± 2 S G l c N S
_
_
O
O
C H 2 O H
N H S O 3
O H
O
O
O X
O HC O O
_
_
O
O
C H 2 O H
N H S O 3
O H
O
O
O X
O HC O O
_
O
O
C H 2 O H
N H S O 3
O H
O
O
O X
O HC O O
I d o A ± 2 S G l c N S I doA±2SGlcNS3S6S
_
_
_
O
O
CH2OSO3
NHSO3
OSO3
O
O
OX
OHCOO _
I doA±2SGlcNS3S6S
_
_
_
O
O
CH2OSO3
NHSO3
OSO3
O
O
OX
OHCOO _
_
_
_
O
O
CH2OSO3
NHSO3
OSO3
O
O
OX
OHCOO _
19 Disaccharides as building units for a hexasaccharide 191919 = 6,859 topologies!
17
New Computational Design Principle
Dual Filter Algorithm
Build Virtual Library of HS Hexasaccharides (‘Average Backbone’
Hypothesis)
Minimize GAG Sequences
(Glycosidic Bond Torsion Constraints)
Scoring (‘Affinity’) Test(10,000 GA iterations,
one run per GAG)
Score in top 1%?
Convergence (‘Specificity’) Test
(100,000 GA iterations; three runs per GAG)
RMSD 2.5 Å?
END
DISCARD
START
YES
YES
NO
NO
High-Affinity, High Specificity Sequence(s)
18
0
200
400
600
800
1000
0 to5
10to15
20to25
30to35
40to45
50to60
65to70
75to80
85to90
95to
100
105to
110
115to
120
07
14212835
95 to100
100to105
105to110
110to115
115to120
Gold Score
Fre
quency
0
200
400
600
800
1000
0 to5
10to15
20to25
30to35
40to45
50to60
65to70
75to80
85to90
95to
100
105to
110
115to
120
07
14212835
95 to100
100to105
105to110
110to115
115to120
Gold Score
Fre
quency
Combinatorial Virtual Library Screening
Raghuraman, Mosier and Desai (2006) J. Med. Chem. 49, 3553-3562.
Application of 1st Filter – the ‘Affinity’ Filter
19
Green = X-rayAtom type = Final 9 of 7000 sequencesPurple = 1 unusual high-affinity sequence
3-O-SO3
2-O-SO3
_
_
6-O-SO3
_
Green = X-rayAtom type = Final 9 of 7000 sequencesPurple = 1 unusual high-affinity sequence
3-O-SO3
2-O-SO3
_
_
6-O-SO3
_
Finding a Needle in a Haystack!
GlcAp (4C1)
I doAp (1C4)
I doAp (2SO)
COOH 2-OSO3
6-OSO3
3-OSO3 2-NSO3
GlcNp (4C1) Color codes:
Sulfate
Acetyl Unsubstituted
LEGEND
3
Glycosidic linkage: (1? 4)
(1? 4)
O
O X
O H
O
O
O
C H 2 O X
N H Y
OX
C O O_
X: SO3/ H Y: NAc/ SO3/ H
_ _
Acetyl Unsubstituted
(1? 4)
(1? 4)
A
B
Application of 2nd Filter – the ‘Specificity’ Filter
20
Arg129Lys125 Lys114
3-O-SO3
_
helix D
helix A
X-ray
Arg129Lys125 Lys114
3-O-SO3
_
helix D
helix A
X-ray
Predictability of Binding Geometry
Geometry Search with Flexible Ligand
