Combinatorial Synthesis and Drug Discovery

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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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Why Combinatorial Synthesis?
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Traditional and Combinatorial Synthesis
? MIXTURE SYNTHESIS

.. 9 products
For example
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? SPATIALLY ADDRESSABLE SYNTHESIS

II)
X
Y

X
Y
III)
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? SPLIT AND MIX STRATEGY
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? SPLIT AND MIX STRATEGY
MIX and SPLIT
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? DECONVOLUTION BY TAGGING WITH GC-ANALYZABLE
MOLECULES
SPLIT
MIX
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? DECONVOLUTION BY TAGGING (continued)
MIX and SPLIT
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? DECONVOLUTION BY TAGGING - Real Example
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? DECONVOLUTION BY ENCODED DNA
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? An Example of Combinatorial Approach in the
Discovery of a Potent Ligand
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-b-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
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? Synthesis of the Library
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? 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.
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? 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.
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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?

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Combinatorial Virtual Hexasaccharide Library

• 19 Disaccharides as building units for a
  hexasaccharide
• 19?19?19 6,859 topologies!

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New Computational Design Principle

• Dual Filter Algorithm

START
YES
NO
NO
DISCARD
YES
High-Affinity, High Specificity Sequence(s)
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Combinatorial Virtual Library Screening

• Application of 1st Filter the Affinity
  Filter

Raghuraman, Mosier and Desai (2006) J. Med. Chem.
49, 3553-3562.
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Finding a Needle in a Haystack!

• Application of 2nd Filter the Specificity
  Filter

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Predictability of Binding Geometry

• Geometry Search with Flexible Ligand