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Graffinity's SPR-based
Fragment Screening Process
Graffinity has pioneered high-throughput SPR-based fragment screening,
an efficient drug discovery paradigm that enables the rapid
identification and development of diverse novel quality leads and drug
candidates.The platform has been validated in a series of projects and
collaborations with leading pharmaceutical and biotechnology companies
and has been successfully applied to over 85 protein targets. |
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Design &
Synthesis |
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2.
Array Production |
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3.
Fingerprinting /
fragment screening |
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4. Data
Analysis |
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5. Hit
Evolution |
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The Five Elements of the Graffinity Drug Discovery Process |
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1. Design & Synthesis
By virtue of the design process, fragments and leadlike compounds
comprise excellent starting points for drug discovery.
Design
Primary screening for drug fragments is an attractive and efficient
paradigm
in the quest for new lead molecules. Compounds of low complexity are
more likely
to match a given binding site and also make better use of their
chemical functionalities
resulting in more efficient binders than those found in traditional
screening libraries.
Due to the higher hit probability, fewer compounds need to be screened
to identify
starting points for chemical optimization. Although generally expected
to be weak
binders, small diverse and information-rich fragments leave more room
for optimization
without the risk of leaving leadlike or druglike chemical space.
Low-affinity screening
with the help of tethered drug-fragments on chemical microarrays is a
highly promising
approach for the rapid discovery and optimisation of such small
molecules.
Graffinity's
Fragment and Leadlike Compound Library
Fragment Space
24,000 Fragments
100 - 300 Da
Leadlike Space
86,000 Displayed Fragments
mean 320 Da
Synthesis
The preparation of high density chemical microarrays for drug discovery
requires a highly parallel and controlled process for the synthesis of
fragment and leadlike compounds. Compound production is achieved by
nanoscale solid phase organic synthesis (40 nmoles/compound). All
compounds are linked to the same proprietary spacer molecule
(ChemTag®), which serves as an attachment point for the covalent
immobilization on the array surface.
After synthesis compounds are cleaved from the solid phase and stored
as stock solutions in microtiter plates each compound is quality
controlled by LC-MS to guarantee highest quality standards. Aliquots of
these stock solutions are further diluted and used in the subsequent
spotting step to generate chemical microarrays. Graffinity’s libraries
contain about 24,000 fragments and 86,000 leadlike compounds. The array
content is key to the success of the drug discovery process and is
continously upgraded.
After synthesis compounds are cleaved from the solid phase and stored
as stock solutions in microtiter plates. Each compound is quality
controlled by LC-MS to guarantee highest quality standards. Aliquots of
this stock solutions are further diluted and used in the subsequent
spotting step to generate chemical microarrays. Graffinity’s
libraries contain about 24,000 fragments and 86,000 leadlike compounds.
The array content is key to the success of the drug discovery process
and is continously upgraded.
2.
Array Production
Nanolitre amounts of compounds are transferred from daughter library
plates onto
the surfaces of microarrays in a highly reproducible, parallel array
production
process using customized spotting technology.
The result of the array
production process is a chemical microarray carrying about 10,000
compounds displayed on top of a self-assembled monolayer (SAM). A thin
gold layer provides basis for SAM formation and SPR detection. The SAM
serves two purposes: (i) it prevents unspecific protein binding to the
array surface, and (ii) it presents anchor molecules that allow
covalent binding of the library compounds to the array surface via the
ChemTag®. The number of displayed compounds is controlled and can be
varied by the ratio of diluent to anchor molecules within the SAM.
3.
Fingerprinting (Detection of Protein Ligand Interactions)
High-throughput, label-free screening of fragment and
displayed-fragment microarrays on
its proprietary SPR platform, allows Graffinity to rapidly identify
novel drug fragments
and leadlike molecules as ligands for a biomolecular target.
The
Plasmon Imager® is based on the phenomenon of surface plasmon resonance
(SPR) which requires the presence of a thin gold metal film on the
array´s glass carrier. Changes of refractive index at the gold/liquid
interface affect the resonance condition of electron energy states in
the metal. In Graffinity´s set up, a wavelength shift that corresponds
to the increase of mass concentration on the chip surface during
binding between the solubilized protein and the immobilized chemical
substances is recorded. Currently, Graffinity's Plasmon Imager® devices
are capable of routinely processing about 10,000 measuring points
simultaneously in a fourfold parallel fashion.
4.
Data Analysis
The evaluation of protein-ligand affinity fingerprints is aided by
proprietary, Java-basedsoftware. Chemical microarray content is
visualized in a point-and-click fashion, individual spots refer to
chemical structures and associated read-out information. During the
process, a specific pattern or "fingerprint" emerges for each target
protein, reflecting the protein´s chemical binding characteristics.
Chemical proximity display, statistic functions and database
cross-queries provide powerful insights into affinity relationships.
Structure-activity relationship (SAR) patterns can be mapped from the
microarray fingerprints and, supported by protein X-ray
crystallography, the process allows the unambiguous determination of
binding mode for multiple ligands.
5.
Hit Evolution
The Graffinity hit evolution program uses array „fingerprint“ data for
rapid compound optimization
by high throughput chemistry, secondary assays and SAR building. The
starting point for the process
is the synthesis of array compound analogs by removal of the Chemtag
spacer subunit. Biochemical
and micoarray data allow the rapid analoguing of the first generation
compounds and the building
of SAR models. |
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