Domainex is a contract research organization located in Cambridge, UK that provides integrated drug discovery services including medicinal chemistry, protein production and biophysics, and screening. They have experience working with many of the top pharmaceutical companies and have produced over 60 patents and 100 scientific papers. Domainex utilizes a comprehensive approach involving multiple disciplines to identify hits against drug targets. As a case study, they used fragment-based screening and x-ray crystallography to identify small molecule fragments that bind the epigenetic target G9a, which they then expanded into lead compounds.

1. Confidential
Hit Identification – Understanding
YourTarget is Key

2. About Domainex
Amongst our clients are:
• 50% of the Top 10 pharma companies
• World’s #2 agrochemical company
• Global biotechnology companies
• Major academic institutions
• Including…
• Based within the Cambridge (UK) bioscience hub
• About 50 scientists
• >2,000 m2 lab space
• Chemistry and biology facilities
2
All staff on one site Dedicated CRO – no internal research projects
• Clinical candidates
• Compounds in pre-clinical
development
• Cited as inventors on more than
60 patents
• Authors on over 100 peer-
reviewed papers

3. Comprehensive medicines research platform
Medicinal
chemistry
Analytical
chemistry
Protein
expression
CDH
X-ray
crystallo-
graphy
Cell
biology
Bio-
physics
Bio-
chemistry
CADD
Chemistry
Protein
Science
Assay
Biology
3

4. Hit Identification at Domainex
Target
Covalent
Binders
Orthosteric
/allosteric
Protein
construct
Protein
folding
Structura
l biology
Functiona
lity
assessme
nt
Protein
labelling
Screening
method
selection
Homolog
y
modelling
Chemistry
Protein
Science
Assay
Biology
4
• Selection of
screening method
based on target
class and desired
chemical class
• Appropriate
labelling and
testing strategy
• Validation using
orthogonal
techniques
• Pharmacologically relevant
protein construct selection.
Not necessarily easiest to
produce.
• Emphasise on structural
biology to guide chemistry
• Careful assessment of
target to recommend most
suitable approach
• Flexible approach towards
strategy and libraries used
• High quality fragment and
virtual library

5. Compound Libraries
Medicinal
Chemistry
5
• Virtual screening platform
• Utilises target structural
information or homology model
• Through confirm docking
• Deliver up to 1000 compounds
selected to cover lead-like space
• Cost effective solution
• Diverse set of ~1100 fragments
• Rule of 3 compliant, includes SP3
rich fragments
• Confirmed solubility at 1 mM
• Regularly QC’d – nitrogen pod
storage
• Covalent fragment set
• Deliver ~5% hits
• Undertake analysis of all available
literature information
• Apply in-house tools and utilise
Domainex “know-how” on multi-
projects experiences
• Devise and informed and timely
work programme

6. IMAC Refolding optimisation AMP agarose affinity
20
30
40
50
60
70
80
100
kDa
Refolding Temp (A) vs. Refolding Time (B)
1
3.875
6.75
9.625
12.5
15.375
18.25
21.125
24
2.0E+07
2.5E+07
3.0E+07
3.5E+07
4.0E+07
4.5E+07
5.0E+07
5.5E+07
6.0E+07
4 6.25 8.5 10.75 13 15.25 17.5 19.75 22
RefoldingTime
(B)
YieldY-Hat
RefoldingTemp(A)
Yield Y-Hat
Surface Plot Refolding Temp (A) vs. Refolding Time (B)
20
30
40
50
60
70
80
100
kDa
20170621 CD73 S200:10_UV 20170621 CD73 S200:10_Fractions 20170621 CD73 S200:10_Inject 20170621 CD73 S200:10_Logbook
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
mAU
0 50 100 150 200 250 ml
1A1 1A4 1A7 1A11 1B3 1B6 1B9 1C1 1C4 1C7 1C11 1D3 1D6 1D9 1E1 1E4 1E7 1E11 1F3 1F6 1F9 1G11G41G7 1G11 1H3 1H6 1H9 2A1 2A4 2A7 2A11 2B3 2B6 2B9 2C1 2C4 2C7 2C11 2D3 2D6 2D9 2E1 2E4 2E7
181.85
QC (DSF)Gel filtration (S200)
apo
+ ligands 1.7Å
Protein Production
• Construct design: Full length vs. truncated, e.g. active core domain only
– Literature and bioinformatics enabled where possible
– Combinatorial domain hunting (CDH) enables soluble expression of challenging targets
• Post-translational modifications effect activity and homogeneity
– Choice of expressions host: E.coli, mammalian, insect cell
– Multi-step chromatography
• QC: purity, activity, compound binding, mass spec analysis

7. Versatile screening technique Microscale Thermophoresis
• Protein requirements: Very low
• Solution based (no immobilization)
• Multiple labelling strategies, directed by structural
information
• Highly sensitive – ideal for fragments and weak
binders
• Broad dynamic range – pM - mM
• Flexibility:
• Wide range of buffers
• Broad range of interactions
• Supports challenging biology
• No microfluidics, no maintenance
Monolith NT.Automated
Dianthus NT.PicoDuo
7

8. Supporting Biophysical Capabilities
Surface plasmon
resonance (SPR)
Orthogonal approach
to confirm binding
Suitable for allosteric
and orthosteric binding
Kon/Koff
Assay development can
take time and requires
immobilization of
protein
Isothermal titration
calorimetry (ITC)
Orthogonal approach
to confirm binding
Resolve stoichiometry
Measures binding
thermodynamics
Assay development can
take time and requires
large amount of
protein
NMR
STD & WaterLOGSY
“Gold standard”
fragment- binding
Fast assay dev
Ligand- protein
structure information
Requires large amount
of protein and
deuterated compound
stocks
Differential Scanning
Fluorimetry (DSF)
Fast assay design and
set-up
Binding Confirmation
Buffer screening for
protein stability
Not all proteins are
monophasic, not
suitable for ternary
complex’s
Structural biology
X-Ray and Cryo-EM
Screening to find
optimal conditions
Determination and
characterisation of 3D
structures
Not all proteins are
amenable (however
CDH can enable)
variable set-up time
Confidential

9. Case Study: G9a
Application of FragmentBuilder

10. Methyltransferase G9a SET domain
• G9a is a lysine methyltransferase (KMT) involved in epigenetic gene
regulation by covalent modification of histones
• G9a catalyses the transfer of methyl groups from S-adenosyl methionine
(SAM) to lysine residues on histone proteins
• Literature supports the role of G9a in mechanisms of carcinogenesis,
making it an attractive oncology target
• Functional G9a SET domain protein produced in-house
10
SEC QC
Apo + SAM
Thermal Shift
Me Me
Me
Me
SAM
SAH
KMT
Chang et al., 2009

11. G9a Lysine Methyltransferase
• Only available assays alphaLISA – unsuitable for high concentration
fragment screening
• Targeting peptide binding site to gain specificity over other SAM
binding KMTs
• Complicated biophysics because a 3-component (ternary) system:
• Virtual screen and fragment screen run in parallel for peptide
binding site
G9a G9a
SubstrateSAM
SAM
G9a
SAM
Me Me
Me
Me
SAM
SAH
KMT

12. • Primary Hit Selection
• Primary screen performed
in duplicate
• Screened against G9a- SAM
• MST enables detection of
false +ve due to
aggregation
• 5 % hit rate
Primary Screening assay
G9a
• False Positive Detection
Aggregation
Aggregation
Acceptable
fluorescence
variation
Auto-fluorescence
Ligand-induced Photobleaching
Photobleaching
Green: DMSO
reference; Blue:
Test compound

13. Fragment binding mode by X-ray crystallography
• In house protein expression and purification of
crystallography grade G9a
• Protein used to validate fragment hits by STD-
NMR
• NMR validated fragment hits were co-crystallised
with G9a and SAM
– Soaking not possible due to protein rearrangement
upon compound/peptide binding
• X-ray structures solved in-house using data
obtained at DLS
Frag ID Kd [µM] LE
STD-NMR
Positive binding
X-ray Structure
Resolution
DMXFRG195 19 0.66  1.5 Å
DMXFRG116 56 0.41 X NA
DMXFRG311 115 0.41  X
DMXFRG331 327 0.54  2.0 Å
DMXFRG132 411 0.53  1.8 Å
DMXFRG176 564 0.44  X
DMXFRG245 718 0.36 X NA

14. Fragment expansion – G9a
FRG-195
Kd 19 µM
Attachment vectors for
growth down peptide
channel
DMX-593
Kd 2.5 µM
L.E. 0.42
Docking followed by
synthesis of focussed arrays

15. Summary
• Integrated approach essential for successful hit ID
• Close working of chemistry, protein production and assay biology to deliver cascade of
assays and
• High quality compound files to deliver lead-like compounds to progress quickly through
hit expansion into lead optimisation
• Appropriate protein delivery and assay ensures translatability into cell models
• High quality, sensitive, physiologically screening assays for hit ID

16. Summary
Domainex offers:
• Integrated suite of drug discovery services
• Established track record of delivering pre-clinical candidates
• One-stop shop for gene-to-hit and hit-to-candidate services
• A partner that is growing strongly and profitable
Our focus is on providing our clients with services that are:
– High-quality
– Cost-effective
– Timely
…and specifically tailored to their needs
Medicinal
Chemistry