Presented by Gary Allenby on 29th April 2020

1. www.aureliabio.com
( +44 (0) 115 8370503
1

2. Biology CRO - Our Services
 Bio-Assay Development, Pharmacological Profiling and
Screening Company – 9 years
Aurelia
Bioscience
• Fee for service – Milestone based
Assay Development
State of the art technology
+
Data Data
Data
Data
Series of compounds with appropriate properties
• Discovery Project - FTE resource based
 Presentation
 Nanoluc assay
 NanoBRET protein-protein interaction assays
 Target Engagement kinase assay
 PROTAC drug discovery
 High Throughput Western blotting
 Future: 3-D cell assays – electrospun material

3. NanoBRET - Protein:Protein Interactions
Competition of an ERK-2 inhibitor binding to ERK-2 and
preventing the binding of Shn-3
Target = Schnurri-3 interacts with ERK-2: Schnurri-3 is thought to regulate bone formation. Schnurri-3
suppresses ERK phosphorylation of GSK-3b leading to suppression of b-catenin. Theory – block Schnurri
– ERK interaction you remove the brake and allow bone formation – Therapy - oesteoporosis
No tool compounds
*
Developed into a 384 well assay

4. NanoBRET 50,000 compound screen
44
Frequency Distribution of Compound Inhibition of the Shn-3 – ERK-2 Interaction
0
0.2
0.4
0.6
0.8
1
1 5 9 13 17 21 25 29 33 37 41 45 49 53
Z factor
ZFactor
Plate Number
Frequency
Percentage Inhibition
0
1000
2000
3000
4000
5000
-30 -15 0 15 30 45 60 75 90 105
Series1
30% cutoff used
(1.3% hits = 233
compounds)
A PPI screen was
performed in 384 well
plates screening 50,000
compounds for inhibitory
activity on Schnurr-3 ERK-
2
Z factors for
assay consistency
and reproducibility
were 0.5 or above
Hit confirmation n=2 retest correlation
Successful identification of active PPI inhibitors

5. NanoBRET Target Engagement
 Competition assay between the tracer (fluorescently
labelled molecule that binds to the kinase)
 Express full length kinase of interest
 Interaction between compound and kinase takes
place in physiological ATP and pH
 Assay takes place within a living cells
 Can look at association and dissociation rates within
the cells – study external factors
5

6. CellNucleus
Plasmid
cDNA
Manufacture plasmid containing target and
Nanoluc enzyme cDNA
NanoBRET Target Engagement

7. CellNucleus
Plasmid
cDNA
Protein (chimera of target
plus Nanoluc)

8. CellNucleus
Plasmid
cDNA
460nm
Add substrate

9. CellNucleus
Plasmid
cDNA
460nm 610nm
Labelled Tracer

10. CellNucleus
Plasmid
cDNA
460nm 610nm
Competing
compound

11. CellNucleus
Plasmid
cDNA
460nm 610nm

12. Kinase screening using Target Engagement
 For a Med Chemist success is
driven by potency – but what
about residency time?
 How long does a compound
need to be bound in order to
have an effect
 How can we perform
residency time experiments
-1 1 -1 0 -9 -8 -7 -6 -5
0
1 0
2 0
3 0
4 0
E ffe c t o f in h ib ito rs o n A B L k in a s e
[c o m p o u n d ] (M )
Ratio
D a sa tinib - IC 5 0 1 1 nM
N ilotinib - IC 50 340nM
F o re tin ib - IC 5 0 E st 2 uM
P o natinib - IC 50 48 0nM
Binding activity of each compound was determined in living cells. Cells were
transfected with each of four kinase; ABL, FGR, EPHA8 and DDR-1. Cells
were treated with exemplar kinase compounds including dasatinib, nilotinib,
foretinib and ponatinib as a dose response for each compound competed
against a fixed concentration of fluorescent tracer K4
Adhere cells to a material that can be moved between
wells – no washing, just change of plate

13. Changing the Paradigm – Move cells plate to plate
0 5 0 1 0 0 1 5 0 2 0 0
0
1 0
2 0
3 0
A s s o c ia tio n R a te A B L K in a s e
T im e (m in s )
BRETRatio
A B L F o rtin ib
A B L D a sa tin ib
A B L N ilo tin ib
A B L P o n a tin ib
A B L D M S O
0 5 0 1 0 0 1 5 0 2 0 0
0
2 0
4 0
6 0
8 0
A s s o c ia tio n R a te D D R 1 K in a s e
T im e (m in s )
BRETRatio
D D R 1 F o rtin ib
D D R 1 D a sa tin ib
D D R 1 N ilo tin ib
D D R 1 P o n a tin ib
D D R 1 D M S O
0 5 0 1 0 0 1 5 0 2 0 0
0
2 0
4 0
6 0
8 0
A s s o c ia tio n R a te E P H A 8 K in a s e
T im e (m in s )
BRETRatio
E P H A 8 F o rtin ib
E P H A 8 D a sa tin ib
E P H A 8 N ilo tin ib
E P H A 8 P o n a tin ib
E P H A 8 D M S O
0 5 0 1 0 0 1 5 0 2 0 0
0
2 0
4 0
6 0
8 0
A s s o c ia tio n R a te F G R K in a s e
T im e (m in s )
BRETRatio
F G R F o rtin ib
F G R D a sa tin ib
F G R N ilotin ib
F G R P o n a tin ib
F G R D M S O

14. PROTAC - Theory
14

15. Degrader Molecule
15

16. WES is a capillary based
automated western blot
instrument. Samples, antibodies
and wash solutions are loaded on
to a pre-filled plates. The western
blot is run along a matrix filled
capillary tube.
Protein Simple WES/JESS

17. 17
U
ntreated
P
ositive
C
on
1
2
3
4
5
6
7
8
9
10
0
25
50
75
100
125
PROTAC Compound Screen
NormalisedChemiluninescence
50
12
66
116
180
230
MolecularWeight
PROTACS
PROTAC data generated on WES / JESS
PROTACS
U
n
tre
a
te
d
2
5
0
n
M
5
0
n
M
1
6
n
M
5
.5
n
M
1
.8
n
M
0
.6
n
M
0
2 5
5 0
7 5
1 0 0
1 2 5
T a rg e t k n o c k d o w n
%ofUntreated

18. 3-D Biology – Electrospun scaffolds
18
EM Image
Fluorescently
labelled
scaffold
HEK-293 cells on scaffolds
Turquoise = Nuclei
Purple = Tubulin
Red = Scaffold material
Optical Coherent Tomography
24hrs
48hrs
72hrs
Cells impregnate
scaffold material

19. To find out more about Aurelia Bioscience go to: http://www.aureliabio.com
Aurelia Bioscience © 2019
Gary Allenby PhD
Chief Scientific Officer
+ allenby@aureliabio.com
( +44 (0) 115 8370503 (UK)
For further information, please contact :-
Aurelia Bioscience