The document describes assays to demonstrate the mechanism of action of SXN101959, a targeted secretion inhibitor being developed for the treatment of acromegaly. It outlines cellular and in vivo assays to show that SXN101959 binds and activates growth hormone-releasing hormone receptors, is internalized into cells, cleaves SNARE proteins via its enzymatic domain, and inhibits pulsatile growth hormone secretion. A battery of assays using receptor expressing cell lines and pituicytes aims to validate the mechanism of action at each step in multiple preclinical species.

1. Identification of assays to address the
mechanism of action of a complex
molecule
Dr Elaine Harper, Head of Pharmacology and Cellular Systems

2. Syntaxin: Company Overview
2
Technology
2
•Novel Platform - Targeted Secretion Inhibitors (TSI)
•Biological therapeutics to treat diseases where vesicular cell secretion is the driver
•Based on re-engineered and retargeted botulinum toxins
Programmes
3
•First molecule outlicensed to Allergan ( AGN-214868) in Phase II in Post Herpetic Neuralgia
and Overactive Bladder
•SXN101959 for Acromegaly – Currently in early preclinical stage
Company
1
•UK Biotech company founded in 2005
•Spin out from the Health Protection Agency ( Originally 12 scientists with 10 years
experience on the technology)
•SME Status (EU)
•Circa 50 staff

3. The basis of the TSI platform
3
TSI
Targeted Secretion
Inhibitor
Targets
Neuromuscular Cells
HC Binding
HN Transport
LC Enzyme,
Multiple Serotypes, A-G
Blocks
Vesicular Secretion
Transports
SNARE cleaving Endopeptidase
TSI are retargeted botulinum neurotoxins that exploit the ability of the toxin to
cleave SNARE proteins and inhibit vesicular secretion
Crystal structure of Botulinum neurotoxin

4. Mechanism of action of Botulinum toxin
1. Targeting
2. Internalisation
3. Translocation
4. Block
4
1
1
2
4
4
3
3
2

5. Rationale design - selectivity of action
5
Binding to specific cell.
Interchangeable between cell types
Pain
Endocrine
Proliferative
Disease
Cells targeted is dependent on targeting domain
Targeting domain changed to target to specific cell types

6. Rationale design - selectivity of action
6
Serotypes A, C and E
Cleave SNAP-25
Serotype C
Cleave Syntaxin
Serotypes B, D, F and G
Cleave Synaptobrevin
Cell secretions are mediated by different
combinations of SNARE proteins
Serotype selected to cleave SNARE(s)
critical to secretion of interest
Enzyme
cleaves SNARE proteins

7. Choosing the targeting domain and serotype
External data sources
• Transcript data
• Proteomic data
• Literature derived
Profiling
• Western blot
• RT-PCR
• Pharmacology
• Microarray analysis
• SNARE knockdown
Transcription Profiles
Disease
Profile
Literature Mining
Profiling
7

8. 8
Showing MOA of a TSI; inferred MOA
TARGETS TRANSPORTSBLOCKS
LinkerLCD (Endopeptidase) SpacerqGHRH(1-40) HND (Translocation)
S S
Stage 3
SNARE CLEAVAGE
Endopeptidase
specifically cleaves
VAMPs, resulting in
inhibition of secretion
of growth hormone
Stage 1
TARGETS PITUITARY
SOMATOTROPHS
Ligand binding to
GHRH receptors
activates receptor
leading to
internalisation of
SXN101959 into
endosomes
Stage 2
ENDOSOMAL ESCAPE
Insertion of
translocation domain
into endosomal
membrane allows
delivery of the
endopeptidase
into the cytoplasm
SXN101959: a TSI to inhibit GH secretion for the treatment of acromegaly

9. 9
Showing MOA of a TSI; requirements of cellular assays
LCD (Endopeptidase) qGHRH(1-40) HND (Translocation)
SNARE CLEAVAGE
Endopeptidase specifically
cleaves VAMPs, resulting in
inhibition of secretion of
human growth hormone
TARGETS
Ligand binding specifically
to GHRH receptors activates
receptor leading to
internalisation into
endosomes
ENDOSOMAL ESCAPE
Insertion of translocation
domain into endosomal
membrane
Intact cells expressing GHRH
receptor, VAMP and secrete
GH
Pituicytes
Pituitary cell lines (GH3 do
not express GHRH-R)
Binding and/or activation –
Cells with GHRH receptor
Cells with other class B
GPCRs
Internalisation – intact cells
with GHRH receptor
Intact cells expressing
VAMP and GHRH receptor
Demonstrate and confirm MOA in in vivo species; rat, cynomolgus macaque and human
Influence of GHRH-R density and SXN101959 intrinsic efficacy
Assay feasibility/ tissue access

10. Demonstration of inferred MOA: Stage 1
10
TARGETS
Stage1
TRANSPORTSBLOCKS
LinkerLCD (Endopeptidase) SpacerqGHRH(1-40) HND (Translocation)
S S
APPROACH Inducible receptor – method of Furchgott (receptor density radioligand binding)
Surface plasmon resonance
SYSTEM CHO-K1 wild type cells and with inducible recombinant GHRH-R of rat, human or macaque
N-terminal GHRH receptor domains of rat, human and macaque
TOOLS Untargeted TSI of equivalent serotype (SXN101655)
Binding Binding and activation Internalisation

11. Demonstration of inferred MOA: Stage 1
11
TARGETS
Stage1
TRANSPORTSBLOCKS
LinkerLCD (Endopeptidase) SpacerqGHRH(1-40) HND (Translocation)
S S
Binding Binding and activation Internalisation
APPROACH Second messenger production (cAMP)
Specificity β-arrestin recruitment assays of type B GPCR
SYSTEM CHO-K1 wild type cells and with recombinant GHRH-R of rat, human or macaque
GH3 cells wild type cells and with recombinant GHRH-R of rat
Rat pituicytes
TOOLS Untargeted TSI of same serotype (SXN101655)
GHRH-R antagonist (JV1-36)

12. Demonstration of inferred MOA: Stage 1
12
TARGETS
Stage1
TRANSPORTSBLOCKS
LinkerLCD (Endopeptidase) SpacerqGHRH(1-40) HND (Translocation)
S S
Binding Binding and activation Internalisation
APPROACH Measurement of endopeptidase domain of SXN101959 in endosomes of cells – HCS with
antibody to light chain of D serotype
SYSTEM CHO-K1 wild type cells and with rat, human or macaque recombinant GHRH-R
GH3 wild type cells and with recombinant rat GHRH-R
Rat and human pituicytes
TOOLS Untargeted TSI of same serotype (SXN101655)
GHRH-R antagonist (JV1-36)

13. Demonstration of inferred MOA: Stage 2 and 3
13
TARGETS TRANSPORTS
Stage 2
BLOCKS
Stage 3
LinkerLCD (Endopeptidase) SpacerqGHRH(1-40) HND (Translocation)
S S
SNARE cleavage and inhibition of GH secretion Endosomal escape
APPROACH SNARE cleavage assays – western blot
GH secretion assays
SYSTEM rat and human pituicytes
GH3 wild type cells and with rat recombinant GHRH-R
TOOLS Untargeted TSI of same serotype (SXN101655)
TSI with catalytically inactive endopeptidase domain (SXN101844)
GHRH-R antagonist

14. 14
STAGE 1 BINDING, ACTIVATION and INTERNALISATION STAGE 2 STAGE 3
STAGE 3
Species Receptor
Density
Affinity Efficacy Internalisation SNARE cleavage In-vitro GH
secretion
Rat cloned GHRH-R
(GH3 cells)
Inducible rat GHRH-R
(CHO-K1 cells)
Rat pituitary
Cynomologus
pituitary
Inducible cynomolgus
GHRH-R
Human GHRH-R
(CHO-K1 cells)
Inducible human
GHRH-R receptor
Human acromegalic
pituitary
Overall strategy to demonstrate MOA in pharmacologically relevant
and responsive species

15. Stage 1: Receptor binding and activation
• SXN101959 causes a dose-dependent increase in cAMP in GH3 cells expressing the rat
GHRH receptor with potency ~ 1 log unit lower than GHRH(1-44)
• Effect is mediated by GHRH receptor; no effect in wild-type cells and reduction in potency of
SXN101959 in presence of GHRH-receptor antagonist (JV-1-36)
SXN101959 + JV1-36
0
20
40
60
80
100
120
-12 -11 -10 -9 -8 -7 -6Basal
log [Ligand] M
%Maximum
GHRH(1-44)-inducedcAMPaccumulation
0
10
20
30
-12 -11 -10 -9 -8 -7 -6Basal
log [Ligand] M
[cAMP](nM)
rat GHRH-RinGH3cells wild-type GH3 cells
GHRH(1-44) SXN101959
15
SXN101959 binds to and activates the rat GHRH receptor

16. SXN101959 has equivalent potency at rat, cynomolgus and
human GHRH receptors
• SXN101959 activates rat, human and cynomolgus macaque GHRH receptor
• The potency of SXN101959 relative to GHRH(1-44) is comparable across species orthologues
of the GHRH receptor
16

17. -11 -10 -9 -8 -7 -6 -5
0
5
10
15
20
log [SXN101959] M
Meanendosomalspotcount
Control + 3 µM SXN101959
• Internalisation of GHRH-R targeted TSI (SXN101742 – his tagged version of SXN101959)
greater in GH3 cells expressing rat GHRH-R than in wild type GH3 cells
• Internalisation of GHRH-R targeted TSI (SXN101742) greater than
non-targeted TSI (SXN101655)
Fluorescence detected
with Anti LC/D antibody
Stage 1: SXN101959 internalisation
17
0, 10, 100, 1000 nM
SXN101959 is dose-dependently internalised into GH3 cells via the rat GHRH receptor

18. Stage 1: importance of receptor activation to internalisation
• Sequential truncation of N-terminus of qGHRH(1-40) in SXN101959 ablates intrinsic efficacy
at the human GHRH receptor expressed in CHO-K1 cells with no effect on affinity
• Sequential truncation of the N-terminus of SXN101959 ablates internalisation into CHO-K1
cells expressing human GHRH receptor
18
Receptor activation is critical for GHRH receptor-mediated internalisation of TSI

19. 0
20
40
60
80
100
-9 -8 -7 -6 -5basal
log [SXN101959] (M)
VAMP2depletion(%)
0
20
40
60
80
100
-9 -8 -7 -6 -5basal
log [SXN101844] (M)
VAMP2depletion(%)
Stage 2 and 3: Endosome escape and SNARE cleavage
• SXN101959 depletes target SNARE protein (VAMP2) in GH3 cells expressing the rat GHRH
Receptor indicating endosomal escape
• SNARE depletion is due to the catalytic activity of the LC of SXN101959; an endonegative
comparator TSI (SXN101844) does not cause dose-dependent SNARE
depletion
Western blot
19
Removal of endopeptidase activity from SXN101959 ablates the ability to cleave VAMP 3 in a
pituitary cell line expressing the rat GHRH receptor

20. Stage 3: Inhibition of pulsatile GH secretion in rat
20
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0
50
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150
Time (24 hr clock)
[ratGH](ng/ml)
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22:00
23:00
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05:00
0
50
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Time (24 hr clock)
[ratGH](ng/ml)
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05:00
0
50
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Time (24 hr clock)
[ratGH](ng/ml)
Vehicle 0.01mg/kg 0.03mg/kg
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02:00
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0
50
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150
Time (24 hr clock)
[ratGH](ng/ml)
19:00
20:00
21:00
22:00
23:00
00:00
01:00
02:00
03:00
04:00
05:00
0
50
100
150
Time (24 hr clock)
[ratGH](ng/ml)
0
100
200
300
400
500
-3 -2 -1 0vehicle
log[SXN101959] (mg/kg)
[GH](AUCng/ml)
0.1mg/kg 0.3mg/kg
SXN101959 produces a dose-dependent inhibition of GH secretion following
intravenous adminstration

21. Preclinical
• Informs on potential for off-target actions that can be explored in in vivo safety and
pharmacology studies
• Informs on the expected effective dose for in vivo studies in different species
CMC
• Informs on the potency assays required for drug product batch release assays
Clinical
• Informs on the selection of first dose in man
21
How does knowing the MOA for a TSI help to develop the
drug