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Identification of assays to address the
mechanism of action of a complex
molecule
Dr Elaine Harper, Head of Pharmacology and Cellular Systems
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
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
Mechanism of action of Botulinum toxin
1. Targeting
2. Internalisation
3. Translocation
4. Block
4
1
1
2
4
4
3
3
2
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
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
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
Showing MOA of a TSI; inferred MOA
TARGETS TRANSPORTS BLOCKS
Linker LCD (Endopeptidase) Spacer qGHRH(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
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
Demonstration of inferred MOA: Stage 1 10
TARGETS Stage1
TRANSPORTS BLOCKS
Linker LCD (Endopeptidase) Spacer qGHRH(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
Demonstration of inferred MOA: Stage 1 11
TARGETS Stage1
TRANSPORTS BLOCKS
Linker LCD (Endopeptidase) Spacer qGHRH(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)
Demonstration of inferred MOA: Stage 1 12
TARGETS Stage1
TRANSPORTS BLOCKS
Linker LCD (Endopeptidase) Spacer qGHRH(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)
Demonstration of inferred MOA: Stage 2 and 3 13
TARGETS TRANSPORTS Stage 2
BLOCKS Stage 3
Linker LCD (Endopeptidase) Spacer qGHRH(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
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
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
% M
axi
mum
GH
RH
(1-4
4)-
induced c
AM
P a
ccum
ula
tion
0
10
20
30
-12 -11 -10 -9 -8 -7 -6Basal
log [Ligand] M
[cA
MP
] (n
M)
rat GHRH-R in GH3 cells wild-type GH3 cells
GHRH(1-44) SXN101959
15
SXN101959 binds to and activates the rat GHRH receptor
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
-11 -10 -9 -8 -7 -6 -5
0
5
10
15
20
log [SXN101959] M
Mean e
ndosom
al spot count
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
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
0
20
40
60
80
100
-9 -8 -7 -6 -5basal
log [SXN101959] (M)
VA
MP
2 d
eple
tion (
%)
0
20
40
60
80
100
-9 -8 -7 -6 -5basal
log [SXN101844] (M)
VA
MP
2 d
eple
tion (
%)
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
Stage 3: Inhibition of pulsatile GH secretion in rat
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150
Time (24 hr clock)
[rat G
H]
(ng/m
l)
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Time (24 hr clock)
[rat G
H]
(ng/m
l)
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Time (24 hr clock)
[rat G
H]
(ng/m
l)
Vehicle 0.01mg/kg 0.03mg/kg
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Time (24 hr clock)
[rat G
H]
(ng/m
l)
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Time (24 hr clock)
[rat G
H]
(ng/m
l)
0
100
200
300
400
500
-3 -2 -1 0vehicle
log[SXN101959] (mg/kg)
[GH
] (A
UC
ng/m
l)
0.1mg/kg 0.3mg/kg
SXN101959 produces a dose-dependent inhibition of GH secretion following
intravenous adminstration
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