01 Elaine Harper

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

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•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

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1

1

2

4

4

3

3

2

Rationale design - selectivity of action

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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

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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

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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


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


TARGETS Stage1

TRANSPORTS BLOCKS


S S


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)


TARGETS Stage1

TRANSPORTS BLOCKS


S S


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


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

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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

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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

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-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

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Receptor activation is critical for GHRH receptor-mediated internalisation of TSI

0

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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

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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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l)

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Vehicle 0.01mg/kg 0.03mg/kg

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-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

01 Elaine Harper

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