DISCOVERY OF NOVEL SMALL-MOLECULE TREATMENT …...BIOLOGICALLY DIVERSE ANNOTATED LIBRARY 5,000 compounds (15h) CHEMICALLY DIVERSE LIBRARY 90,000 compounds (30% 15h, 70% 72h) ... ACTIVE

DISCOVERY OF NOVEL SMALL-MOLECULE TREATMENT OPTIONS FOR FSHD

Joris De Maeyer, PhD.

26TH FSHD INTERNATIONAL RESEARCH CONGRESS

JUNE 19-20, 2019 MARSEILLE, FRANCE

DISCLAIMER

This presentation is for informational purposes only. The information contained in this presentation (“the Information”) is not complete without a verbal explanation. The Information is based on information obtained by Facio Therapies BV (“Facio”). Facio regards the Information as reliable but does not accept any liability in connection with possible inaccuracies.

2Facio Therapies BV Discovery of Novel Small-Molecule Treatment Options for FSHD

ESTABLISHING A CHAIN OF TRANSLATABILITY

Facio Therapies BV Discovery of Novel Small-Molecule Treatment Options for FSHD 3

Phenotypic Drug Discovery to Account for Incomplete Understanding of the Complexity of FSHD

HIGH-CONTENT SCREEN IN

PRIMARY MUSCLE CELLS

MOLECULAR PHENOTYPING

IN VIVO VALIDATION

PK/PD

Nat Rev Drug Discov. 2017 16:531-543


4



MAXIMUM PREDICTIVE VALIDITY DISCOVERY MODEL

1. assay readout proximity to disease pathophysiology

2. disease-relevant assay system

3. disease-relevant stimulus

Facio Therapies BV Discovery of Novel Small-Molecule Treatment Options for FSHD

ASSAY READOUT PROXIMAL TO DISEASE

5

11-100 KpnI fragments of 3.3 kb

D4Z4

D4Z4 4q35

HEALTHY

FSHDDeletion D4Z4 units (<11)

REPRESSIVE CHROMATIN

RELAXED CHROMATIN

DUX4 DE-REPRESSION IS A VALIDATED MARKER

OF EPIGENETIC CHANGES IN FSHD MUSCLE

Physiology signature

Disease signature


DISEASE-RELEVANT ASSAY SYSTEM & STIMULUS

6

MHC, DUX4, DAPI

PRIMARY FSHD CELLS PROPRIETARY DUX4 DETECTION METHOD

PCA RNAseq data different cell lines

DUX4 ABcontrol DUX4 ABcontrol

Initialdetectionmethod

Optimizeddetectionmethod

Num

ber

ofD

UX4

pos

itiv

enu

clei

Z’:0.5

Z’:0.4

o Increased sensitivity o Largely reduced plate illumination time

Upregulated genes: 231 (including DUX4)Downregulated genes: 63

DESMIN

33%

25%

HEALTHYFSHD

FSHD

FSHD

FSHD

FSHD

HEALTHYHEALTHY

HEALTHYHEALTHY

o Retained endogenous regulatory mechanismso Dexa, Insulin: orthogonal assay requirement! (e.g. p38)


AUTOMATED HIGH-CONTENT SCREENING ASSAY

7

SCRIPT-BASED IMAGE ANALYSIS

Myotubearea

Myotubeskeleton

Myotubewidth

Nucleiidentification Myotube identification

Nucleiin- andoutsideofmyotubes DUX4positivenuclei

Day -1 Day 3

differentiation medium

Fixation & Imaging

Myotubearea

Myotubeskeleton

Myotubewidth

Nucleiidentification Myotube identification

Nucleiin- andoutsideofmyotubes DUX4positivenuclei

READOUTS

DUX4-positive nuclei within myotubes

DUX4 intensity

Fusion index

Myotube area, skeleton and width

Nuclei count (cell loss, toxicity)

Primary FSHD

myoblasts

384 well

DUX4 expression

15h TREATMENT

72h TREATMENT


HIGHLY PERFORMANT SCREENING ASSAY

8

DUX4-POSITIVE NUCLEI DUX4 INTENSITY FUSION INDEX

Z’ > 0.4 Z’ > 0.5

Plate position effect

Fusion Index differences are directly

reflected in primary DUX4 readout

ASSAY VALIDATED FOR SINGLICATE SCREENING

Inner wells 1st outer wells 2nd outer wells

Risk mitigation to exclude potentially

false positives1 of 3 independent assay validation experiments Fusion Index correlates well with myotube area readout


BIOLOGICALLY DIVERSE ANNOTATED LIBRARY 5,000 compounds (15h)

CHEMICALLY DIVERSE LIBRARY90,000 compounds (30% 15h, 70% 72h)

Replicate plots - Blue: confirmed hits (> 3 sigma)

DUX4 COUNT DUX4 INTENSITY

NUCLEI COUNTZ’ SCREENING PLATES

DU

X4

CO

UN

T IN

HIB

ITIO

N

DUX4 INTENSITY INHIBITION

DENSITY OF NORMALIZED ASSAY SIGNAL

HISTOGRAM OF HITS

DUX4 inh FUSION inhZ’ > 0,6 Z’ > 0,8

DUX4 inhibition

Hit Calling @40%(3sigma)

Pos control

FUSION INDEX

HIT IDENTIFICATION HIT CONFIRMATION606 CONFIRMED HITS WITHOUT

FUSION INHIBITION

DIVERSE SCREENING CAMPAIGNS


HIT TRIAGING, H2L AND LO

10

PROFILING SAR BUILDINGMOLECULAR

PHENOTYPINGSPECIFIC ASSAYS HIGH-QUALITY

LEADS

Target ProfileCK1 inhibitor

Risk Profilesβ2AR agonist (formoterol) BET inhibitor (JQ1)

15h

p38 inhibitor (losmapimod)

72h15h 72h 72h 15h 72h15h

#03_7 #35_3 #01_3

15 h

72 h

Chen et al. 2019; Kim et al., 2018 Perdiguero et al., 2007

hERG/ Nav1.5/CaV1.2KinomeScan,BromoscanCerep Diversity PanelAffinity ChromatographyPROTAC target KD

QPCRMultiple donorsRNAseqCell paintingProteome analysisBio-informatics (annotated screen)

Physicochem propertiesAqueous solubilityPAMPA, CACO2CytotoxMetabolic stabilityPlasma protein binding

Solid stockConcentration-responseTreatment kinetics



11





CELL PAINTING

12

Compounds with similar biological profiles tend to

modulate similar pathways without necessarily having similar chemical

structures

cytoskeleton

mitochondria

ER

nuclei

segmentation

Z-Sc

ore

Feature

Computing hundreds of phenotypic parameters per cell

CLUSTER BY PHARMACOLOGY


HIT TRIAGING BASED ON PHENOPRINT


HDAC

EPIGENETIC READER DOMAIN

TOX

HYPOTHESIS BUILDING FOR UNKNOWN

COMPOUND MoACOMPARING ALL COMPOUND SIGNATURES

SELECTED HIT LOCATIONS

Similarity to references

TRANSCRIPTOME PROFILING

Low expression

High expression

H: Healthy myotubesF: FSHD myotubeCK1: FSHD myotube + CK1 inhibitor

DUX4Trim43ZScan4

Log concentration

Fold

ch

ange

rel

ativ

e to

ve

hic

leQPCR

MYOGENIC MARKER EXPRESSION VS. MYOTUBE FUSION

Cell CompoundTimepointDiffStage DiffTime DiffTreat MYOG MYH1 MYH2 MYH3 MYH4 MYH6 MYH7 MYH8 MYH9 MYH10 MYH11 MYH13 MYH14

16MB003 DMSO 24 MB 0 0_24 37.71 1.13 1.13 90.38 1.13 0.00 4.68 7.02 252.24 32.69 2.34 2.26 0.00

16MB003 DMSO 24 MT1 24 24_24 382.68 1.82 3.36 160.00 0.00 0.00 3.64 15.52 214.01 20.84 5.32 3.50 0.00

16MB003 DMSO 48 MT2 48 48_48 1233.47 1.46 1.46 872.82 0.00 0.68 19.01 85.52 167.56 9.24 2.14 2.03 0.68

16MB003 DMSO 24 MT2 72 72_24 736.16 19.03 4.69 1867.45 3.37 0.86 74.92 177.96 154.52 7.26 1.26 3.43 0.00

16MB003 DMSO 72 MT2 72 72_72 765.02 9.11 4.55 1921.65 0.00 0.00 56.92 198.08 154.82 22.77 2.28 2.28 2.28

MBMT 24hMT 48hMT 78h

Note: no uniform correlation between compounds inhibiting myogenic fusion index & their effect on myogenic marker expression

H F CK1

CK1 inhibitor

Heat map based on DUX4 target genes

from Yao et al. 2014


15

TARGET

E3

Nature Chem Biol 2015, 11: 634–635 (2015)

ACTIVE DEGRADER > E3 targeting warhead

INACTIVE DEGRADER> methylated warhead can’t bind E3

GLOBAL PROTEOME PROFILING

CK1

FT000333 ACTIVE VS INACTIVE PROTAC

FT000333FT000631* Degrader inactive (N-Me)

DM

SO

Pro

tac-

01

Pro

tac-

16

Pro

tac-

15

Pro

tac-

06

Pro

tac-

10

Pro

tac-

14

Pro

tac-

12

WA

RH

EAD

1

WA

RH

EAD

2

**

Vinculin

CK1ε

CK1δ

PROTAC COMPOUNDS WESTERN BLOT

CHEMICAL TARGET KNOCK-DOWN

2-fold difference


UNRAVELING MOLECULAR MODE OF ACTION

16

EC50 ⇨ EC50 ⇦

INACTIVE DEGRADER ACTIVE DEGRADER

INCREASED SELECTIVITYDEGRADATION AT LOW

COMPOUND CONCENTRATIONS

Promiscuous PROTAC

Selected downregulated proteins: CK1δ, CK1ε, DEFB103 (others not disclosed)Upregulated proteins involved in transcriptional regulation, PRC1 complex, p53 pathway

HINTS ON MOLECULAR

MODE OF ACTION

PROTACS with large potency shift



17




IN VIVO VALIDATION

PK/PD

Nat Rev Drug Discov. 2017 16:531-543


da

y

1

day

2

d

a

y 3

d

a

y

4

da

y 5

d

a

y

6

d

a

y

14

0 .0

0 .1

0 .2

0 .3

0 .4

re la t iv e h D U X 4 e x p re s s io n in h u m a n c e lls

fold

ch

an

ge

re

l. t

o

hu

ma

n-s

pe

cif

ic H

KG

F S H D m y o b la s ts

h ea lth y m y o b las ts

DUX4 Expression in Human cells

Fold

ch

ange

rel

ativ

e t

o

hu

man

-sp

eci

fic

HKG

XENOGRAFT WITH PRIMARY MUSCLE CELLS

t = day 3 t = day 4t = day 1 t = day 2

Oral treatment with compound or placebo ensuring 24h or 72h free compound exposure in muscle 10x EC50

Placebo30mg/kg

1 mg/kg

6mg/kg

FT

'439 3

0 m

g/k

g

FT

'439 6

mg

/kg

FT

'439 1

mg

/kg

veh

icle

0 .0 0

0 .0 5

0 .1 0

0 .1 5

0 .2 0

0 .2 5

E v o 1 8 1 0 1 : re la t iv e h D U X 4 e x p re s s io n

fold

ch

an

ge

re

l. t

o

hu

ma

n-s

pe

cif

ic H

KG

F S H D m y o b la s ts

h e a lth y m y o b la s ts

No effect on hMYOG, hMYH2, mPAX7

huLamin A/C, Laminin,

DAY 14

FSHDHealthy

Fold

ch

ange

rel

ativ

e to

h

um

an-s

pec

ific

HKG

NOD/SCIDBaCl2

FSHDHealthy

DOSE-DEPENDENT DUX4 REPRESSIONDUX4 EXPRESSION IN HUMAN CELLS


SUMMARY

19




IN VIVO VALIDATION

PK/PD

Multiple Series of

Novel Scaffolds

Novel Modes of Action

In vivo PoP


Acknowledgements

20

STRATEGIC PARTNER FSHD FOUNDATIONS


PATIENT MATERIAL

Documents

DISCOVERY OF NOVEL SMALL-MOLECULE TREATMENT …...BIOLOGICALLY DIVERSE ANNOTATED LIBRARY 5,000 compounds (15h) CHEMICALLY DIVERSE LIBRARY 90,000 compounds (30% 15h, 70% 72h) ... ACTIVE