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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
ESTABLISHING A CHAIN OF TRANSLATABILITY
4
HIGH-CONTENT SCREEN IN
PRIMARY MUSCLE CELLS
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
Facio Therapies BV Discovery of Novel Small-Molecule Treatment Options for FSHD
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)
Facio Therapies BV Discovery of Novel Small-Molecule Treatment Options for FSHD
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
Facio Therapies BV Discovery of Novel Small-Molecule Treatment Options for FSHD
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
Facio Therapies BV Discovery of Novel Small-Molecule Treatment Options for FSHD
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
Facio Therapies BV Discovery of Novel Small-Molecule Treatment Options for FSHD 9
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
Facio Therapies BV Discovery of Novel Small-Molecule Treatment Options for FSHD
ESTABLISHING A CHAIN OF TRANSLATABILITY
11
HIGH-CONTENT SCREEN IN
PRIMARY MUSCLE CELLS
MOLECULAR PHENOTYPING
Facio Therapies BV Discovery of Novel Small-Molecule Treatment Options for FSHD
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
Facio Therapies BV Discovery of Novel Small-Molecule Treatment Options for FSHD
HIT TRIAGING BASED ON PHENOPRINT
Facio Therapies BV Discovery of Novel Small-Molecule Treatment Options for FSHD 13
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
Facio Therapies BV Discovery of Novel Small-Molecule Treatment Options for FSHD 14
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
Facio Therapies BV Discovery of Novel Small-Molecule Treatment Options for FSHD
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
Facio Therapies BV Discovery of Novel Small-Molecule Treatment Options for FSHD
ESTABLISHING A CHAIN OF TRANSLATABILITY
17
HIGH-CONTENT SCREEN IN
PRIMARY MUSCLE CELLS
MOLECULAR PHENOTYPING
IN VIVO VALIDATION
PK/PD
Nat Rev Drug Discov. 2017 16:531-543
Facio Therapies BV Discovery of Novel Small-Molecule Treatment Options for FSHD
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
Facio Therapies BV Discovery of Novel Small-Molecule Treatment Options for FSHD 18
SUMMARY
19
HIGH-CONTENT SCREEN IN
PRIMARY MUSCLE CELLS
MOLECULAR PHENOTYPING
IN VIVO VALIDATION
PK/PD
Multiple Series of
Novel Scaffolds
Novel Modes of Action
In vivo PoP
Facio Therapies BV Discovery of Novel Small-Molecule Treatment Options for FSHD
Acknowledgements
20
STRATEGIC PARTNER FSHD FOUNDATIONS
Facio Therapies BV Discovery of Novel Small-Molecule Treatment Options for FSHD
PATIENT MATERIAL
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