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www.valentiabiopharma.com
[email protected]
Valentia Biopharma - In vivo Drug Discovery
Research and development of new drugs for human
diseases
Currently focused on finding lead
compounds to achieve an effective
treatment on Myotonic Dystrophy
Type 1 (DM1) disease
Valentia Biopharma - In vivo Drug Discovery
Drosophila melanogaster, a new technology for Drug
Discovery
Valentia Biopharma R&D
Generation of disease models
Generation of genetic/biochemical models
• Genetic diseases
• Other disease
mechanisms
In vivo High Throughput Screening
• Phenotypic assays
• Biochemical assays
Why Drosophila?
75% of human disease causing genes are conserved in
Drosophila
Short time frame from drug dosing to results
No ethical issues.
Large number of genetic tools available
Low variability at low costs
(Flies are CHEAP)
Not only Drosophila:
Drug delivery can be problematic and not all human systems can be easily modeled. (pulmonary
disease…)
So we always validate any positive result with mouse models and human cell lines
Drosophila models. Screening experimental approaches
(1) Phenotypic assay (viability)
Viability model
Toxic in mushroom bodies
(neuronal cells)
200
160
120
80
40
0
n=191
n=67
Control DM1 fly
(2) Biochemical assay (gene-reporter) by fusing known human
genes involved in DM1 pathologies to a reporter
TRANSGENIC FLY
PROMOTER-CONSTRUCT
Gene
Reporter
FLY
INJECTION
In vivo automated HTS platform
Our high-throughput screen (HTS) platform allows testing
thousands of compounds per week with the advantage of working
in in vivo conditions.
TOXICITY AND ACTIVITY EVALUATION
IN THE SAME EXPERIMENTAL
APPROACH
In vivo automated HTS platform
Screening platform - Steps in red are automated
1. FLY CROSSES
2. DRUG PLATES
PREPARATION
(Robot with stackers)
F0:Adults
3. SEEDING
(Sorter Cytometry)
4. HOMOGENIZATION
(Robot with stackers)
5. READING
(Envision Reader
/Scanner)
6. ANALYSIS
F1:Embryo/Larvae
F1:Adults
By genetic modification we have been able to develop transgenic flies models that reproduces some
aspects of the genetic human disease Myotonic Dystrophy. This has been the first model we developed in
and now it is being used with our High Throughput Screening (HTS) technology. Our HTS platform allows
the testing of thousands of compounds per week, with the differential advantage of working with in vivo
models. Large scale in vivo compound testing on Drosophila provides important early information on
multiple key parameters of drug discovery
DM1 Drug Discovery Pipeline
> 15,000 small molecules
screening
>8,000
screening analyisis
and validation
30 candidates
Secondary assays
validation
Research
Exploratory
4 hits
Development
Hit to lead
Optimization
Pre-clinic
*VLT001
(ABP1)
VLT002
VLT003
VLT004
VLT005
*ABP1 was discovered by an academic group performing manual screening in the DM1 Drosophila model
Clinic
The disease: Myotonic Dystrophy Type 1 (Steiner disease)
Rare Disease. Overall worldwide prevalence: 1 / 8,000. Higher in
some populations like in Quebec (Canada).
High penetrance
Multifactorial disease. Mainly a muscular disorder:
Myotonia, progressive muscular wasting and weakness.
But also cataracts, hypogonadism, ECG changes,
infertility, cognitive dysfunction, mental retardation…
Source:http://omim.org/entry/160900
Harper 2001
DM1 DNA mutation: repeat expansion
(A)
(B)
(C)
Size of CUG repeat
DM1 Phenotype
“Premutation” Asymptomatic
Normal
CAP
5’
50 - 400
38 - 49
5 - 37
(CUG)n
3’ (AAA)
n
5’ UTR DMPK Gene (Coding region) 3’ UTR
(A) In DM1, the repeat involved is a CTG tract located in the 3´UTR region of the DMPK gene. In normal
population we have two alleles between 5-37 CTG repeats. In patients the length of one allele is
expanded from more than 50 repeats up to even thousands of repeats. (B) Mutant transcripts form
stable CUG hairpins that avoid their normal transportation to cytoplasm, with the ability of forming
nuclear aggregates and sequester RNA-binding factors such as Muscleblind-like-1 protein (MBLN1),
which plays an important role in alternative splicing and gene expression regulation. (C) Thus, in DM1
patients several aberrant splicing events in many genes (spliceopathy) have been characterized, most of
them dependent of MBLN1. A few of these aberrant splicing event have already been linked to distinct
DM1 clinical symptoms.
VLT001 (ABP1) active compound
In vivo discovery of a peptide that prevents CUG–RNA hairpin formation
and reverses RNA toxicity in myotonic dystrophy models
Amparo García-López, Beatriz Llamusía Mar Orzáez, Enrique Pérez-Payá, and Ruben D. Artero
PNAS U S A. 2011 Jul 19;108(29):11866-71.
Summary Results:
1. Orally administered ABP1 increased adult viability and reversed muscle
degeneration phenotypes in DM model flies in a dose-dependent manner.
2. Aberrant ribonuclear CUG foci diminished and Muscleblind (the
Drosophila functional homolog of human MBNL1) misdistribution in CUG foci
was improved in model flies taking ABP1 orally.
3. Intramuscular administration of ABP1 suppressed muscle histopathology
signs and reversed missplicing events in DM model mice up to one month
after administration. Expression of Clcn1, which is low in DM1 model mice,
recovered in ABP1 injected muscle.
4. Binding and destabilizing CUG repeat RNA hairpins in vitro suggest a
mechanism of action of ABP1 located very upstream of the disease pathway
suggesting the molecule should be therapeutically active against many of the
clinical signs.
ABP1 suggested mechanism of action
MBNL1
MBNL1
sequestration
Splicing
misregulation
CUG
G
C
U U
CG
GC
U U
CG
GC
U U
CG
GC
U U
CG
GC
U U
CG
GC
U U
CG
GC
U U
CG
GC
U U
CG
GC
U U
CG
GC
U U
CG
GC
U U
CG
GC
UC GU
GC
U U
CG
DMPK 3’ UTR
ABP1
A)n
CUG
G
C
U U
CG
GC
U U
CG
GC
U U
G C
C
G
U
U
G
C
C
G
U U
CG
GC
U U
CG
GC
U U
CG
GC
U U
CG
GC
U U
CG
GC
U U
C
G
G
C
U
U
CUGCUGC
GCUGCUG
MBNL1
released
Normal Splicing
DMPK 3’ UTR
ABP1 binds to CUG repeat RNA and induce a switch to a single-stranded conformation,
releasing MBLN1 and decreasing CUG toxicity.
Business case for DM
Among rare diseases, Myotonic Dystrophy presents an attractive
commercial opportunity on multiple fronts:
 Clinical state (urgent unmet medical need, lack of suitable
treatments)
 Scientific rationale (solid understanding of disease pathology,
druggable targets, and proof of concept for therapeutic
intervention leading to clinically meaningful benefit in animal
models)
 Logistics/marketing considerations (significant disease
prevalence/potential market size, accessibility of patients, wellorganized global research community)
Source: 2010 Marigold therapeutic strategies for myotonic dystrophy