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Research and development of new drugs for human diseases

Valentia Biopharma - In vivo Drug Discovery

Currently focused on finding lead

compounds to achieve an effective

treatment on Myotonic Dystrophy

Type 1 (DM1) disease

Drosophila melanogaster, a new technology for Drug Discovery

Generation of disease models Generation of genetic/biochemical models

• Genetic diseases • Other disease mechanisms

In vivo High Throughput Screening • Phenotypic assays • Biochemical assays

Valentia Biopharma - In vivo Drug Discovery

Valentia Biopharma R&D

Why Drosophila?

75% of human disease causing genes are conserved inDrosophila

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)

Drug delivery can be problematic and not all human systems can be easily modeled. (pulmonarydisease…)So we always validate any positive result with mouse models and human cell lines

Not only Drosophila:

Drosophila models. Screening experimental approaches

(1)Phenotypic assay (viability)

(2)Biochemical assay (gene-reporter) by fusing known human genes involved in DM1 pathologies to a reporter

PROMOTER-CONSTRUCT

Reporter Gene

FLYINJECTION

TRANSGENIC FLY

Toxic in mushroom bodies(neuronal cells)

n=191

n=67

Control

Viability model

DM1 fly

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

1. FLY CROSSES

2. DRUG PLATES PREPARATION(Robot with stackers)

3. SEEDING (Sorter Cytometry)

4. HOMOGENIZATION (Robot with stackers)

5. READING (Envision Reader /Scanner)

6. ANALYSIS

F0:Adults F1:Embryo/Larvae F1:Adults

Screening platform - Screening platform - Steps in red are automated

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

Research Development

Exploratory Hit to lead Optimization Pre-clinic Clinic

*VLT001 (ABP1)

VLT002

VLT003

VLT004

VLT005

> 15,000 small molecules

>8,000

30 candidates

4 hits

screening

screening analyisis and validation

Secondary assays validation

DM1 Drug Discovery Pipeline

*ABP1 was discovered by an academic group performing manual screening in the DM1 Drosophila model

The disease: Myotonic Dystrophy Type 1 (Steiner disease)

Multifactorial disease. Mainly a muscular disorder:

Myotonia, progressive muscular wasting and weakness.

Harper 2001

Rare Disease. Overall worldwide prevalence: 1 / 8,000. Higher in

some populations like in Quebec (Canada).

High penetrance

But also cataracts, hypogonadism, ECG changes,

infertility, cognitive dysfunction, mental

retardation…

Source:http://omim.org/entry/160900

3’5’(CUG)n(CUG)n (AAA)

n

CAPCAP

Size of CUG repeat

50 - 400

5 - 37

38 - 49

5’ UTRDMPK Gene (Coding region) 3’ UTR

DM1 Phenotype

“Premutation” Asymptomatic

Normal

DM1 DNA mutation: repeat expansion

(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.

(A) (B) (C)

VLT001 (ABP1) active compound

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.

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.

MBNL1 sequestration

MBNL1

DMPK 3’ UTRA)n

ABP1

C U G C U G C

CC G

G

CGC G

U U

U UG C U G C U G

CGC G

U U

CGC G

U U

CGC G

U U

CGC G

U U

U

UU

CC G

GU

CC

G

G

C

UC G

U UC G

CG

G

CGU U

C GU U

DMPK 3’ UTR

C

UC G

U UC G

CG

G

CGU U

C GU U

CGC G

U U

CGC G

U U

CGC G

U U

CGC G

U U

CGC G

U U

CGC G

U U

CGC G

U U

CGC G

U U

CGC G

U UCG

C GU UC G

Splicing misregulation

MBNL1released

Normal Splicing

ABP1 suggested mechanism of action

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, well-organized global research community)

Source: 2010 Marigold therapeutic strategies for myotonic dystrophy