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Drug Discovery Process
Massimiliano Beltramo, PhD
SP1999 target validation summaryReceptor identification• SP1999 is a receptor for ADP, belong to the purinergic receptor family and has been named
P2Y12
Expression profile• mRNA is present in brain and platelets (northern blot, PCR, and in situ hybridization)• The protein is expressed in several brain region and is localized in microglia cells
(immunocytochemistry)• Gene expression is increased in some disease models (in situ hybridization)
Functional activity• Activation of Gi protein (GTPγS)• Modulation of microglia activation (live cell imaging)• Role in platelet aggregration (Genetic analysis and in vitro assay)
Potential role in diseases• Bleeding disorder/Thrombosis• Chronic pain
Target validation
Target validation flow-chart
Biology of the target
Expression Profile
Expression in relevant areas
Expression in pathologic state
Expression consistent between human and animal
model
Functional role
Role in human disease
In vivo studies
In vitro studies
Scientific rationale definition
Predicted side effect
profile
Predicted additional indication
s
Platelet activation cascade
X
Your drug
Scientific rationale
Resting platelet
ThrombinCollagen
ADP TxA2
PAFEpinephrine
GP IIb/IIIa
Activated platelet
Resting platelet
ThrombinCollagen
ADP TxA2
PAFEpinephrine
GP IIb/IIIa
Activated platelet
Scientific rationale
Increase expression in microglia after inductionof chronic pain
Reduction of pain in P2Y12 KO mice
What is the link between microglia andpain reduction??
Chronic pain
Do we need an agonist or an antagonist?
Gi
ACATP
cAMP
Stimulate AC with forskolin
↓Add test
compound↓
Detect modulation of cAMP level
fsk stimulation
[Drug]cA
MP
leve
l
agonist
100%inactive/ antagonist
inverse agonist
A New Paradigm for Drug Discovery
Gene ofUnknownFunction
Determination of Cellular Function
Validation as anAttractive DrugTarget
High-ThroughputScreening AssaysEstablished againstTarget Protein
Expression/ProteinPurification
Identify LeadInhibitor
Chemical Structure-Activity Analysis•Therapeutic Index•Pharmacokinetics
- Gene-to-Function -
R&D process for a new drug
Exploratorydevelopment
Fulldevelopment
IDEA
DRUG
CANDIDATE POCTARGET
Therapeuticresearch
Exploratoryresearch
Hit to lead
Discovery of new active molecules
Biologicaltarget
Assay Test to assess target biological activtiy
Chemical compoundlibrary
Hit
Lead
Drug candidate
HIGH THROUGHPUT SCREENING
(HTS)
Hit to lead
How to maximize the chance of success:
• High diversity of chemical library compounds
• Capacity to screen rapidly a large number of compounds
• Robust and standardized assay
• Low false positive and false negative rate
Hit to lead
Chemical library
Derived from combinatorial chemistry
Collection from previous project
Commercially available
Hit to lead
• The Old Way
–One chemist, one compound, 1 gram recrystallized and stored forever
• A chemist might make 50-100 compounds a year
• Combinatorial Synthetic Methods
–“Mix and Split” Solid Phase Synthesis
– Multi-Parallel Synthesis and Purification
– Automation of Synthetic and Analytical Methods
• A chemist today might make > 10,000 compounds/year
Compound synthesisHit to lead
Combinatorial chemistry
R1
R2
R3
R4
Scaffold or pharmacophore
20 R120 R213 R310 R4 20 X 20 X 13 X 10 = 52,000 compounds !!
Substituents
N
Hit to lead
High Throughput Screening (HTS) is the process
through which a high number of small molecules
(>50,000-100,000 per day) are tested on the target of
interest.
To perform an HTS is necessary an adequate in vitro
assay
High throughput screening
Hit to lead
Require the expression of the target:
recombinant cell lines
native systems
In vitro assay development
Two types:
1. “cell-free”
2. “cell-based”
Hit to lead
General requirements
- Target pharmacology must be preserved
- Signal have to be: 1. robust 2. reproducible 3. easy to measure
- experiment should require minimal handling
- miniaturized
- automatized
- low cost
In vitro assay development Hit to lead
