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Biological Discovery

High VolumeScreening

CombinatorialDiversity

Structure, Design, Informatics

Lead Series

Biodisposition

Toxicity

Efficacy

Pharmacokinetics

INDIND

Lead Discovery Iterative PreclinicalResearch Process R&D

FULL TOOLBOX REQUIRED TO COMPETE

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BIOCHEMICAL APPROACHES TO DRUG DESIGN

• Receptor-based Approachstructure, kinetics, thermodynamics

• Gene- based Approachintroduction of gene(s), whole cell(s) or macromolecular assembly

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Receptor-based Drug Design

Structure of a Receptor

Structure

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Receptor-based Drug Design

Structure

O

OH

OH

O

COO

O

O

CH2OSO3

NHSO3

OSO3

O

O

CH2OSO3

NHSO3

OH

O

OSO3

OH

O

COOO

O

CH2OSO3

NHSO3

OH

_ _ _ _

__

_ _ _ _

Arg129Lys125 Lys114

Arg46Arg47

Lys11Arg13Asn45

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0

1

2

3

4

5

Lys11 Arg13 Asn45 Arg46 Arg47 Lys114 Lys125 Arg129

Receptor-based Drug Design

Thermodynamics

GO (

kcal

/mo

l)

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Receptor-based Drug Design

Kinetics

P + L P:L

P + L P:L

P*:L

k1

k-1

k2k-2

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Gene-based Approach

Gene Therapy

Transfer of a therapeutic gene into the target tissue and maintenance of the gene function for an acceptable time

Potential Target Diseases for Gene Therapy

Disease Deficient Gene Affected Tissue

Cystic Fibrosis CFTR Lung, intestine Familial

hypercholesterolemia LDL Receptor Liver

Emphysema 1-AT Liver Hemophilia A and B Factor VIII and IX Blood plasma Duchenne muscular

dystrophy Dystrophin Muscles

-thalassaemia -globin Erythrocytes Phenylketonuria PAH Liver

Cancer Various Various Parkinsons Dopamine synthesis Brain Alzheimers Apo E/amyloid inhibition Brain

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Gene-based Approach

Gene Therapy – Basic Steps

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Gene-based Approach

Gene Therapy – Basic Steps

• Discover Genes

• Design Replacements

• Deliver to cell / body

• Ensure Incorporation

• Detect Function

• Ensure Stability

• Test Toxicity

• Test long-term effects

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Gene-based Approach

Gene Therapy – Delivering Genes

Basic Methods for Gene Transfer in Cell Culture

Method Efficacy Stability

Physical Methods Electroporation Moderate Short/long Microinjection High Short/Long

Particle Bombardment High Short

Chemical Methods Calcium phosphate Low/moderate Short/long

DEAE-dextran Low short

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Delivering Genes – Viral Gene DeliveryAdvantages of using Viruses for Gene Delivery 1. Specific cell-binding and entry properties 2. Efficient targeting of the transgene to the nucleus of the cell 3. The ability to avoid intracellular degradation

Types of Viruses Used in Gene Delivery 1. Retroviral Vectors RNA based viruses …. HIV-1 and HIV-2 families Good gene expression … stable for long term … may integrate into host chromosome …….. Easy to produce

2. Adenovirus DNA based viruses Second most commonly used in clinical trials Commonly infect humans, but not dangerous do not integrate into host chromosome

3. Herpes Simplex Viruses DNA based virus Extrachromosomal DNA Ability to establish long-lived asymptomatic infections in neurons

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Delivering Genes – NON-Viral Gene Delivery

Advantages of using NON-Viral Gene 1. No danger of deleterious effects of viruses 2. Reduced immune response

Types of Viruses Used in Gene Delivery 1. Liposomes Two lipid species, a cationic amphiphile and a neutral phospholipid High affinity for plasma membranes and DNA

2. DNA-ligand conjugates Two components – a DNA binding domain and a ligand for cell surface Cell targeting is possible

3. Naked DNA Purified DNA …. Simplest approach … low transfection efficiency

4. Ballistic Gene Delivery Physical method of using microcarriers (Gold particles) coated with DNA …. Uses a gene gun … low efficiency method

5. Calcium phosphate precipitation method

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Delivering Genes – Cells