Chapter 12

Target disease Priority for the Pharmaceutical Industry: Can the profits from marketing a new drug outweigh the cost of developing and testing that drug? Questions to be addressed: Is the disease widespread? (e.g. cardiovascular disease, ulcers, malaria) Does the disease affect the first world? (e.g. cardiovascular disease, ulcers) Are there drugs already on the market? If so, what are their advantages and disadvantages (e.g. side effects) Can one identify a market advantage for a new therapy?

Drug targets A) Lipids Cell membrane lipids B) Proteins Receptors Enzymes Carrier proteins Structural proteins (tubulin) C) Nucleic acids DNA RNA D) Carbohydrates Cell surface carbohydrates Antigens and recognition molecules

Between species: Antibacterial, antifungal and antiviral agents Identify targets which are unique to the invading pathogen Identify targets which are shared but which are significantly different in structure Within the body: Selectivity between different enzymes, receptors etc. Selectivity between receptor types and subtypes Selectivity between isozymes Organ and tissue selectivity

Bioassays (test systems) Tests are required in order to find lead compounds and for drug optimisation Need to be quick, easy and relevant Tests can be in vivo or in vitro A combination of tests is often used in research programs

Carried out on live animals or humans Measure an observed physiological effect Measure a drug’s ability to interact with its target and its ability to reach that target Can identify possible side effects Rationalization may be difficult due to the number of factors involved Transgenic animals - genetically modified animals Drug potency - concentration of drug required to produce 50% of the maximum possible effect Therapeutic ratio/index - compares the dose level of a drug required to produce a desired effect in 50% of the test sample (ED 50 ) versus the dose level that is lethal to 50% of the sample (LD 50 ) In vivo Tests

Tests not carried out on animals/humans Target molecules (e.g. isolated enzymes or receptors) Cells (e.g. cloned cells) Tissues (e.g. muscle tissue) Organs Micro-organisms (for antibacterial agents) More suitable for routine testing Measure the interaction of a drug with the target but not the ability of the drug to reach the target Results are easier to rationalize - less factors involved Does not demonstrate a physiological or clinical effect Does not identify possible side effects Does not identify effective prodrugs In vitro tests

High Throughput Screening NMR Enzyme Inhibition tests Identify competitive or non competitive inhibition Strength of inhibition measured as IC 50 IC 50 = concentration of inhibitor required to reduce enzyme activity by 50% Receptor tests Not easy to isolate membrane bound receptors Carried out on whole cells, tissue cultures, or isolated organs Affinity - strength with which compounds bind to a receptor Efficacy - measure of maximum biochemical effect resulting from binding of a compound to a receptor. Potency - concentration of an agonist required to produce 50% of the maximum possible effect. Testing drugs

The lead compound A compound demonstrating a property likely to be therapeutically useful The level of activity and target selectivity are not crucial Used as the starting point for drug design and development Found by design (molecular modelling or NMR) or by screening compounds (natural or synthetic) Need to identify a suitable test in order to find a lead compound Active principle - a compound that is isolated from a natural extract and which is principally responsible for the extract’s pharmacological activity. Often used as a lead compound.

A) The natural world B) The synthetic world C) The virtual world Plantlife (flowers, trees, bushes) Micro-organisms (bacteria, fungi) Animal life (frogs, snakes, scorpions) Biochemicals (Neurotransmitters, hormones) Marine chemistry (corals, bacteria, fish etc) Chemical synthesis (traditional) Combinatorial synthesis Computer aided drug design The lead compound

A) Isolation and purification solvent-solvent extraction chromatography crystallization distillation B) Structure determination elemental analysis molecular weight mass spectrometry IR spectroscopy UV spectroscopy NMR ( 1 H, 13 C, 2 D) spectroscopy X-ray crystallography Identification of the lead compound

Plants Lead compounds from the natural world

Poppy capsule Morphine Lead compounds from the natural world

OPIUM - MorphineOPIUM - Morphine CINCHONA BARK - QuinineCINCHONA BARK - Quinine YEW TREE - TaxolYEW TREE - Taxol Lead compounds from the natural world

Willow tree - salicylic acid Coca bush - cocaine Aspirin Procaine Lead compounds from the natural world

The beatons of pennycross Lead compounds from the natural world

CURE for EPILEPSY Heat a cauldron of water until warm to the touch Add a liberal sprinkling of juicy spiders Produce one pedigree dog Flavour with essence of dog turd Simmer for 20 minutes then enjoy THE BEATONS OF PENNYCROSS

Herbal remedies of old Majority may have worked through a placebo effect Lead compounds from the natural world Lead compounds from the natural world

Rain forests Lead compounds from the natural world

Coral and marine chemistry CURACIN Lead compounds from the natural world

Micro-organisms

Penicillin Cephalosporins Tetracyclines Streptomycin Chloramphenicol Lead compounds from the natural world Micro-organisms

Venoms and toxins Captopril (anti-hypertensive) Teprotide Lead compounds from the natural world

Venoms and toxins Lead compounds from the natural world

Tubocurarine (from curare) Lead compounds from the natural world Venoms and toxins

Atracurium (Neuromuscular blocker) Tubocurarine (from curare) Venoms and toxins Lead compounds from the natural world

Endogenous compounds Natural ligands for receptors Agonist Agonist Lead compounds from the natural world

Antagonist Antagonist Endogenous compounds Natural ligands for receptors

Endogenous compounds Natural substrates for enzymes Enkephalins Enkephalinase inhibitors Peptides Protease inhibitors Lead compounds from the natural world

Lead compounds from the synthetic world Lead compounds from the synthetic world SYNTHETIC COMPOUNDS

PRONTOSIL Lead compounds from the synthetic world Lead compounds from the synthetic world

SULFANILAMIDE Lead compounds from the synthetic world

TNT Lead compounds from the synthetic world

Rubber industry Antabuse Lead compounds from the synthetic world Lead compounds from the synthetic world

Organic synthesis 4.4 Lead compounds from the synthetic world

Automated synthetic machines Combinatorial synthesis 4.4 Lead compounds from the synthetic world

RESIN BEAD AMINO ACID AMINO ACIDS X PEPTIDE Combinatorial synthesis - peptide synthesis 4.4 Lead compounds from the synthetic world

N N YO CHR 1 R 2 RESIN BEAD Combinatorial synthesis - heterocyclic synthesis 4.4 Lead compounds from the synthetic world

RESIN BEADN N O R2R2 R3R3 H Combinatorial synthesis - heterocyclic synthesis 4.4 Lead compounds from the synthetic world

N N O O EtO O R2R2 R3R3 R4R4 R5R5 RESIN BEAD Combinatorial synthesis - heterocyclic synthesis 4.4 Lead compounds from the synthetic world

N N N NHNNHN O R2R2 R3R3 R4R4 R5R5 RESIN BEAD Combinatorial synthesis - heterocyclic synthesis 4.4 Lead compounds from the synthetic world

N N YN O R2R2 R3R3 R4R4 R5R5 HNHNHNHN RESIN BEAD Combinatorial synthesis - heterocyclic synthesis 4.4 Lead compounds from the synthetic world

The Past Lead Compound TargetsTargets Lead compounds The Present and Future 4.5 Lead compounds - impact of the human genome project

4.5 Lead compounds - impact of the human genome project

4.5 Lead compounds - impact of the human genome project

Lead compounds - impact of the human genome project

4.5 Lead compounds - impact of the human genome project

4.5 Lead compounds - impact of the human genome project

4.6 Lead compounds - de novo design

X-RAY CRYSTALLOGRAPHY

PROTEIN STRUCTURE 4.6 Lead compounds - de novo design

Receptor 4.6 Lead compounds - de novo design

Scaffold CO 2 - HO IONICBOND H-BOND VDWBOND Scaffold H3NH3N + O CH 3

N HNHNHNHN O S O O NHNHNHNHN N N H2NH2NH2NH2N S O OON Thymidylate kinase inhibitors Anticancer agent Lead compound Optimisation 4.6 Lead compounds - de novo design

4.7 Design of lead compounds using NMR spectroscopy NMR spectroscopy

Binding Site Protein 4.7 Design of lead compounds using NMR spectroscopy

Protein No observable biological effect 4.7 Design of lead compounds using NMR spectroscopy

13 C NMR C C CH CH CH CH 2 CH Design of lead compounds using NMR spectroscopy

CH 2 CH 3 13 C NMR C C CH CH CH CH 2 CH Design of lead compounds using NMR spectroscopy

Protein Optimiseepitope

Protein OptimiseepitopeOptimiseepitope

Protein OptimiseepitopeOptimiseepitope Link

LEAD COMPOUND 4.7 Design of lead compounds using NMR spectroscopy

Me Design of a lead compound as an immunosuppressant Epitope A Epitope B 4.7 Design of lead compounds using NMR spectroscopy

Me Design of a lead compound as an immunosuppressant Lead compound 4.7 Design of lead compounds using NMR spectroscopy