Drug Discovery & Development PHC 311 Lec. 1

Reem Al-Wabli Office no.; S67 E-mail; ralwabli@ksu.edu.sa

Course Description: This course is designed to give pharmacy students an overview of the process that involves the discovery of new drugs. The course covers the drug discovery (DD) process from the beginning through the final stages of clinical trials. The various stages of identifying and selecting a target, selecting and optimizing a lead compound, carrying out of in-vitro and in-vivo testing to determine biological activity and/or toxicity. The course will focus on the role of the pharmacist, as a scientist involved in discovering and developing new drugs in the pharmaceutical industry.

References 1. Gareth Thomas Medicinal Chemistry, An Introduction, 2nd Edition. Wiley-Interscience (2008) 2. Graham L. Patrick, An Introduction to Medicinal Chemistry, 3ed Ed.; Oxford University Press (2005))

Credits Type 25 Midterm I 1 Midterm II 2 10 Term Activity* 4 40 Final exam 5 *Homework and Classroom Assignments and Discussion

Topics to be covered Introduction to Drug Discovery Overview of the Drug Discovery Process Target ID & Selection Lead ID and Optimization Fragment-Based, Structure-Assisted Lead Generation Assays and Animal Models in Drug Discovery In-vitro & In-vivo Testing ADME Evaluation Clinical Trials Epidemology Pharmacogenetics

Medicinal Chemistry Folklore Medicinal chemistry is the science that deals with the discovery of therapeutic chemicals and their development into useful medicines Medicinal chemistry has been practiced for thousands of years The earliest written records of the African, Chinese, Indian, South American, and Mediterranean cultures describe the therapeutic effects of various plant parts. Man has search for cures by chewing on bark, roots, leaves and berries.

Medicinal Chemistry is defined as an interdisciplinary science situated at the interface of organic chemistry and life sciences (such as biochemistry, pharmacology, molecular biology, immunology, pharmacokinetics and toxicology) on one side and chemistry-based disciplines (such as physical chemistry, crystallography, spectroscopy and computer-based information technologies) on the other. Medicinal Chemistry Chemistry based disciplines Organic Chemistry Life Sciences

Medicinal Chemistry Terms more or less synonymous with medicinal chemistry Pharmacochemistry Molecular pharmacochemistry Drug design

Definition and Objectives Medicinal chemistry relates to the design and production of compounds that can be used in medicine for the prevention, treatment or cure of human and animal diseases. Medicinal chemistry covers three critical steps: A discovery step: consisting of the identification and production of new active substances usually called lead compounds. Leads can originate from synthetic organic chemistry, from natural sources or from biotechnological processes. An optimization step: that deals mainly with the synthetic modification of the lead structure in order to improve potency, selectivity and lessen toxicity. Its characteristics are the establishment and analysis of structure-activity relationships (SARs).

Definition and Objectives (cont.) A development step consisting of: the optimization of the synthetic route for bulk production. modification of the pharmacokinetic and pharmaceutical properties of the active substance to render it suitable for clinical use. This may cover optimization of properties associated with: Chemical formulation Solubility Elimination of unpleasant taste or irritation Reduction of pain at site of injection

Drugs are strictly defined as chemical substances that are used to prevent or cure diseases in human, animals and plants. The word drug, therefore, imposes an action-effect context within which the properties of a substance are described. For example when a drug is defined as an analgesic, it means that it is used to treat pain ….. Thus a drug may described as having analgesic, vasopressor, anticonvulsant, antibacterial, …….…etc properties. Medicinal chemist must excel in organic synthesis and understanding modern approaches to structure-activity analysis. Medicinal chemist are involved in the design, synthesis, optimization and selection of new lead compounds

Classification Systems Drugs can be classified under the following categories: 1) The origin of the drug 2) The mode of action 3) The nature of the illness 4) The chemical structure

1) The origin of the drug Classification Systems (cont.) Drugs of natural origin can come from 4 sources. Minerals: iodine, phosphates, calcium, sodium , iron Animal kingdom: insulin, fish oils, biliary salts Vegetable or plant origin: alkaloids, cardiac glycosides, anticancer agents Genetic engineering and fermentation products.

Classification Systems (Cont.) 2) The mode of action (3 flavors) Medicine that treat the cause of the disease termed “true drugs” or etiological drugs. Antibacterials, antifungals, antivirals and antiparasitics (chemotherapeutic drugs). Activity resides in their selective toxicity or the ability to destroy the invader without destroying the host. This includes also vaccines and preventive therapies.

2) The mode of action (cont.) Medicine that compensate for the deficiency (Substitutive drugs) to take the place of missing substances (vitamin therapy, insulin, i.v. rehydration during hemorrhages and diarrhea, long term treatment in Addison’s disease. Medicine that alleviate the symptoms (Symptomatic treatments) to attenuate or neutralizes a disorder in a disease state (i.e. anesthestics, ibuprofen). As a rule this mode does not cure the patient but rather to render daily life more comfortable.

Classification Systems (Cont.) 3) The nature of the illness The world health organization (WHO) in 1968 adopted this physiological classification which classifies drugs by the body system on which they act. EX. Cardiovascular, Diuretics, immunomodulators……….etc. 4) The chemical structure A great area for synthetic chemist to learn

Practical Classifications In practice the most powerful and useful system developed is known as the anatomical-therapeutic-chemical (ATC) system. The system is divided into 4 general groups according to the body system on which they act.

Common Medicinal Classifications 1) Pharmacodynamic agents: Affecting normal dynamic processes of the body. 2) Chemotherapeutic agents: Drugs inhibiting the development of various kinds of infesting host. Agents acting on metabolic diseases and on endocrine function. 4) Agents acting on the central nervous system: Psychotropic and neurological drugs.

Common Classification (Continued) 1) Pharmacodynamic agents—affecting normal dynamic processes of the body Antiarrhythmics Antianginals Vasodilators Antihypertensives Antithrombotics Antiallergic drugs

Common Classification (Cont.) 2) Chemotherapeutic agents: drugs inhibiting development of various kinds of infesting host protozoa, microbes, fungi, virus Antibiotics included in this class 3) Agents acting on metabolic diseases and on endocrine function Anti-inflammatories Antiarthritics Antidiabetics Hypolipidemic Peptides Steroid hormones

Common Classification (Continued) 4) Agents acting on the central nervous system: psychotropic and neurological drugs: Common drugs are: Anti-depressants Anti-psychotics Anxiolytics Anticonvulsants Sedatives Hypnotics Analgesics Anti-Parkinson’s drugs

Drug names: (nomenclature) Chemical 6-Chloro-3,4-dihydro-7-sulfamoyl-2H-1,2,4-benzothiadiazine 1,1-dioxide Trade Hydrodiuril®, Hydroaquil®, Esidrex®, Urozide®, Novohydrazide® etc. Many others Generic Hydrochlorothiazide

Sources of 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 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.

Methods for Drug Discovery Without a lead compound (Serendipity). Lead discovery: Random screening. Clinical Observations. Drug Metabolism studies. Rational approaches to lead discovery. Computer Aided Design

Drug Discovery Without a Lead (Serendipity) Penicillins: 1928 - Fleming Bacteria lysed by green mold mold spore contaminates culture dish left dish on bench top while on vacation weather was unseasonably cold particular strain of mold was a good penicillin producer Structure of penicillin elucidated in 1944 - X-ray crystal structure by Dorothy Hodgkin (Oxford)

Recently, the product of genetic engineering (e. g Recently, the product of genetic engineering (e.g. recombinant insulin) through Recombinant DNA Technology. It involves gene transfer to bacteria, production of the peptide product formed by expression of the transferred gene, and yielding the peptide product.

Finding a Lead compound??? 1. Random screening Disadvantage: Quite complex structures is extremely difficult to synthesize , we have to design simpler analogues. I- High throughput Screening (HTPS). II- Virtual Screening.

I- High throughput Screening (HTPS). Very rapid and sensitive method. Can test a large number of compounds. It is a biological method.

High-throughput screens (HTSs) are very rapid and sensitive in vitro screens that can be carried out robotically in 1536- or 3456- well titer plates on small (submicrograms) amount of the compounds. 1536- or 3456- well titer plates

II- Virtual Screening. Virtual screening (in silico screening) is an interesting approach in lead discovery. It involves the screening of chemical databases to identify compounds that are fitting and interacting with the receptor of study. It is a computational screening.

A. The plant kingdom A rich source of lead compounds (e.g. morphine, cocaine, digitalis, quinine, tubocurarine, nicotine, muscarine and many others). Recently, the anticancer agent Paclitaxel (Taxol)®, and the antimalarial agent artemisinin have been separated from the yew tree, and a chinese plant, respectively. eg. Morphine from Papaver somniferum Quinine from Cinchona bark.

Paclitaxel e.g. Artemisinin: antimalarial isolated from shrub, containing extremely unstable looking trioxane ring (no chemist would dream to synthesize) Sildenafil the anti-impotence drug vasodilator in the penis more than in the heart

B. The Microbiological World Bacteria have provided rich pickings for drugs and lead compounds e.g. Penicillins Soil and water samples were collected from all around the world, led to discovery of cephalosporins, tetracyclins, aminoglycosides, rifamycins and chloramphenicol. Fungal metabolite: Asperlicin, isolated from Aspergillus alliaceus, antagonize cholecystokinin hormone and used to control appetite, and treat anxiety). Lovastatin (lower cholesterol level). Cyclosporins (immune suppressant in transplantation).

C. The Marine World Coral and sponges have a wealth of biologically potent chemicals, e.g. A potent antitumor agent. Curacin A, is obtained from a marine cyanobacterium.

D. Animal Sources A potent analgesic compound called epibatidine was obtained from the skin extract of the Ecuadorian poison frog.

E. Venoms and toxins From animals, plants, insects, and microorganisms. e. g. Teprotide is a peptide isolated from the venom of the Brazilian viper and was the lead compound for the development of the antihypertensive agents Captopril and Cilazapril (ACE inhibitors). Captopril

F. ENDOGENOUS COMPOUNDS NATURAL LIGANDS FOR RECEPTORS Agonist Agonist (Serotonin)

F- ENDOGENOUS COMPOUNDS (cont.) NATURAL LIGANDS FOR RECEPTORS Antagonist Antagonist

F- ENDOGENOUS COMPOUNDS (cont.) - NATURAL SUBSTRATES FOR ENZYMES Enkephalins Enkephalinase inhibitors Peptides Protease inhibitors

Lead compounds with activity – Flow of Drug Discovery Target screening – 1,250,000 compounds Lead compounds with activity – 2,500 compounds Clinical trials – 1 compound

SYNTHETIC COMPOUNDS SYNTHETIC COMPOUNDS G-Lead Compounds from the Synthetic World SYNTHETIC COMPOUNDS SYNTHETIC COMPOUNDS MOR008C.WAV

i- Screening synthetic banks Chemicals or intermediates synthesized by the pharmaceutical companies or purchased from research groups for studying a new target. e.g. INH is more active intermediate than the target structure isonicotinaldehyde thiosemicarbazone.

PRONTOSIL

SULFANILAMIDE

TNT

ii- COMBINATORIAL SYNTHESIS Automated solid phase procedure aimed at producing as many different structures as possible in a short time as possible. AUTOMATED SYNTHETIC MACHINES

Combinatorial chemistry Is an approach that provides efficient synthesis of a large collection of molecules Generates a wide diversity of compounds and reduces the cycle time for drug screening An intentionally created collection of differing molecules which can be prepared either synthetically or biosynthetically

A1-B1 A2-B1 A3-B1 A1-B2 A2-B2 A3-B2 A1-B3 A2-B3 A3-B3

New compounds with improved biological activity Compounds + biological activity QSAR New compounds with improved biological activity

2. Clinical Observations of Side Effects of Drugs An existing drug may have an undesirable side effect, which might be used as a lead compound based on its side effects. e.g. 1. Sulphonamides are used as antibacterial agents possess convulsive effect brought on by hypoglycemia, and has a diuretic effect in large dose. Structural modifications were made to the sulphonamide to enhance both the hypoglycemic activity and the diuretic effect

e.g. 2. Enhancing the sedative side effect of promethazine (antihistamine), developed the neuroleptic agent chlorpromazine. Sildenafil the anti-impotence drug vasodilator in the penis more than in the heart

Clinical Observations (cont.) e.g.3. Sildenafil (Viagra) Developed for angina and hypertension by Pfizer Lack of potency in Phase II trials sent the drug back to Phase I trials to determine highest tolerated dose. Volunteers in second trial reported increased erectile function Became first treatment for erectile dysfunction [see also: tadalafil (Cialis) and vardenafil HCl (Levitra).

3. Drug Metabolites as Leads for Drug Discovery

Drug Metabolism Studies (cont.) Terfenadine HCl (Seldane) caused arrhythmia in some patients also taking certain antifungals Fexofenadine HCl (Allegra) is a metabolite of terfenadine that is also a non-sedating antihistamine and can be metabolized in presence of antifungals

4. Rational Approaches The key information that permits rational approaches to drug design is: knowledge of the etiology of a given disease or at least the biochemical processes that are disturbed. Try to find a proper method for inhibition of the life cycle of this organism (For example; enzyme inhibition). Design of lead compound by computational and/or biological methods. Examples of the rational approach are the discovery of the inhibitors of the angiotensin-converting enzyme. Captopril

e.g. 2) the oral contraceptive 17 α-ethynyl estradiol is prepared by analogy to 17 β-estradiol and it was found to possess stronger activity and longer duration.

e.g. 3) Serotonin the inflammation mediator was used as a lead for antiinflammatory agents, from which Indomethacin was developed. Serotonin Indomethacin

5. Computer Aided Design A detailed knowledge of a target binding site aids the design of novel lead compounds intended to bind with that target. If enzymes or receptors can be crystallized, it is possible to determine its structure and its binding site by X-ray crystallography, molecular modeling soft ware programs can then be used to study the binding site and design molecules which will fit and bind.

Very few lead compounds are ideal. Most are likely to have: If the lead compound has useful biological activity. Why bother making analogues? Very few lead compounds are ideal. Most are likely to have: Low activity Poor selectivity Significant side effects So optimization of the interaction between the drug and its target will allow higher activity and selectivity

DRUG DESIGN AND DEVELOPMENT Stages 1) Identify target disease 2) Identify drug target 3) Establish testing procedures 4) Find a lead compound 5) Structure Activity Relationships (SAR) 6) Identify a pharmacophore 7) Drug design- optimising target interactions 8) Drug design - optimising pharmacokinetic properties 9) Toxicological and safety tests 10) Chemical development and production 11) Patenting and regulatory affairs 12) Clinical trials

1. 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 there advantages and disadvantages? (e.g. side effects) Can one identify a market advantage for a new therapy?

2. 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

Drug targets Drug Binding site Binding regions Binding groups Intermolecular bonds Drug Binding site Macromolecular target Drug Bound drug Macromolecular target Unbound drug

- Messenger binding Induced fit Binding site is nearly the correct shape for the messenger Binding alters the shape of the receptor (induced fit) Altered receptor shape leads to further effects - signal transduction

Induced fit - Binding site alters shape to maximise intermolecular bonding Phe Ser O H Asp CO2 Phe Ser O H Asp CO2 Induced Fit Intermolecular bonds not optimum length for maximum binding strength Intermolecular bond lengths optimised

Overall process of receptor/messenger interaction Signal transduction Binding interactions must be: - strong enough to hold the messenger sufficiently long for signal transduction to take place - weak enough to allow the messenger to depart Implies a fine balance Drug design - designing molecules with stronger binding interactions results in drugs that block the binding site - antagonists

Agonists Agonist binds reversibly to the binding site Similar intermolecular bonds formed as to natural messenger Induced fit alters the shape of the receptor in the same way as the normal messenger Receptor is activated Agonists are often similar in structure to the natural messenger E Agonist R E Agonist R Agonist R Induced fit Signal transduction

Competitive (reversible) antagonists Antagonist binds reversibly to the binding site Intermolecular bonds involved in binding Different induced fit means receptor is not activated No reaction takes place on antagonist Level of antagonism depends on strength of antagonist binding and concentration Messenger is blocked from the binding site Increasing the messenger concentration reverses antagonism

Non competitive (irreversible) antagonists OH OH X O X Covalent Bond Irreversible antagonism Antagonist binds irreversibly to the binding site Different induced fit means that the receptor is not activated Covalent bond is formed between the drug and the receptor Messenger is blocked from the binding site Increasing messenger concentration does not reverse antagonism

Overall process of enzyme catalysis E + P S E ES P E EP Binding interactions must be; - strong enough to hold the substrate sufficiently long for the reaction to occur - weak enough to allow the product to depart Implies a fine balance Drug design - designing molecules with stronger binding interactions results in enzyme inhibitors which block the active site

Competitive (reversible) inhibitors Inhibitor binds reversibly to the active site Intermolecular bonds are involved in binding No reaction takes place on the inhibitor Inhibition depends on the strength of inhibitor binding and inhibitor concentration Substrate is blocked from the active site Increasing substrate concentration reverses inhibition Inhibitor likely to be similar in structure to the substrate

Non competitive (irreversible) inhibitors OH X OH X O Covalent Bond Irreversible inhibition - Inhibitor binds irreversibly to the active site - Covalent bond formed between the drug and the enzyme - Substrate is blocked from the active site - Increasing substrate concentration does not reverse inhibition - Inhibitor likely to be similar in structure to the substrate

Non competitive (reversible) allosteric inhibitors (open) ENZYME Enzyme Induced fit Active site unrecognisable Active site ACTIVE SITE (open) ENZYME Enzyme Allosteric site Allosteric inhibitor - Inhibitor binds reversibly to the allosteric site - Intermolecular bonds are formed - Induced fit alters the shape of the enzyme - Active site is distorted and is not recognised by the substrate - Increasing substrate concentration does not reverse inhibition - Inhibitor is not similar in structure to the substrate

TARGET SELECTIVITY Between species Within the body Antibacterial 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 selectivity

3. TESTING DRUGS Tests are required in order to find lead compounds and for drug optimisation Tests can be in vivo or in vitro A combination of tests is often used in research programs

3.1 in vivo Tests 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 Rationalisation 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 (ED50) versus the dose level that is lethal to 50% of the sample (LD50)

3.2 in vitro 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 Used in high throughput screening Measure the interaction of a drug with the target but not the ability of the drug to reach the target Results are easier to rationalise - less factors involved Does not demonstrate a physiological or clinical effect Does not identify possible side effects Does not identify effective prodrugs

3.2.1 Enzyme Inhibition Tests Identify competitive or non competitive inhibition Strength of inhibition measured as IC50 IC50 = concentration of inhibitor required to reduce enzyme activity by 50% 3.2.1 Enzyme Inhibition Tests

3.2.2 Testing with Receptors 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.

Dose = amount of drug administered to the patient Dose Response Relationships Dose = amount of drug administered to the patient Response = effect in the body produced by the drug Drug + Receptor  Drug-Receptor Complex  Response

Log Drug Concentration [Molar] 100 3 4 Response 50 2 1 ED50 Log Drug Concentration [Molar] KEY PARAMETERS 1. Dose required to produce any effect at all. 2. ED50 = effective dose to produce 50% response 3. Dose required to produce maximum effect 4. Dose that produces a toxic response.

Efficacy (or Intrinsic Activity) – ability of a bound drug to change the receptor in a way that produces an effect; some drugs possess affinity but NOT efficacy

Log Drug Concentration [Molar] Potency vs Efficacy Potency – how much drug is required to produce a certain effect. 100 Response 50 2 1 ED50 Log Drug Concentration [Molar]

Efficacy – how large an effect the drug produces. Potency vs Efficacy Efficacy – how large an effect the drug produces. 100 Response 50 2 1 ED50 Log Drug Concentration [Molar]