Drug Discovery PY366

What are medicines?

chemical preparation containing 1/+ drugs, administered with intention of having a therapeutic effect

What are medicines in antiquity?

• Naturally sourced medicine not based on scientific knowledge but rather serendipity

• Suboptimal outcomes because theres no structured development

What are two approaches to drug discovery?

1. Traditional (empiric)

2. Rational

What does the approach involve?

1. Isolate & purify the active substance from natural sources

2. Screen isolated compounds in bioassays to test their activity

3. Structural modification/optimisation of compound

What is the process?

1. Test molecule is synthesised on mg to g scale

2. Sent for bioassay testing

3. Data is returned and analysed

4. Structural changes are made and new molecules is synthesised and tested

Limitations of traditional approach

• Identification of active agent in natural products is complex

• The validity of the result of bioassay

• Purification of active constituent often involves separation of biological activity of natural product

What is rational drug discovery?

• Development of drugs that act against specific physiological or biochemical target

• Identification of failures at an earlier stage of the discovery process

What is the germ theory of disease?

• Diseases are caused by microorganisms that invade the body

• Specific organism are causative agents for infectious disease

What did Ehrlich call Salvarsan and why?

• Magic bullet

• Its highly toxic to treponema (causative agent for syphilis) but not humans

What are the 4 steps of rational drug discovery process?

1. Target identification

2. Target validation

3. Lead discovery

4. Lead optimisation

Explain Step 1

• Knowledge of physiology and pathology and biochemistry

• Elucidation of biochemical pathways and regulatory systems

• Pharmacology

• Genomic libraries

  • Identify receptors, enzymes and ion channels

  • Gene profiling - over or under expressed in pathological conditions

  • Gene engineering tech - gene knock ins or knock outs

Explain Step 2

• Structure of target complements lead molecule

• Role of target in disease progression

• Involvement of target in other processes (suitability of targeting it)

• Interaction between target and other drugs

• Dose response assay measuring activity of drug on target

Explain Step 3

• Natural sources

• Chemical libraries

  • Structure based design

  • Cell-based design

  • In-silico approach

Explain Step 4

• Optimisation of pharmacological profile of lead molecules

• Drug candidates with optimal properties:

  • Chemical

  • Pharmacodynamic

  • Pharmacokinetic

  • Toxicological

What are all 6 steps sin drug discovery?

1. Target identification

2. Hit identification

3. Lead optimisation

4. Pre-clinical development

5. Clinical candidate

6. Clinical trials

What is a target?

• Any system that can potentially be modulated by a molecules to produce a beneficial effect

• Biological macromolecule or complex that is critical for the disease e.g enzyme critical in life cycle of a virus

What is target validation?

• A form of risk assessment

• Better the validation, lower the risk in advancing the project is

• Mitigates selection of wrong target and wrong patient population

How do we identify the correct target?

• Is the target critically involved in the disease and normal biology

  • If yes to normal biology, its not a target we can use as normal physiology is affected if targeted

• The location of the target

  • One not evenly distributed throughout body to minimise SE e.g. receptors in brain for stroke but they’re not present elsewhere

• Can target manipulation lead to desired effect?

• Can target be effectively studied?

• Is the target able to be easily used in high throughput screening

Target Types

• Cells - vaccines

• DNA

• Viral surface proteins

• Transporters

• Ion channel blockers

• Receptor agonist/antagonist

• Enzyme activators/inhibitors

Can you name 3 major components of target identification

1. Tissue expression

   1. Target protein is expressed in desired organ

   2. mRNA expression is altered in desired disease tissue

   3. Protein expression is altered in disease tissue

2. Genetics

   1. Genetic association of a variant with a disease

   2. Genetic polymorphisms linked to disease state

3. Clinical experience

   1. Known ligand affecting target pathway or protein has shown efficacy in disease

Name 3 technologies used to identify targets

1. DNA microarrays - screen for gene changes between physiological and diseased tissue

2. Proteomics - Post translation mods

3. RNA knockdowns - gene knockouts

What happens when a target is validated?

• Literature survey and competitor info taken

• Has a pathophysiologically relevant molecule target been identified

• Analyse of molecular signalling pathways

• Molecular pharmacology of variants

• Determination of DNA and protein structure

• Elucidation of function and mechanism of protein

What is target validation pharmacology?

1. Pharmacological tool moderates disease associated pathway in vitro

2. Ligand with intended mode of action modulates disease associated pathways ex vivo

3. Ligands with intended mode of action modulate disease associated pathway in vivo with target engagement activity relationship established

What is antisense tech?

• identifies targets

• siRNA blocks synthesis of targeted protein

  • Small interfering RNA

  • Creates dsRNA to stop protein expression

What is the purpose of transgenic animals?

• Inhibits specific proteins in every cell in body which can determine role in biology and identify SE

• Specific protein expression is inhibited in every cell

• Identification of role of protein is not just in disease also normal biology

• Tissue restricted and/or inducible KO are now more desirable with advancements of drug delivery

What is ligand pharmacology?

• Determine whether a specific ligand against identified target have pharmacological effect in disease state

• This is beneficial where there isn’t lockout gene available or mRNA is hard to knock down

Why are animal models of disease significant?

• essential in bridging translational gap between preclinical and clinical research

• Need to check how close it is to human condition in order for results to be extrapolated

Name T1D model and explain

• Multiple low dose streptozotocin model

  • Toxin destroying B-cells so immune response is triggered

  • Chemically induced T1D

  • REAL T1DM NOT CHEMICALLY INDUCED

Name another 2 T1D model and explain

• Non-obese diabetic mouse

  • Genetic

  • Closer to human model

• Virally induced

  • Close to human model as virus role is established in development of diabetes

Name 3 T2D models

1. Genetically obese models

   1. Treatments improve insulin resistance or improve beta cell function

2. Induced obesity

   1. FAT RATS FAT RATS

3. Non-obese models

   1. Insulin resistance model

How can animal models fail to identify effective treatment

1. Disease model does not fully reflect disease

   1. Key cellular elements in animal model may not always be major players in human disease

   2. Model is quantitatively wrong with species different in capacity and sensitivity

2. Incorrect translation of intervention to pt., target is relevant but not appropriately engaged pharmacologically

   1. Incorrect dosing regimen because of species differences in pharmacokinetics

   2. Physiology of primary target is different between species, or altered in human disease

3. Clinical endpoints differer in animal models

   1. May be beneficial biochemically but have no effect on pt. symptoms

   2. QoL is an important endpoint in clinical trials and is measured usually by questionnaire which cannot be done with animals

   3. Animal models use an easy to measure objective endpoint for disease but these may not be the same endpoints for clinical trials

Why may drug fail even if animal model is correct?

• Clinical trial design is not appropriate to test intervention under investigation

• Negative clinical trials risk abandoning a hypothesis

  • Wrong dosing regimen

  • Study too short

  • Wrong patient population

Standard drug development approach:

1. Identify target

2. Develop assay for high-throughput molecular screen

3. Mass screening and/or directed synthesis programme

4. Select one or more lead structures

What molecular biology, in vitro studies and computer tech cannot do:

• Integrated response

• Reveal the unexpected

• Determine therapeutic index

• Assess importance of multiple mediators

• Determine pharmacokinetics

• Assess safety and toxicology

• Set clinical dose range

What is pharmacodynamics?

• Study of biochemical and physiological effects of drugs

• Specifically those actions for which drug was designed

• Information obtained includes

  • Lead optimisation

  • Efficacious dose range and therapeutic

  • Specificity

What is pharmacokinetics?

• Determination of fate of substances administered externally to a living organism

  • ADME

Explain Absorption

• Process where drug proceeds from site of admin to site of measurement in body

• Varies due to route of admin

  • Oral is delayed and incomplete

  • IV drug is immediate and complete

Explain Distribution

• Partitioning across membranes

• Binding to tissue and blood components

Explain Metabolism

• Determined by:

  • Location

  • Pro-drug

  • 1st pass metabolism

Explain Excretion

• Either unchanged or drug metabolites

• Associated with chemical modification of drug with overall goal of getting rid of drug

• Main process to remove unwanted substances

Types of Toxicology profiling

• Single dose and repeated dose

• Genotoxicity

• Carcinogenicity

• Reproductive and developmental toxicity

• Local tolerance

• Environmental issues

Molecular mechanisms of toxicity

1. Allergic response

   1. Leads to anaphylactic shock

   2. Deplete blood cell types

2. Receptor/ion channel/enzyme mediated

   1. Animal toxins can block channels

3. Biochemical pathways

   1. Inhibition of mitochondrial function (oxidative stress)

4. Organ directed toxicity

   1. Hepatotoxicity

   2. Nephrotoxicity

5. Mutagenesis and carcinogenesis

6. Teratogenicity

Testing methods

• Preliminary toxicity testing

  • LD50 - test the dose of drug which kills 50% of treated animals within a specified short amount of time

Single dose studies

• Effect of single dose

• Designed to support repeated dose study

• Identifying non toxic doses

• Carried out on two different species and observed for 14 days

Repeated dose studies

• Two mammalian species

• Long duration studies

• Dose is dependant on dose escalating studies

• Drug administered by clinical route

• Parameters monitored include:

  • Behavioural

  • Physiological

  • Biochemical

  • Histological

Local toxicity studies

• Route of admin dependant e.g:

• Dermal toxicity studies

  • Local signs (oedema, erythema)

  • Histological studies

• Rectal tolerance studies

  • Signs of pain, blood or mucus

  • Histological studies

• Parenteral drugs

  • For IV, IM, ID, SC

  • Sites of injection examined grossly and microscopically.

Types of allergenicity/hypersensitivity toxicological studies

• Guinea pig maximisation

  • Evaluation of oedema and erythema

  • Determination of maximum non-irritant or minimum irritant dose

• Local lymph node assay

  • Drug given on mouse ear skin

  • 5 day treatment followed by auricular lymph node dissection

Carcinogenicity and oncogenicity studies

• Lifetime bioassays

• Drug used for >6 months or frequent intermittent use for chronic diseases

• Chemical structure of drug indicates carcinogenic potential

What are clinical signs of toxicity

1. Respiratory

   1. Abdominal breathing, gasping

2. Motor activity

   1. Loss of righting reflex, tremors

3. Reflexes

   1. Pineal, righting

4. Ocular signs

   1. Lacrimation, iritis

5. Cardio-vascular signs

   1. Bradycardia, tachycardia, vasodilation

6. Autonomic signs

   1. Para/sympathomimetic actions or blockers

7. Other signs

   1. Salivation, piloerection, GIT signs

Two types of preliminary toxicity testing

• NOAEL (no observed adverse effects level)

  • Highest conc that does not produce a toxic response

• LOAEL (lowest observed adverse effects level)

  • Lowest conc that produces a toxic response

What is the use of NOAEL?

Determining it and then converting to human equivalent dose

Target related safety

1. Tissue distribution

   1. Target is highly expressed un organs other than those intended for therapeutic modulation

2. KO animals

   1. Phenotypes observes in genetically manipulated animals are valuable in identifying potential issues

3. siRNA approach

   1. Silencing the target in specific organs can help identify toxicities

   2. Confirm role of target in a toxicological outcome

4. Inactive enantiomers

   1. Inactive structure is a mirror image of the active isomer, however potential for chemistry related toxicity is equivalent

   2. Determines that the target is cause of the toxicity

Chemistry related safety

• Chemical series

  • Identify structural features associated with adverse effects

• Metabolites

  • Is the drug metabolised into a chemical that causes adverse effects

• Isomers

  • Does the drug have an isomer and does it show the same activity/toxicity

• Impurities

  • During the synthetic pathway, what impurities are formed

  • What % remain in the final formulation

  • Do they cause any adverse effects?

Identifying physiological parameters for clinical monitoring of potential adverse effects

• Adverse effects in animals for specific organs

• Tissue expression of the target

• Target itself

Hazard integration and risk assessment

• Regarding patient safety

  • Co-morbs

  • Co-meds

  • Age

• Risk benefit

  • Effect of drug on symptoms

  • Effect of drug on disabilities

  • Prognosis balanced against unwanted effects

What is central dogma?

• Flow of genetic info within a biological system

• Once info is passed into protein, it cannot get back out

• Curved red arrow around RNA

• Red arrow going to DNA: DNA synthesised using mRNA template

What is molecular biology?

• Study of biological molecules, their reactions and interactions

• Generally understood to mean: study of molecular basis of genetics, including DNA, RNA and control of gene expression

Types of ‘omes

• Genome - Total genetic content of cell/organism

• Transcriptome - all mRNA molecules currently present in a cell/organism

• Proteome - Total set of proteins currently present in a cell/organism

Define genome

• Largely, the same between cell types within an individual

• Includes

  • Protein coding genes

  • Introns and regulatory sequences

  • RNA coding sequences (siRNA, ribosomal components, tRNA etc.)

  • Junk DNA

• Studied by transcriptomic and proteomic profiling

• Epigenetics

Genotype vs Phenotype

• Genotype

  • What is in the DNA

  • Inherited from parents

  • Deduced from gene sequence

  • E.g. genes encoding eye colour

• Phenotype

  • What is observed in an individual

  • Protein expression

  • Environmental factors

  • Epigenetics

  • E.g. an individual with brown eyes

What is genetic polymorphism?

• Difference in DNA sequence between individuals

• Can involve:

  • Chance mutations

  • Induced mutations

Give examples of induced mutations

• Viral infections

• Chemical agents - can be mutagenic/carcinogenic

  • Can be linked to epigenetic changes

  • E.g. smoking increases methylation of DNA which can trigger tumour growth

• Ionising radiation

How are gene disorders caused? with examples

• They are caused by abnormalities in genome

1. Single gene mutations - point mutations and/or indels

   1. E.g. CFTR gene mutations, sickle cell disease (beta-globin gene)

2. Chromosomal disorders - numerical and/or structural

   1. E.g. down syndrome (trisomy 21)

3. Complex/ multifactorial disorders

   1. E.g. Alzheimer's disease, some cancers

What are Genome wide association studies (GWAS)?

• Collect very large data sets on individuals' genetic makeup

• Look for associations between genetic variations and different disease states

What are clinical implications of GWAS?

• Can establish correlation but not necessarily causation

• Can be used for diagnosis and prognosis studies

• Starting point for further biological study

  • What is the rile of the proteins involved?

  • What it the associated pathophysiology for those with the SNP variants

What are transcriptomes?

• Complete set of mRNA molecules in a cell or organism

• Will vary:

  • Between different types of cells - different cells have different functions

  • With environmental conditions

If mRNA goes up, does the amount of protein go up too?

1. Ribosomes recognise mRNA for translation, but efficiency varies based on mRNA sequences and structures.

2. Interactions with proteins and factors influence mRNA stability and translation.

3. mRNA degradation varies, affecting protein production; some mRNA have short half-lives, limiting translation rounds

How to calculate mRNA abundance and protein abundance?

• RNA sequencing

• DNA microarrays

Protein abundance:

• Antibody based:

  • ELIZA assays

  • Mass spec

What is the experimental side of DB searching?

1. Protein mixture treated with trypsin which cleaves polypeptide chain at argenine and lysine. This generates tryptic fragment with ends dealinated by arg and lys residues

2. sample is fed into the mass spectrometer

3. mass spec lists peptide masses from sample

What is the computer side?

• Has DB of potential proteins where you look for relevant microorganism that you used

• Using theoretical trypsin digestion gives you list of possible masses that could’ve been produced

• Compare detected masses to theoretical for protein ID

Why is molecular biology useful for drug discovery and development?

• Targets are critical to drug discovery and can be identified in various ways

• Likely to involve the comparison of healthy vs diseased tissue states

  • Literature searches

  • Classical biochemistry

  • Genomes

  • Transcriptomes

  • Proteomes

1. Genetic association studies

   1. Look for the presence of DNA sequence variation in diseased vs healthy individuals/cells

   2. Identify mutations and polymorphisms associated with disease

2. Gene expression studies (transcriptomics)

   1. Looks for differences in gene expression in diseased vs healthy individuals (i.e. what genes are "on" and "off")

3. Protein expression studies (proteomics)

   1. Look for differences in protein expression and modification in diseased vs healthy individuals (i.e. which proteins are made/not made/made more/made less/modified/not modified/modified differently)

4 characteristics of good targets

1. Unique to the pathogen/cell type (e.g. cancer) otherwise there will be higher chance of side effects

2. Essential (inhibition or antagonism needs to affect the cell or tissue)

3. Non-redundant (i.e. no other molecules with a similar function otherwise the other gene/protein may compensate when then target is inhibited)

4. Susceptible to modification - drug like molecules need to modify activity

Validation is done in two key steps: Explain them

1. Reproducibility once identified via a specific technique

2. Introduction of variation to ligand (drug) - target environment

   1. Modulation of drug molecule activity results in changes in affinity for target

   2. Variation in cell or tissue type should/ should not vary the effect

   3. Mutation of binding domain of protein target should result in loss or modulation of drug effect

Altering amounts of gene/protein expression

• Overexpression = making more of the protein than normal

• Gene knockout = delete the gene and therefore stop protein production

  • Can also knock down the expression of mRNA using RNAi

Making the target

• Unlikely that we can insolate or purify much of the protein from the source

• Expression of the recombinant protein in a suitable host

  • E.g. E.coli - cheap, fast, <100kDa, no post translational modifications

  • Saccharomyces cerevisiae (an other yeasts) - slightly slower, more expensive, larger proteins possible, some posttranslational modifications (but not necessarily the right ones)

  • Cells in culture (e.g. insect cells, mammalian cells) - slower, expensive, no size limit, PTM's can be correct

Advantage of recombinant DNA techniques

• You can essentially get unlimited amounts of protein which can enable structural and functional studies as well as screening

• You can make altered forms (change the DNA to change the resulting protein) which means that isolated domains and regions can be studied

  • Alter single amino acid residues (site-directed mutagenesis) and test hypotheses about drug binding etc

Lead Discovery

Process of identifying active new chemical entities (leads) where subsequent modification can transform them into clinically useful drug

Lead generation

Strats developed to identify compounds which possess a desired but non-optimised biological activity

Why may lead activity molecules not automatically be good drug molecules?

• May be peptidic

  • Poorly absorbed and rapidly cleared

• May have poor “drug like” properties (PK)

• Final drug may only slightly be similar to original substance

What are strategies for discovery of lead activity molecules?

1. Exploitation of biological information

2. Improvement of existing drugs

3. Systematic screening

4. Planned research and rational drug design

Exploitation of biological information

• Study of natural products and indigenous medicine

• Clinical obs of SE in medicines

• Obs made in other scientific studies

• Activities of industrial chemical products

Examples of natural products

• Medicines e.g opium

• Toxins and poisons e.g. snakes

• Hallucinogens e.g. Alcohol

What is Bothrops jararaca?

• Snake venom which peptides are used for ACEI

• Lowers BP

• Studies on peptide fragments showed that pro or aromatic amine acid in C-terminus were most effective

Examples of ethnopharmacology (indigenous medicines)

• Neuromuscular blocking agents

  • Curare - arrow poison

• Artemisinin

  • Active substance in TCM

  • Used for chloroquine

  • Artemether - methyl ether derivative

SE observation in medicine

• Most drugs have SE

• SE can be result of activity at other targets

• Amplifying SE/minimising intended effects = New drug, target different receptor

E.g. procaine (local anaesthetic):

• Rapidly metabolised

• Has CNS effects

Procainamide (antiarrhythmic)

• Resistant to esterases

• Less lipid soluble

• Fewer CNS effects

Observations made in other scientific studies

• Anticancer alkaloids tested for anti-diabetic activity

• Rats died of septicaemia due to leukopenia which told us vincristine, vinblastine were potent anti-leukaemia drugs

Activities of industrial chemical products

• Nitroglycerin observed to give workers headaches

• Potent vasodilator

• Isosorbide mononitrate now used in angina treatment

How to improve existing drugs

• Improving:

  • Potency

  • Selectivity

  • Safety

  • Duration of action

  • Formulation more easily handled by HCP

  • Formulation more acceptable to patient

• Why may competing with market leader may be financially advantageous?

  • Target is already defined

  • Market is already established

  • Clinical trials have good reference

• ß-lactams - side chain mods to penicillins allow for different selectivity of activity

What does systematic screening seek to test

• As many different compounds as possible

• Sources of test compounds can be

  • Natural products

  • Chemical libraries

• Need for assays that are fast and reliable

What are 2 drugs derived from screening dyes?

• Suramin - Trypanocide

  • Trypanosomiasis

  • Sleeping sickness in African

  • Chagas disease in south America

  • Protozoal parasite

• Chloroquine - antimalarial

Name 5 other drugs derived from screening dyes?

1. Sulphonamides - antibacterial

2. Dapsone - antileprotic

3. Bendroflumethiazide and frus - diuretics

4. Chlorpropamide and Gibenclamide - antidiabetics

5. Mesalazine and olsalazine - UC

Drugs derived from screening organic chemical libraries

• Promethazine - antihistamines

• BDZ - anxiolytics and hypnotics

• TCA’s

What is high throughput screening used for?

• Screen large numbers of small molecule compounds for interaction with target system

• Hope to find at least ONE hit on target system

• Assay system immobilised

• Many replicates in well-plates

• Highly automated

  • Liquid handling

  • Detection of response

  • Presentation and analysis of data

• Large robotic systems

How to create molecular libraries?

• Combinatorial synthesis is a process to prepare large sets of organic compounds by combining sets of building blocks

• Goal: Synthesise and test as many molecules as possible in as few steps as possible

What is combinatorial analysis?

• 3 points of diversity can generate:

  • Nr1X, Nr2X and Nr3 molecules

  • Each term is the number of different substituents at each position

• Judicious selection of building blocks can mean that very large libraries can be constructed quickly

What are problems with combinatorial chemistry?

• Working out what you have in the library

• Working out which are active molecules

• Low hit rates

• High costs

• Low quality hits

• Chemically reactive

• Problems with the chemistry

  • Individual chemical reactions should not yield alternative products

  • Should be high yielding at each step

  • Multistep reactions are problematic.

What are some current successes from high throughput screening/combinatorial chemistry?

• Insulin mimetics

• Thrombin inhibitors

• Neuropeptide Y5 receptor antagonists

• Selective COX-2 inhibitors

What is fragment based lead discovery

• Libraries of fragments of drug molecules are screened

  • RMM <300

  • LogP<3

  • H bond acceptors & donors <3

• Fragments that show binding to a target are selected for further development

• Has better success rate than combinational chemistry

What is planned research and rational drug design?What is planned research and rational drug design?

• Identify a target or biochemical system

• Attempt to address the target

• Use endogenous molecules as a starting point

Antimetabolites

• Hitchings and Elion studied DNA synthesis

  • Thought to be possible to block growth of bacteria, protozoa or tumours by interfering with synthesis of nucleic acids

  • Looked of molecules that might be dihydrofolate reductase inhibitor