drug discovery case Flashcards
what was the Black Death and what treatments were used?
started around 1348-1350
killed 1.5 million people.
treatments that was used were • Bezoar stones, from animal stomachs
• Goa stones made from clay, silt, shells, resin and musk
– Scrapings were drunk as a remedy for ailments – Also used to counteract suspected poisoning
– Highly valued and expensive
what are opium alkaloids?
Obtained from the latex of Papaver somniferum L.
• Known since antiquity for its analgesic and sedating properties
• Opium is the dried latex of the opium poppy, containing all the alkaloids
what is morphine?
first isolated in 1804. it is an analgesic antitussive reduced gastric motility respiratory depressant
what are some of the traditional drugs and medicines?
Herbal medicines
• Poisons e.g. from plants, minerals
• Toxins e.g., snake, spider and scorpion venoms
• Either as a purified substance or a mixture of substances
what were some key observations when making a drug?
• Possible to isolate active substances
• Frequently >1 chemically similar active
– Structurally related (but different) molecules from natural sources frequently have similar (but subtly different) biological activities
• E.g. different cardiac glycosides have similar activity but different durations of action
what is the traditional approach to drug discovery?
Isolation and purification of active substance
• Structural modifications to active substance
• Some screening of groups of molecules
– Dyes
– Plant extracts
• Test molecule is synthesised on mg to g scale
• Sent for bioassay testing
• Data returned and analysed
• Structural changes made and new molecule synthesized, tested etc
what considerations is needed in the drug discovery process
The drug target • A lead activity molecule • Drug candidates (new chemical entities, NCE) having appropriate properties – Pharmacodynamic – Pharmacokinetic – Toxicological – Chemical
how do you go about searching for new drug targets?
Knowledge of physiology / pathology
– Elucidation of biochemical pathways and regulatory systems
• Knowledge of mechanisms of actions of drugs, poisons, toxins and venoms
• Structural similarities in biomolecules suggesting new targets, e.g. G-protein coupled receptors have conserved sections in their structure
• Genomics
– Search for new receptors, enzymes, ion channels on the basis of amino acid sequence homology with similar known targets
what are the drug targets?
Humans are our primary area of concern • Animals also need medicines • Principles are broadly similar – Mammals – Birds – Reptiles – Other animals
Infectious diseases • Viruses
• Bacteria
• Fungi
• Parasites • Prions
how do you exploit a drug target?
Need information on
– Structural nature of the target
– Possible mechanism of interaction between the target and potential drugs • Need to develop an assay that accurately measures the activity of the target
– Dose dependent response
– Capable of comparison with other ligands
Once scientisits discovered this cycle, they were able to inhibit or stimulate different pathways
• Renin, ACE, AII, Aldosterone
how do you go about finding targets?
Physiology and biochemistry
• Pharmacology – drug effects
• Toxins and venoms from the natural world • Human genome
what are the steps in drug discovery?
- Target identification/validation
- Hit (pre-lead) identification – what the drug does • Lead optimization
- Pre-clinical development
- Clinical candidate
- Clinical trials
what are the two types of target identification strategies?
Target Deconvolution
– Start with an efficacious drug and identify the target retrospectively
• Target discovery (obviously more logical)
– Identify a target and develop a drug to affect it
what is meant by a target?
Any system that can potentially be modulated by a molecule to produce a beneficial effect • Biological macromolecule or molecular complex that is critical for the disease
o E.g. an enzyme critical in the life cycle of a virus
list the types of targets
Enzymes - inhibitors (competitive or non-competitive, reversible or irreversible) or activators
• Receptors - agonist or antagonist
• Ion channels - blockers or activators
• Transporters - inhibit or stimulate
• Viral surface proteins - blocking the entry to cell
• DNA-intercalating agents, minor groove binders, anti-sense drugs
• Cells-vaccines
what is meant by target validation
Form of risk assessment, the better the validation the lower the risk in advancing the project
• Mitigates selection of wrong target and wrong patient population
what is meant by target identification
Is the target critically involved in disease?
• Is the target critically involved in normal biology? – if so, you don’t really want to do anything to it (is it going to harm the patient if we temper with it?)
• Location of the target
– Ideally one that is not evenly distributed throughout the body (to minimize side effects) • Does manipulation lead to desired effect?
• Can the target be effectively studied?
• Is the target amenable to High Throughput Screening?
• The proposed target has a favorable intellectual property status?
what is high throughput screening?
is a drug discovery process that allows automated testing of large numbers of chemical and/or biological compounds for a specific biological target
what are the major components of target identification
tissue expression
- targets protein is expressed or active in the desired organ
- target mRNA expression is altered in the desired disease tissue
- target protein expression is altered in diseased tissue
Genetics
– Genetic association of a variant with a disease
– Genetic polymorphisms linked to a disease state
• Clinical experience
– Known ligand affecting target pathway or protein has shown efficacy in the disease
what are the different methods in identifying targets?
Many different technologies can identify novel targets including: • DNA microarrays • Proteomics – Post-translational modifications – Protein isoforms – Antibodies interacting with the traget • RNA knockdown – Gene knockouts – Antisense technology
what are DNA microarrays used for?
the DNA microarray is a tool used to determine whether the DNA from a particular individual contains a mutation in genes
what is meant by proteomics?
it is a large scale study of proteomes which is a set of proteins produced in an organism, system or biological context.
it is used to investigate when and where portions are expressed, rate of protein production, degradation and steady state abundance
what is meant by knockdown in biology?
deletion or inactivation of genes is known as knockdown or knockout. in most organisms, the genes are encoded in DNA. DNA is transcribed into RNA and RNA is translated into protein which become the building blocks and catalysts for the whole organism.
what are validated targets?
- Literature survey and competitor information
- Identification of a pathophysiologically relevant molecule target e.g. an enzyme, receptor, etc
- Analysis of molecular signaling pathways
- Molecular pharmacology of variants
- Determination of the DNA and protein structure
- Elucidation of the function and mechanism of the protein
- Proof of therapeutic concept in animals
what is target validation in pharmacology?
Pharmacological tool moderates disease associated pathway in vitro
• Ligand with intended mode of action modulates disease associated pathways ex vivo or in native tissue
• Ligands with intended mode of action modulate disease associated pathway in vivo with target engagement-activity relationship established
what is antisense technology
it is a tool for controlling gene expression in a cell. using synthetic antisense oligonucleotides, it targets the gene at the level of mRNA rather than DNA and prevents them from producing proteins
- siRNA blocks synthesis of the targeted protein
- Example-blockage of the P2X3 receptor was able to lower pain sensitivity
- Advantage is that this is reversible model so can be sure that it is the target that is responsible for the effect
what is meant by transgenic animals
means that 1 or more DNA sequence from another species have been introduced by artificial means. animals are usually made transgenic by having a small sequence of foreign DNA injected into a fertilised eggs or developing embryo
Specific protein expression inhibited in every cell
• Identification of role of the protein not just in disease but also in normal biology • Tissue-restricted and/or inducible knockouts are now more desirable with the advancements of drug delivery
what is ligand pharmacology
Determine whether a specific ligand against the identified target have a pharmacological effect in the disease state
what is meant by animal models of disease
- Animal models are essential to bridge the translational gap between preclinical and clinical research
- Is there a suitable animal model of the disease you want to treat?
- How close is it to the human condition?
give an example of animal model of disease
Colitis
– DSS model
– IL-10 KO mouse • Arthritis
– Subdermal collagen injection • Hemorrhagic shock
– Blood removal and replacement • Septic shock
– LPS injection i.p.
• Adult respiratory distress syndrome
– Intratracheal LPS application • Myocardial infarction
– Tie off left ventricular artery
what is diabetes animal model
Type 1 – Auto-immune disease – Genetic component • Type 2 – Obesity – Insulin resistance
give an example of is type 1 diabetes model
Multiple-low-dose-streptozotocin model (MLDS)
– Chemically induced
– Fast see data after 21 days – Not a very good model
• Non-Obese diabetic (NOD) mouse
– Genetic
– Incidence increases after 5 weeks
– 80% of female mice develop diabetes only 20% of male mice (not gender generalizable) – Experiments run to 30 weeks (very long)
– Also not a very good model • Virally-induced
– Role of viruses in development of diabetes
give an example of type 2 diabetes model
enetically obese models
– ZDF rats or OB/OB mice
– Treatments to improve insulin resistance or improve beta cell function • Induced obesity
– High fat feeding mice or rats • Non-obese models
– GK rat
why can animal models fail to identify effective treatments
Disease model does not truly reflect the disease
– Key cellular elements in animal models are not the major players in human disease
– The model is quantitatively wrong with species differences in capacity, sensitivity or rate constants governing the relationship between elements are not captured
• Incorrect translation of the intervention to the patient so target is relevant but not appropriately engaged pharmacologically
– Incorrect dosing regimen because of species differences in pharmacokinetics
– Physiology of the primary target is different between species, or altered in human disease
• Clinical endpoints differ in animal models
– Quality of life is an important endpoint in clinical trials and is measured usually by questionnaire which cannot be done with animals
– Animals models use an easy to measure and objective endpoint for disease but these may not be the same endpoints for clinical trials e.g. tumor size in animal models of a cancer drug while overall survival is the endpoint in clinical trials
why do drugs fail even though the animal models is correct
Clinical trial design not appropriate to test the intervention under investigation. Negative clinical trials risks abandoning a correct hypothesis
– Wrong dosing regimen
– Study too short
– Wrong patient population
what is the standard drug development approach
Identify target (e.g., enzyme, receptor, ion channel, transporter) • Develop assay for high-throughput molecular screen • Mass screening and/or directed synthesis program • Select one or more lead structures
why do we need to perform bioassays??
Bioassay: A bioassay is an analytical method to determine the concentration or potency of a substance by its effect on living animals or plants, or on living cells or tissues.
• To predict some type of therapeutic potential either directly or by analogy, of test compounds
define target deconvolution?
The subsequent identification of the molecular targets that underlie an observed phenotypic response
what is affinity chromatography
• Identify proteins that bind to the drug by linking the drug so it is immobile, incubate with protein extracts, wash away the unbound proteins before decoupling the attached protein from the drug and identifying the target
what is meant by expression cloning
Phage display: present potential target proteins on the surface of a phage (bacterial) in the form of a phage library. Expose the library to the drug and the phages with the target are bound to drug.
• Three hybrid system: DNA-binding domain linked to a ligand-binding domain, ligand linked to drug molecule and transcriptional activation domain fused to a potential drug target. If the drug attaches to the target a trimeric complex is formed resulting in expression of the reporter gene.
what is protein microarray?
Protein targets immobilized on a glass slide, incubated with a labelled drug molecule which will indicate successful binding
what is meant by transfected cell microarray?
Living microarrays’ with cells overexpressing potential target proteins incubated with labelled drug
what is meant by biochemical suppression?
Does not depend on a molecule’s affinity for its biological target. Firstly, a protein extract is mixed with a molecule that is known to inhibit the activity of interest. An uninhibited protein extract is then fractioned and introduced to the inhibited extract, it is then possible to determine if any of the fractions suppress inhibition.
in vitro/ex vivid studies what does the target discovery show
enzyme/receptor assays – Cell viability – Drug-drug interaction studies – Metabolite identification/elucidation – Plasma protein binding – Genotoxicity
what is meant by pharmacodynamics
Does the drug affect the target at effective concentrations that can be reached clinically • Does the drug modify the disease process in an animal model
what is a screening assay?
High through-put in vitro assay • Specific assay
– Human target • Cost
• Specificity
• Commonly used in vitro enzyme/ receptor assays to test specificity in plate-based format • Examples of assays to test for new drugs include:
– Cell growth for anti-cancer medications
– Coagulation time for anti-coagulants
– IC50 determination for drugs inhibiting enzymes (concentration that inhibits the activity
by 50%)
define assay?
an assay is a process of analyzing a substance to determine its composition or quality.
define animal models
Test leading compounds in animal model of the disease to determine if it has the desired effect
what is pharmacokinetics in relation to drug discovery
Only very basic measurements can be made using bioassays
• Full animal studies are required to get the full pharmacokinetic profile for: – Absorption
– Distribution
– Metabolism
– Excretion
what does drug -drug interactions studies show
Using pooled human liver microsomes or other in vitro metabolism models
• Determine the effects of your new compound on metabolizing enzyme activities and hence potential interactions/side effects
what is metabolite identification / elucidation?
- Using liver microsomes/cells
- Using LC-MS etc to identify possible metabolites both mass and structure • Synthesize metabolites to test both for toxicity as well as efficacy
what is plamsa protein binding
Using human and other species plasma
• Use equilibrium dialysis or ultrafiltration
• Also determine the stability of the drug in plasma
what is toxicology
Some identification of toxicological issues with a new drug can be identified using bioassays
• You can study some of the potential undesirable pharmacodynamic effects of a substance so called safety pharmacology.
what is cytotoxicity
- MTT assay – mitochondrial respiration
- LDH assay – assess the plasma membrane damage in a cell population.
- Apoptosis and necrosis measurements – apoptosis: gently dies and is carried away. Necrosis: cell explodes and spreads contents everywhere
what is cardiovascular toxicity
All drugs require hERG testing as if its ability to
conduct electrical current across the cell membrane is inhibited or compromised then potentially long QT syndrome may occur
– hERG is a potassium ion channel in the heart
what is meant by Genotoxicity - the ability of harmful substances to damage genetic information in cells.
• Bacterial reverse mutation (Ames) test
– Use bacteria which have mutations in genes involved in histidine synthesis
– Need histidine to grow, if they grow without histidine indication of mutations induced by drug – If your drug fails the Ames test, usually don’t
take it any further – genotoxicity!
describe the process of DNA damage- comet assay
- living cell from culture media, blood or tissue
- cells with damaged DNA having single stranded/ double stranded breaks
- single cells are embedded on agarose-coated slide and lysed
- after electrophoresis and fluorescent staining, the damaged DNA is separated from the intact DNA (head) and generates a comet (tail)
describe ex vivo studies
Test compounds on tissue taken from a living organism
• Can be used to test effectiveness of your drug but also to identify potential side effects • E.g. Isolated heart and aorta preps to determine cardiovascular effects
describe in vivo studies
• Alongside the testing of your drug in animal model of disease can look at the key systems for possible toxicity
– Cardiovascular system – CNS
– Respiratory system
– Hepatic system
– Renal system
• Biochemical
– Blood levels of ALT, AST, ALP, CK, BUN, Creatine, etc.
• Histopathology
• Physiological observations
– Breathing, heart rate, urine, faeces, etc.
• Observation of behavior when drug is administered
what is meant by central dogma
Flow of genetic information within a biological system
• Once info is passed into a protein, it cant get back out
• Curved red arrow around the RNA: RNA replication
• Red arrow going to DNA: DNA synthesized using an mRNA template
what is molecular biology
The study of biological (macro)molecules, their reactions and interactions
• Generally understood to mean: the study of the molecular basis of genetics, including DNA, RNA and the control of gene expression
- Tools and techniques for studying these molecules and processes
what are some components in molecular biology
DNA sequencing • Gene “cloning” and identification • Protein over-expression • Gene expression studies • Gene knock-out (no gene expression) and knock-downs (reduced gene expression) • Protein structure determination • Genomics and proteomics
what is meant by omics
Omics”
• Study all of a particular type of molecule in the cell/tissue/organism
• Generates a lot of data
• Typically experimentally and computationally demanding
what is traditional biochemistry omic
• Study one protein, DNA sequence or small group of interacting biomolecules • Typically study several aspects (e.g. kinetics, structure, cellular role)
define genome
total genetic content of cell/organism
define transcriptome
all mRNA molecules currently present in a cell/organism (like a snapshot in time)
define proteome
total set of proteins currently present in a cell/organism (like a snapshot in time) • Also lipidome (all lipids), glycome (all carbohydrates), metabolome (all metabolites), reactome (all reactions)…
what entails in a genome
The complete DNA sequence of the cell or organism
• Largely the same between cell types within an individual, includes: ⇒ Protein coding genes
⇒ Introns, regulatory sequences
⇒ RNA coding sequences (siRNA, ribosomal components, tRNA, etc) ⇒ “Junk DNA” (much of which probably isn’t rubbish…)
• Expression of genomic information to determine phenotypes is of central importance for basic and translational life science research
⇒ Studied by transcriptomic and proteomic profiling.
what is entailed in transcripomes
The complete set of mRNA molecules in a cell or organism
• Will vary:
⇒ Between types of cells (e.g. liver and brain cells express very different sets of mRNA
molecules)
⇒ With environmental conditions (including stressors, hormones, toxins, drugs etc)
what is meant by mRNA abundance
the amount of specific mRNA in a cell
RNA sequencing
DNA micro assay
what is protein abundance
Traditionally Ab-based
• ELISA assays or quantitative western blots
• Needs antibodies!
• Lacks highly parallel methods and universal external standard • Mass spectrometry (MS)
what is proteomes
The complete set of protein molecules in a cell or organism
Will vary:
• Between types of cells
• With environmental conditions
• Post-translational modification important
• Proteolytic cleavage
• Covalent modification (phosphorylation, methylation, glycosylation etc)
define mass spectrometry
Method to accurately measure mass of proteins and protein fragments (peptides). • Requires proteins/peptides are ionised to allow detection.
• High sensitivity, accuracy and resolution.
define biological mass spec
- A mass spectrometer generates gas-phase ions from a sample, separates them according to their mass-to-charge ratio (m/z) and generates a record of their abundance, allowing:
- MW determination.
- Amino acid sequencing.
- PTM detection.
- Relative quantification
what are some of the considerations for omics
Omics’ studies generate (very) large amounts of data
• They can take a lot of time (and money)
• Picking conditions in transcriptomics and proteomics may be critical • Risk of errors (often n=1)
• Amount of data often exceeds amount of understanding…
what is meant by DNA sequencing
- Nucleic acid sequencing has also become common in diagnostics.
- Sequencing of genes amplified by PCR allows identification e.g. of microorganisms.
what is the human genome project
• The human genome project identified 20-25,000 genes in the human genome along with many repetitive and non-coding regions (~2% of the total genome)
• Genome and proteome information is essential in modern drug discovery:
✓ target identification
✓ characterisation of the target’s sequence and structure
✓ proof of concept studies
✓ assay development
• ‘Omics also impact on basic science, biomarker discovery, vaccine target identification and personalised medicine
what are the target identifications
Drug targets can be identified in a variety of ways
• Likely to involve the comparison of healthy vs disease tissue states
• Literature searches.
• Classical biochemistry.
• Transcriptomes.
• Proteomes.
what is meant by genetic association studies
Look for the presence of DNA sequence variation in diseased vs healthy individuals/cells • Identify mutations and polymorphisms associated with disease
what is meant by gene expression studies for transcriptomics
Look for differences in gene expression in diseased vs healthy individuals (i.e. which genes are “on” and which are “off”)
what is meant by Protein expression studies (proteomics)
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)
what are good drug targets
Unique to pathogen/cell type (eg cancer)
• Otherwise, higher risk of side-effects
Essential
• Inhibition/antagonism needs to affect cell or tissue Non-redundant (i.e. no other molecules with similar function) • Otherwise the other gene/protein may compensate Susceptible to modification
• Drug-like molecules need to modify activity
what are the two ket steps of validation
Two key steps:
• Reproducibility
• Once identified via specific technique • or literature review
• Introduction of variation to ligand (drug)-target environment
• Modulation of drug molecule activity results in changes in affinity for target
• Variation in cell or tissue type should/should not vary effect
• Mutation of binding domain of protein target should result in loss or modulation of drug effect
what is meant by over expression, gene knockout
Overexpression: Make more of the protein than normal
• Gene knockout: Delete the gene (and therefore stop protein production) • Can also “knock down” expression of mRNA using RNAi
how is over expression and gene knockout critical for disease
Overexpression: normally, increased speed of onset, severity • Gene knockout: disease will not occur
• Gene knock-down: disease less severe
what is Animal models of human disease
Biochemistry of many organisms is similar – so they often suffer similar diseases, respond to same drugs
• Animal “models” can be used to understand diseases and how organisms respond to drug-like molecules, for example:
• Rats, mice, rabbits etc.
• Pigs
• Fruit flies, nematode worms, snails, spiders
• Animal models can be genetically modified so that they develop human disease, e.g. – Knockouts: • BRCA1, BRCA2 (breast cancer) • Apolipoprotein E (atherosclerosis) – Transgenes: • Human APP (Alzheimer’s disease) • LDL receptor (Hypercholesterolemia)
how do you go from target ID to structure
determine the DNA and protein sequence
elucidate structure and functions of that target
DNA and protein sequences can be downloaded from databases
• X-ray crystallography and NMR enable structure determination of proteins
• Alternatively comparison of protein sequences allows “homology models” to be built
hw do you make the target
Unlikely that we can isolate/purify much of the protein from the source
• Express recombinant protein in a suitable host:
• Escherichia coli: Cheap, fast; <100 kDa; no post-translational modifications (PTMs)
• Saccharomyces cerevisiae (and other yeasts): Slightly slower, more expensive etc; larger proteins possible; some PTMs (but not necessarily the right ones)
• Cells in culture (insect cells, mammalian cells): Slower, expensive; no size limit; PTMS can be correct
what are the advantages of recombinant DNA techniques
Get (essentially) unlimited amounts of protein
• Enables: Structural studies, Functional studies, Screening
• Can make altered forms (change DNA to change resulting protein)
• Study isolated domains/regions
• Alter single amino acid residues (site-directed mutagenesis) and test hypotheses about drug binding etc
what are biomarkers
Identification through molecular biology methods • ‘Omics methods can be especially useful here
• Multiple markers often better
what are vaccine targets
molecular biology can also help identify these
• Recombinant expression/purification for basic science, testing and development • Some vaccines are recombinant proteins
• Recent developments in Covid-19 vaccines:
• DNA and mRNA vaccines
• Adenovirus vector vaccines etc.
