drug discovery case Flashcards

Question

what is target validation in pharmacology?

Answer 1

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

Answer 2

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

Answer 3

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

Answer 4

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

Answer 5

* 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?

Answer 6

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

Answer 7

``` Type 1 – Auto-immune disease – Genetic component • Type 2 – Obesity – Insulin resistance ```

Answer 8

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

Answer 9

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

Answer 10

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

Answer 11

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

Answer 12

``` 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 ```

Answer 13

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

Answer 14

The subsequent identification of the molecular targets that underlie an observed phenotypic response

Answer 15

• 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

Answer 16

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.

Answer 17

Protein targets immobilized on a glass slide, incubated with a labelled drug molecule which will indicate successful binding

Answer 18

Living microarrays’ with cells overexpressing potential target proteins incubated with labelled drug

Answer 19

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.

Answer 20

``` enzyme/receptor assays – Cell viability – Drug-drug interaction studies – Metabolite identification/elucidation – Plasma protein binding – Genotoxicity ```

Answer 21

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

Answer 22

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%)

Answer 23

an assay is a process of analyzing a substance to determine its composition or quality.

Answer 24

Test leading compounds in animal model of the disease to determine if it has the desired effect

Answer 25

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

Answer 26

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

Answer 27

* 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

Answer 28

Using human and other species plasma • Use equilibrium dialysis or ultrafiltration • Also determine the stability of the drug in plasma

Answer 29

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.

Answer 30

* 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

Answer 31

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

Answer 32

• 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!

Answer 33

- 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)

Answer 34

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

Answer 35

• 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

Answer 36

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

Answer 37

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

Answer 38

``` 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 ```

Answer 39

Omics” • Study all of a particular type of molecule in the cell/tissue/organism • Generates a lot of data • Typically experimentally and computationally demanding

Answer 40

• Study one protein, DNA sequence or small group of interacting biomolecules • Typically study several aspects (e.g. kinetics, structure, cellular role)

Answer 41

total genetic content of cell/organism

Answer 42

all mRNA molecules currently present in a cell/organism (like a snapshot in time)

Answer 43

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)...

Answer 44

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.

Answer 45

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)

Answer 46

the amount of specific mRNA in a cell RNA sequencing DNA micro assay

Answer 47

Traditionally Ab-based • ELISA assays or quantitative western blots • Needs antibodies! • Lacks highly parallel methods and universal external standard • Mass spectrometry (MS)

Answer 48

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)

Answer 49

Method to accurately measure mass of proteins and protein fragments (peptides). • Requires proteins/peptides are ionised to allow detection. • High sensitivity, accuracy and resolution.

Answer 50

* 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

Answer 51

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...

Answer 52

* Nucleic acid sequencing has also become common in diagnostics. * Sequencing of genes amplified by PCR allows identification e.g. of microorganisms.

Answer 53

• 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

Answer 54

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.

Answer 55

Look for the presence of DNA sequence variation in diseased vs healthy individuals/cells • Identify mutations and polymorphisms associated with disease

Answer 56

Look for differences in gene expression in diseased vs healthy individuals (i.e. which genes are “on” and which are “off”)

Answer 57

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)

Answer 58

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

Answer 59

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

Answer 60

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

Answer 61

Overexpression: normally, increased speed of onset, severity • Gene knockout: disease will not occur • Gene knock-down: disease less severe

Answer 62

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) ```

Answer 63

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

Answer 64

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

Answer 65

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

Answer 66

Identification through molecular biology methods • ‘Omics methods can be especially useful here • Multiple markers often better

Answer 67

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.

drug discovery case Flashcards

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