Cell Bio Cellular Models for Drug Discovery

Question 1

a brief overview of drug discovery

Answer 1

• ongoing field of study
• for one drug eventually clinically used - ~6000-10,000 new chemical compounds are synthesized (available for potential preclinical screening)
• not done in patients –> lab testing (cells)
• from time target drug is identified ~12 more years until used clinically
• total cost ~$1 billion - ~half in lab research; ~half in clinical trails
• for one drug successfully clinically trialed ~19 have failed clinical trials

*can we do better safely?

Question 2

model systems - example criteria

Answer 2

• “druggable” target
• pick a model with target that can show a suitable response
• validate the target/model combo as an appropriate stand-in

Question 3

What are scientifically, ethically, fiscally, medically & pharmacologically appropriate stand-ins (models) for patients eventually treated with the medications?

Answer 3

model systems

Question 4

druggable” target

Answer 4

*biological identity that interacts with a drug

• a receptor activated by binding insulin –> diabetes
• an enzyme inhibited by ibuprofen –> inflammation
• microtubules affected by taxol –> cancer metastasis (assay) before animal + clinical tests
• just want to know physical association = what is the appropriate model

Question 5

pick a model with target that can show a suitable response

Answer 5

• how do you test binding of insulin derivative to receptor?
• what’s a valid model for testing enzyme inhibition?
• what’s needed to test new anti-metastasis drug?
• will any one preclinical test prove efficacy of new drug?
• ex. if we only want to know Kd vs. if we want to turn that into biological response of glucose uptake

*scientific readout you want

Question 6

validate the target/model combo as an appropriate stand-in

Answer 6

• does model have relevant target regarding delivery, metabolism, binding of drug?
• can you simplify/downsize/reduce use of typical research models (lab mice/rats etc.)
• thousands to be tested

*increase throughput

Question 7

drug discovery models

Answer 7

*fish, flies, fungus, & farmacy

• zebra fish
• drosophila (fruit fly)
• yeast (fungus)

Question 8

zebra fish

Answer 8

• pre-clinical drug discovery
• easy to monitor embryogenesis, vertebrate cell physiology & gene homologues, small adult size (~1.5 in) allows hi thru-put
• b-amyloid (alzheimer’s-associated) protein –> defective movement
• in aquarium, small so many fit, similar vertebrae model

Question 9

drosophila (fruit fly)

Answer 9

• before clinical testing with people
• numerous behavior & physiology mutants mapped to specific genes, numerous human gene-equivalents identified
• Parkinson’s-associated gene –> loss of dopaminergic neurons
• model nerve degeneration = monitor flight patter, etc. to reflect motor neuron function

Question 10

yeast (fungus)

Answer 10

• extreme example
• eukaryote with metabolism mapped to conserved genes
• SBDS protein associated with bone marrow failure in humans; protein function was unknown
• a study in yeast showed protein’s function crucial for ribosome function providing a “druggable” target to improve the translation
• model stands in for bone marrow

Question 11

human cells in petri dishes as models

Answer 11

• ~45 years ago
• sensitivity of human cancer cells to anticancer drugs in petri dish tests was directly related to drug success in treating patient tumor
• does not always occur this way
• at least for the compounds tested
• NOT a universally guaranteed approach; that is part of the validation process
• depends on cancer type, drug, etc.
• how did we get to that point?
• how do we go beyond it to make improvements?

Question 12

cell culture in the 1950’s

Answer 12

• get cells to replicate in lab
• henrietta lacks & george gey
• cervical carcinoma cells attached to test tube, mitotically active, split to additional tubes (hela cells)
• 1st continuous human cell line
• her cells were immortal and perpetuated

Question 13

cell culture in 2020

Answer 13

cultured cells from diverse sources in addition to sometimes patient tissue

• if you can get them to grow in petri-dish environment
• hela and 100’s of other cancer-derived and normal tissue-derived cell types (muscle, skin, cardiac, liver, etc.) used in hi thru put metabolism studies, cancer gene identification, gene sequencing, pre-clinical drug testing

Question 14

Imetelstat example of preclinical studies for drug effectiveness

Answer 14

• imetelstat inhibits telomerase (RNA/DNA) and stops cell growing, which decreases cells in petri dish
• imetelstat treated plate slows growth of cancer cells –> cancer cell growth inhibited in presence of antisense oligo imetelstat
• sense oligo is the same length/sequence but it cannot interact with RNA template –> cancer cells grow in presence of control (sense) oligo

Question 15

tying topics together

Answer 15

tying topics together

• DNA replication & telomerase
• cells in culture (petri dish) for testing of new cancer drugs
• cancer stem cells (to come later in semester)

*normal stem cells need telomerase (no replication senescence)

Question 16

addressing thru-put - petri dishes

Answer 16

• 6000-10,000 new potential drugs per year (many chemicals to screen)
• need to increase thru-put for repeats, different concentrations (dose-response), different exposure times
• downsize to gelatin drop with cells, nutrients (glucose), and test compounds “printed” on microscope slide
• reduces amount needed of test compound, target cells, cost

Question 17

designing the cell culture model

Answer 17

*interpret what happens to cells

• what cells?
• what’s to be determined?
• what scale of testing is to be done?

Question 18

What cells?

Answer 18

*do they appropriately model target?

• normal and cancer cells (test on normal to see if you want to treat cancer –> if it is toxic it might be bad)
• validate cells retain relevant processes (ex. cyp450 enzymes)
• demonstrate target is present (ex. receptor)

Question 19

What’s to be determined?

Answer 19

*what’s the endpoint

• survival (maintain number) and/or replication (increase number) - test with neutral red
• metabolism (biotransformation, ex. in liver) of drug
• cell migration, intracellular movement
• cell death (necrosis vs. apoptosis); toxicity - test with neutral red (lethal effect)

Question 20

What scale of testing is to be done?

Answer 20

• biological repeats within assay
• technical repeats of entire assay
• varied times of exposure (many days)
• dose - response correlation
• cell biol meets bio-engineering

*each variation expands thru put

Question 21

cell culture - drug testing

Answer 21

*neutral red (NR) assay - lysosomes

• NR weak cationic dye - readily penetrates cell membrane
• more neutral red –> more cells grow with lysosome (healthy)

Question 22

NR in healthy cells

Answer 22

• retained within lysosomes in healthy cells (selectively accumulates)
• NR binds anionic proteins in lysosome
• amount is therefore related to cell number
• assay endpoint; experimentally measure amount of dye
• replaces counting individual cells
• dye incorporated into cells is detected visually (microscope) or extraction from cells at end of incubation
• red dye measured by spectrometry at 540nm
• dye amount gives quantifiable response to drug
• lysate cell (break open with red dye) to give an objective number
• saves time of counting individual lysosomes

Question 23

NR in stressed/damaged lysosome

Answer 23

• dye leaks from stressed/damaged lysosome
• drug toxic effects stress cell or directly damage lysosome
• decrease in intracellular (lysosomal) or extractable dye reflects cell damage and/or dying cells
• less red = less absorbed

Question 24

neutral red (NR) assay - putting it to use

Answer 24

• individual cell (accumulated red) to increase the concentration of candidate drug in cell causes increased damaged cells
• damaged cells do not spread around the bottom of the well
• do many times to accumulate repeatable data
• max redness when all alive (quantitative readout)

Question 25

addressing multiple organ interactions

Answer 25

- most drug delivery ultimately has systemic distribution - test new colon cancer drug: drug effects on other organs (possible bone marrow tox) OR other organs (cells) affecting drug? (possible liver metabolism of drug?) - cells-on-chip technology = microscale systems biology (organ-on-chip) - very small - cells in the chamber (organs) connecting for the circulatory system - -/+ liver metabolism, marrow toxicity, colon target

Question 26

assay potential chemo drug: cell survival

Answer 26

- liver chamber empty and full is the same output - no liver cells with drug - and liver cells and drug *impact on tumor cells

Question 27

your interpretation of toxicity? any negative effect on bone marrow cells? regarding effectiveness (reducing tumor cell number

Answer 27

- many cancer chemotherapy drugs decrease WBC counts - all bone marrow cells survived with and without the liver but with parent compound or predicted metabolite (drug) - the drug has an increased impact on colon cancer cells in presence of liver because fewer live tumor cells (metabolize to a more active form) *would not have seen without a complex model system (whole physiology)