Cellular Drug Discovery, Cell Cycle, & Apoptosis Flashcards
Drug discovery
ongoing field of study, for one drug eventually clinically used - about 6000-10,000 new chemical compounds are synthesized (available for potential preclinical screening), not done in patients so lab testing (cells), from time target drug is identified ~12 more years until used clinically, total cost ~$1 billion, half in lab research and half in clinical trails, for one drug successfully clinically trialed about 19 have failed clinical trials
Model systems - example criteria
“druggable” target, pick a model with target that can show a suitable response, validate the target/model combo as an appropriate stand-in
Model systems
Scientifically, ethically, fiscally, medically and pharmacologically appropriate stand-ins (models) for patients eventually treated with the medications
“Druggable” target
biological identity that interacts with a drug, a receptor activated by binding insulin so diabetes, an enzyme inhibited by ibuprofen so inflammation, microtubules affected by taxol so cancer metastasis (assay) before animal and clinical tests, just want to know physical association = what is the appropriate model
Pick a model with target that can show a suitable response
- 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?
Validate the target/model combo as an appropriate stand-in
- 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
Drug discovery models
*fish, flies, fungus, & farmacy
- zebra fish
- drosophila (fruit fly)
- yeast (fungus)
Zebra fish
- pre-clinical drug discovery
- easy to monitor embryogenesis, vertebrate cell physiology & gene homologues, small adult size (about 1.5 in) allows high throughput
- b-amyloid (alzheimer’s-associated) protein so defective movement
- in aquarium, small so many fit, similar vertebrae model
Drosophila (fruit fly)
before clinical testing with people, numerous behavior & physiology mutants mapped to specific genes, numerous human gene-equivalents identified, parkinson’s-associated gene so loss of dopaminergic neurons, model nerve degeneration = monitor flight patter to reflect motor neuron function
Yeast (fungus)
extreme example, eukaryote with metabolism mapped to conserved genes, SBDS protein associated with bone marrow failure in humans; protein function was unknown, study in yeast showed protein’s function crucial for ribosome function providing a “druggable” target to improve translation, model stand in for bone marrow
Human cells in petri dishes as models
- about 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 and drug
Cell cycle 1950s
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
Cell cycle 2020s
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 high through put metabolism studies, cancer gene identification, gene sequencing, pre-clinical drug testing
Imetelstat example of preclinical studies for drug effectiveness
- imetelstat inhibits telomerase (RNA/DNA) and stops cell growing, which decreases cells in petri dish
- imetelstat treated plate slows growth of cancer cells so cancer cell growth inhibited in presence of antisense oligo imetelstat
- sense oligo is the same length/sequence but it cannot interact with RNA template so cancer cells grow in presence of control (sense) oligo tying topics together
- DNA replication & telomerase
- cells in culture (petri dish) for testing of new cancer drugs
Addressing through-put - petri dishes
- 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
Designing the cell culture model
*interpret what happens to cells
- what cells?
- what’s to be determined?
- what scale of testing is to be done?
What cells?
*do they appropriately model target?
- normal and cancer cells (test on normal to see if you want to treat cancer so if it is toxic it might be bad)
- validate cells retain relevant processes (ex. cyp450 enzymes)
- demonstrate target is present (ex. receptor)
What’s to be determined?
*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)
What scale of testing is to be done?
- biological repeats within assay
- technical repeats of entire assay
- varied times of exposure (many days)
- dose - response correlation
- cell biology meets bio-engineering
*each variation expands through put
Cell culture - drug testing
*neutral red (NR) assay - lysosomes
- NR weak cationic dye - readily penetrates cell membrane
- more neutral red so more cells grow with lysosome (healthy)
NR in healthy cells
- 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
NR in stressed/damaged lysosome
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
Neutral red (NR) assay - putting it to use
individual cell (accumulated red) to increase concentration of candidate drug in cell causes increased damaged cells, damaged cells do not spread around bottom of well, do many times to accumulate repeatable data, maximum redness when all alive (quantitative readout)
Addressing multiple organ interaction
most drug delivery ultimately has systemic distribution, test new colon cancer drug: drug effects on other organs (possible bone marrow toxic) OR other organs (cells) affecting drug?, cells-on-chip technology = microscale systems biology (organ-on-chip), very small, cells in chamber (organs) and connecting for circulatory system, +/- liver metabolism, marrow toxicity, colon target
Assay potential chemotherapy drug: cell survival
liver chamber empty and full is same output, no liver cells with drug, and liver cells and drug
*impact on tumor cells
Interpretation on toxicity? any negative effect on bone marrow cells? regarding effectiveness (reducing tumor cell number)
many cancer chemotherapy drugs decrease WBC counts, all bone marrow cells survived with and without liver but with parent compound or predicted metabolite (drug), drug has increased impact on colon cancer cells in presence of liver because less live tumor cells (metabolize to more active form)
*would not have seen without complex model system (whole physiology)
Cell cycle steps
M: mitosis, G1: gap 1, G0: quiescent, S: synthesis, G2: gap 2
Mitosis
nuclear & cell division (cytokinesis) - relatively short in time compared to interphase
G1: Gap 1
hours to days or more (some are so long that they may not go back into cycle)
G0: Quiescent
Apparently non-dividing cells - long term temporary or permanent (extended G1)
S: synthesis of DNA
Remember, most cells will wind up with shortened telomeres (need nutrients, growth factors, etc.)
G2: gap 2
Completion of G1 replication and replicated genome ready to undergo mitosis
Interphase
Metabolically active with euchromatin & heterochromatin observed, G1 to G2 (no mitosis)
Gap
named because apparent gap in activity under microscope, seems like this but actually replicating and actively metabolizing (intracellular), G1, G0 and G2 are still metabolically, biochemically active, etc., G1 variation on theme (become longer or shorter), permanent G0 (cells with extended G1 phase) - post-mitotic and will not reenter cell cycle
Cell replication
central to wound healing, normal cell replacement, tumor growth (via cell cycle)
Cell cycle - M phase sub-steps
after interphase starts prophase, prometaphase, metaphase, anaphase, then telophase
Prophase
chromosome condense) - daughter chromosomes attached and mitotic spindle formation
Prometaphase
(nuclear membrane breakdown) - dissolution begins
Metaphase
(chromosomes align) - daughter chromosomes align at metaphase plate (equator of spherical cell) and attach to microtubule structure (depolymerization of microtubule to drive separation)
Anaphase
(chromosome separate) - pull at poles
Telophase
(nuclear membrane reforms) - cleavage furrow forms with microfilaments
*complete chromosome separation then reformation of nuclear membrane then 2 daughter cells
Cytoskeletal proteins in M Phase Sub-Steps
intermediate filament, nuclear lamins, microtubule, microfilament
Intermediate filament
depolymerization (phosphorylation) throughout cell and repolymerization (dephosphorization) in daughter cells
Nuclear lamins
under nuclear membrane need to depolymerize (IF)
Microtubule
depolymerization to pull spindles
Microfilament
polymerization for cleavage furrow to separate cells (contractile ring)
Permanently stopped cells
*post-mitotic
“terminally differentiated” - maturation/specialization over, upper layers of epidermis, many neuronal cells, skeletal muscle, RBC, RBCs as characteristic terminally differentiated because they do not have a nucleus
*never enter the cell cycle
Indefinitely stopped cells
quiescent = G0 extended but can be stimulated again, liver cells (liver damage/tissue loss would stimulate), some WBC (right stimulus would start cell cycle again in lab conditions), some can be triggered to divide with the right signal
Routinely stop & go cells
certain epidermal (lower level mitosis but never upper layers) - every 28 days, gut lining epithelial cells (highly active and continual replacement), bone marrow (blood cell progenitors)
Myeloid stem cell
ability to enter cell cycle always, myeloblasts have shorter term ability to enter cell cycle, RBCs and platelets (no DNA/nucleus) do not go into cell cycle, granulocytes are mostly post-mitotic
Cell cycle control
*some of the signals (dozens of + and - signals)
- external signals then internal signals then consequences
External positive signal (just one of many examples)
EGF - epidermal growth factor (protein promotes skin cell replication), outside cell then inside response, many non-skin tissues produce & respond to EGF (stimulate cycling of cells), kidney, salivary gland, prostate, thyroid, bone marrow, lung, breast, uterus, & colon, large precursor protein processed to small active peptide, increased production in many cancer cells
*breast cancer treatment targeting balance (over stimulation of EGF)
EGF receptor
*transmits signal across membrane
transmembrane glycoprotein with 3 sub-regions, one projects from cell surface & binds EGF (extracellular), one spans across lipid bilayer, one projects into cytoplasm & has kinase activity (cytoplasmic portion activation) - attaches PO4 groups to tyrosine in itself & other proteins, kinase domain hyperactive from mutations present in cancers (excess cell growth), enzymatic activity built in
External positive signals ex. EGF to internal consequences
ligand binding & dimer formation (2 receptors) - EGF binds extracellularly, activation of receptor kinase & self-phosphorylation at cytoplasmic tail (self and trans), cytoplasmic proteins associated w/ receptor are phosphorylated, intracellular kinase activated & phosphorylate other cytoplasmic proteins (signal cascade), move to nucleus & cause transcription of genes encoding cell cycle promoting proteins cyclins & Cdk’s once phosphorylated, multiple proteins activated due to increase levels of cyclin/Cdk activity
EGF increases CDK expression
internal positive signals - phosphorylation during signal cascade post EGF-R leads to transcription of cell cycle-promoting genes, cyclin - regulatory subunit amounts increase and decrease during cell cycle, Cdk - catalytic subunit phosphorylates proteins; possible cancer drug target (cyclin dependent kinases), MPF’s - mitosis promoting factors: combine activity of cyclins & Cdk promotes G2 to M, short cyclin protein half-life leads to its degradation (and inactive), Cdk is active part to phosphorylate (only active with cyclin)
*protein functional activation via phosphorylation
Cancer therapeutics of Cdk
inhibitors of cyclin-dependent kinases as cancer therapeutics, easier to target (even if bound to cyclin), stop mitosis of cell, selectively target one CDK (many different CDKs), did not succeed in clinical trials, important + legit target but nothing successful
External negative signals
myostatin (muscle stopping) - just one example of many factors for inhibitor, organism evidence - myo-/- mice leads to more muscle mass and human mutation then increased muscle (2 myostatin mutations resulting in non-functional myostatin regulation), myostatin gene cut out of genome then much larger and more muscular due to overgrowth, petri dish evidence - protein secreted into liquid from non-muscle cells stops replication of muscle cells (evidence that stopping factor is secreted from other cells)
Myostatin mechanism - external negative signals
myostatin present = normal tissue size (think of balance of + & - signals), myostatin absent = excess tissue growth (imbalance due to loss of -)
External negative signals - consequences
immediate consequences
- myostatin binding (secreted) to receptor dimerization
- recruits transmembrane ALK (kinase) not on receptor
- ALK phosphorylates Smad
- Phospho-Smad to nucleus (only phosphorylated)
- binds promoter and increased transcription of CC inhibitors p21 & p53
downstream consequences
- p21 binds to & inactivates CDK (CDK promotes cell cycle)
- p53 binds DNA & slows/stops DNA replication
Do you ever want to block myostatin?
- increase muscle growth - muscular “dystrophy” diseases
- muscle wounds/tissue void
p27, p21, p53 nomenclature
protein + molecular weight = p(number)
Internal negative signals: quantitative & qualitative effects
p27 and p21 - bind & inactivate cyclins, block entry to S phase, frequently mutated in cancers to no brake: excess cycling
p53 - blocks cell cycle if DNA is damaged (DNA binding protein), binds DNA slowing topoisomerase progress along helix; overall DNA replication slowed, more time for correction of mutated DNA bases by “proofreading” function of DNA polymerase
Loss of p53 protein function
gene mutation to inactive p53 protein (cancer), HPV protein degrades p53 protein in infected cervical cells (cancer), no p53 to no stopping cell cycle + mutations
*excess cell growth contributing to cancer
Cell cycle checkpoints
cell cycle checkpoints integrate multiple criteria, as opposed to individual positive or negative control proteins, stopping points, G1, G2 and metaphase checkpoint
G1 checkpoint
pass checkpoint if: (normal cell conditions?), growth factors (external positive signals) present, adequate cell size (sufficient components to distribute to daughter cells), nutrients available
*then: cell move through checkpoint
- differing length of G1 (G0)
G2 checkpoint
pass checkpoint if:
- adequate cell size
- chromosome replication is complete (ensure no loss of genes to daughter cells) - during S phase (DNA synthesis)
Metaphase checkpoint
pass this checkpoint if:
- all chromosomes are attached to functional mitotic spindle
- if not, non-equal distribution to daughter cells
- ultimately leads to 2 daughter cells
Cell cycle control - benefits to organism
increase efficient use of nutrient/energy resources (especially at G1)
- cell replication is huge metabolic commitment (not continuing if not successful)
- energy use, synthesis of protein, DNA
- nutrients, energy supply use limiting
arrested cell cycle if DNA is damanged
- damage may = mutation or missing chromosomes (p53 binding arrest cell cycle)
- cell cycle checkpoints are activated by mutagens (to stop cell cycle)
- UV light, radiation, free radicals (peroxisomes and OH)
- ensure integrity of genome (especially at G2 & metaphase)
- division is delayed until DNA is repaired or division is completely halted preventing passage of errors to next cell generation
Discovery of apoptosis
- 1972
- nuclear & cytoplasmic condensation
- breaking up of cell into membrane-bound fragments
- participates in (normal) cell turnover & therapeutically induced tumor regression (chemotherapy)
- “p” is silent
*understand this morphology & correlation info to understand therapy effect
Apoptosis meaning
- from greek meaning “falling off”, as leaves from a tree
- a process of controlled death; aka programmed cell death (enzymatically driven)
- participates in normal cell removal and therapeutically induced tumor regression
Tissue homeostasis
- number of cells is tightly regulated by controlling cell division and cell death
- pharmacologically controlling program might restore balance
imbalance - new cells = cancer (hyperplastic growth) - would want to promote apoptosis
- cell death = stroke (oxygen deprivation) = prevent apoptosis and rescue tissue
Apoptosis examples
fetal limb development, developing nervous system, adult liver (anti-convulsant drug consequences), phenobarbital
Fetal limb development
tissue between fingers & toes starts out webbed, individual digits separate as cells between them die
Developing nervous system
matching number of nerve cells to number of target cells requiring innervation, unmatched nerve cells die
Phenobarbital
stimulates liver cell replication (side effect - normally extended G0), stop phenobarbital then cells die off until liver returns original size (normal balance via apoptosis)
Necrosis
death from external injury, cells swell, burst & empty contents into surrounding area (spill) - attractant for WBC + secrete pro-inflammatory signals, loss of membrane integrity, typically induces inflammation via WBC
Apoptosis
death from internal process but started by an external signal, caspase enzymes are activated - breakdown everything, nuclear & cytoplasmic cytoskeleton collapses (condensed nuclear material), nuclear DNA is digested, actin under cell membrane degraded (no more structure), membrane shed as intact “blebs”, blebs endocytosed by macrophages (neighboring cells), limits/prevents inflammation
Apoptosis - modeled in petri dish
retraction, blebbing for several hours, 1st to 2nd cell retraction, about 30 min, secondary necrosis about 9hrs, large balloon-like swellings, cells lose cell membrane integrity & release contents to surroundings, secondary necrosis prevented in vivo by phagocytosis - no surrounding cells in the environment because in the lab
*because experimental setting (chemical induced apoptosis) so no surrounding cells to consume
Apoptosis pathways - activation signals
*both triggered by extrinsic/external signal
intrinsic pathway
- consequences start inside cell
- radiation/chemotherapy
extrinsic pathway
- target outside of cell (transmembrane protein)
- receptor for extracellular signal
- Fas/CD95 TNF-R1
*branch, continue, and move to other pathway - enzyme(s) of initiators (upstream) and effectors (downstream)
Apoptosis enzymes
*caspases
- a family of cysteine aspartic acid-specific proteases (protein degrading enzymes)
- highly specific for what proteins are degraded ex. actin, organelles, translation apparatus
- cleave protein after recognizing a 4 amino acid repeat in substrate protein (like actin) - cysteine and aspartic acid
- normally present in healthy cells as inactive precursor enzymes (zymogens) with little or no protease activity
Caspase family
- about 14 members; 2 major groups
*present as zymogens and different activations (sequential activation)
initiators - able to auto-activate (enzymatic) & initiate proteolytic processing of other caspases - via some upstream signal
- important in controlling start of process (earlier –> similar to checkpoint)
effectors - activated by upstream initiator caspases (sought out)
- important in carrying out majority of substrate digestion during apoptosis (later)
*2-step process from initiator & effector caspases - keeps tight control over apoptosis
Extrinsic: receptor-mediated apoptosis
peptide binds receptor
- peptide secreted from neighboring damaged cells
- receptor shape change signals initiator caspase auto-activation (internal consequences) -conformation change is sufficient signal
“fork” in pathway
- caspase 8 (initiator) auto-activation, then activation of downstream effector caspases and other proteins
- cleavage of BID (proteolytic cleavage) so intrinsic pathway
- BID truncated to tBID
- tBID has similar effect as Bax; disrupts mitochondrial membrane integrity
- insertion triggers apoptotic signal in mitochondria
Intrinsic: mitochondria pathway
*fork in road or chemotherapy drug
- signals target mitochondria
- certain apoptosis signals (radiation, drugs) get into cell & cause Bax and/or tBID insertion into mitochondria membrane (proportion)
- mitochondria membrane disrupted leading to release of several proteins & Ca++ (stored in mitochondria)
- Ca++ activates initiator caspase (caspase 9)
- continued degradation of nuclear and cytoplasmic structural proteins
- initiator caspase activation of effector enzymes leads to apoptosis
Apoptosis pathways - integrate signals & consequences
- effector caspases so proteolytic breakdown
- converted from zymogen to active form by initiator caspases
Enzyme cascade (post initiator caspases)
- effector caspases digest inhibitors of DNAase (ICAD) - prevents DNA degradation
- DNAse (CADs) move to nucleus; digest DNA between nucleosomes (DNA backbone)
- effector caspases move to nucleus & digest lamins (nuclear lamins are IF) - nuclear bleb (compromises shape of nucleus and bulge due to lamin digestion)
- effectors digest microfilaments - cell-substrate retraction and no stress fibers
- effectors digest adhesion plaque proteins
- effectors digest translation factors - promote translation (auxillary) *cessation of protein synthesis
Blc- & BAX-targeting drugs
- Blc stop apoptosis
- BAX drive apoptosis (cancer)
*clinical promise
