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PHAR 503 > 2. Drug-Target Characterization > Flashcards

2. Drug-Target Characterization Flashcards

1
Q

why can we not determine affinity of a drug in a cell culture assay?

A

too much stuff in cell –> not isolated system

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2
Q

why do we express protein in bacteria? how do we express protein in bacteria (3 steps)?

A

to allow for protein purification

  1. cDNA encoding for protein of interest is ligated into a plasmid for bacterial expression
  2. harvest bacteria
  3. lyse cells then extract protein
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3
Q

what is the main way to extract protein?

A

chromatography

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4
Q

what are the 2 phases of chromatography?

A
  1. stationary phase –> inert or active solid phase
  2. mobile phase –> bacterial lysate and buffer
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5
Q

what is the benefit for chromatography that is automated?

A

allows for a standardized method so purification is always the same

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6
Q

what are 2 types of chromatography?

A
  1. size exclusion chromatography
  2. affinity chromatography
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7
Q

why are beads cross-linked in chromatography?

A

so they don’t react with anything

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8
Q

what is unique about beads in size exclusion chromatography?

A

they have pores

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9
Q

describe size exclusion chromatography

A

buffer with bacterial lysate will constantly flow thru
- LARGE molecules will not enter the pores and are eluted first
- SMALL molecules will enter the pores and will be eluted last –> will have to cross bigger volume to eventually get eluted

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10
Q

in general, how are proteins separated in affinity chromatography?

A

separated by biophysical traits

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11
Q

what are 4 examples of ways we can purify proteins with affinity chromatography?

A
  1. hydrophobicity
  2. ligands
  3. metals (some proteins bind metal)
  4. protein tags
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12
Q

what are 3 examples of ligands that can be used in affinity chromatography?

A
  1. lectins
  2. protein A/G
  3. collagen
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13
Q

how do we purify using protein tags?

A

doesn’t purify by endogenous property –> protein is tagged with something that can bind to another molecule attached to the beads

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14
Q

what are 4 examples of protein tags used in affinity chromatography?

A
  1. histidine binds Ni-NTA-beads
  2. GST binds glutathione-sepharose/agarose-beads
  3. MBP binds amylose resin-beads
  4. myc, flag, V5 binds antibodies against the tags
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15
Q

what is it called when we use antibodies in affinity chromatography?

A

immunoprecipitation

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16
Q

what is the general idea for the radioligand competition assay?

A

drug is radiolabeled (1 atom replaced by isotope) and added to purified protein target

filter this solution –> radioligand bound to protein is too big to cross the filter so it will stick to the filter paper and can measure radioactivity

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17
Q

describe the 4 steps of the radioligand competition assay

A
  1. start with only radioligand –> radioligand will saturate all purified protein target
  2. then add normal ligand in increasing concentrations
  3. normal ligand will compete and replace the radioligand so radioactivity on the filter paper will decrease
  4. continue until no more radioligand
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18
Q

how do we analyze the radioligand competition assay? describe the graph

what value does the curve give us?

A

look at changes in inhibition curves

Y = amount of radioligand bound
X = concentration of normal ligand

curve starts with high radioactivity (binding site saturated with radioligand), then decreases with increasing doses of normal ligand

gives us IC50 –> concentration of normal ligand that displaces 50% of the radioactive ligand –> tells us affinity and proves specificity

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19
Q

what is an alternate way of using the radioligand competition assay? explain

A

can see if 2 different drugs bind at the same or different binding sites

1 drug is radiolabeled, 1 drug is not

  • if both drugs bind at the same site –> reduced radioactivity bc competition
  • if each drug binds at a different site –> radioactivity is maintained bc no competition
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20
Q

describe how elution is also a competition

A

if we have a drug bound to a GST-tagged protein for example: it will bind glutathione-beads

if we add free glutathione, it will compete with the beads to bind to the protein, allowing us to elute the protein

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21
Q

describe the instrument used in surface plasmon resonance

A

instrument has flow chamber that sits above the gold part of a plastic chip –> the flow chamber has a volume of 2nL

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22
Q

why is the volume in the flow chamber important?

A

its only 2nL so it’s helpful when you have very little drug

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23
Q

describe the gold chip in surface plasmon resonance

A

functional groups (ex. disfulfide) stick off the gold part, allowing protein of interest to be COVALENTLY bound and immobilized on the surface

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24
Q

what happens to high affinity drugs in buffer solution flows thru the chamber?

A

high affinity drugs have greater intermolecular forces with the receptor, so they will bind

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25
what happens to low affinity drugs in buffer solution flows thru the chamber?
low affinity drugs have less intermolecular forces with the receptor so they will bind less well
26
what is the principle of surface plasmon resonance? (describe the light) 3 steps
1. infrared light strikes the gold at an angle of total reflection (fully reflected out) 2. gold layer becomes activated via EVANESCENT WAVE and some energy from the light is absorbed 3. decrease in light intensity causes a shadow at a certain angle
27
what are the 4 steps of surface plasmon resonance?
1. drug flows over gold surface and binds to protein 2. this changes the mass of what is bound to the gold layer 3. the evanescent wave depends on the mass of what's on the gold surface so the change in mass changes the angle of the shadow 4. the angle change can be measured and plotted using a sensorgram
28
describe the sensorgram curve (3 time points)
t=1: buffer with drug flows thru t=2: association curve as drug binds to protein, changing the shadow angle until equilibrium t=3: stop buffer flow causing dissociation curve
29
why can drugs associate AND dissociate?
binding is not permanent!!!!
30
what 4 things does SPR let us measure?
1/2. association and dissociation rates over time 3/4. association and dissociation equilibrium constants
31
what is Ka? what does low kA mean? what is the equation and units?
Ka = association tendency low Ka = low affinity
32
what is Kd? what does low kd mean? what is the equation and units?
Kd = dissociation tendency low Kd = high affinity
33
what does it mean when a drug is given at a concentration equal to Kd?
50% of the receptors are occupied
34
difference btwn competition assay and SPR
competition assay gives us IC50 and proves specificity SPR gives us affinity
35
what is Isothermal Titration Colorimetry?
like SPR but protein and drug are free-floating in water the energy released after evanescence wave causes the water to warm up --> can measure drug binding based on water temp may be better than SPR
36
what does NMR measure?
binding
37
what does ELISA measure? what is its downside?
competition and binding assay but less accurate
38
what is an assay?
way to find a measurement
39
what are 2 benefits of kits vs 1 downside?
kits allow science to be faster and more flexible but they are more expensive
40
what is a protocol?
stepwise instructions
41
describe off-target effects
if compound is added to cells, may get most binding to the target but some can bind elsewhere and cause problems (unlikely to fully mute the therapeutic effect) usually has lower affinity
42
what do we use to identify off-target effects?
TRIFUNCTIONAL COMPOUNDS
43
what is a trifunctional compound?
drug fused to: 1. moiety with AZIDE group (3 N's) which is linked to: 2. BIOTIN
44
what is the role of the azide group?
when you shine UV light, it will cross-link to nearby protein --> therefore we stabilize the low affinity off-target effect to allow us to later identify it
45
what is the role of biotin?
binds streptavidin beads very strongly (10^-14 M --> nearly covalent) so we can purify the stabilized off-target effect
46
how do we purify the off-target protein?
biotin binds to streptavidin beads very strongly so we can wash very harshly with high salt buffers, strong detergents, etc. to remove other interactions
47
what do we do once the off-target protein has been purified?
identify the protein with MS
48
before MS, what do we do to the protein? how does this work? how does this let us identify the protein?
digest protein using TRYPSIN --> cleaves only after LYSINE and ARGININE residues which is approximately every 10 amino acids and is a good size for MS every protein that is digested gives a specific MS fingerprint that is unique and identifiable --> we know the whole genome so we know all the proteins
49
what are the 3 parts of MS?
1. ion source --> ionization lets them fly 2. mass analyzer --> ion separation 3. detector --> amplifies/multiplies electrons for ion detection
50
what is fingerprint analysis?
1. generate list with exact peptide masses measured in the spectrum 2. compare list to database of "in silico" cleavage of known proteins 3. can determine which protein you have
51
once you identify the protein, how do you validate/sequence it?
collide peptides with air so they randomly break at their different peptide bonds, giving each peptide - then using MS, will see Y ion (C-terminal ion) peaks and the distance between each peaks = an amino acid
52
what is a fluorophore?
molecule that absorbs light of LOW wavelength and emits light of HIGH wavelength
53
besides wavelength, how does excitation light differ from emission light?
they are perpendicular
54
what does the Stoke's shift explain?
explains why emission is always at higher wavelength than excitation
55
describe the Stoke's shift (4 steps)
1. fluorophore is in the ground state 2. add photon to excite the fluorophore --> energy increases 3. fluorophore stays in excited state for some time, losing some energy via non-radiative transition/internal conversion 4. then emits photon at ground state
56
why is there higher wavelength for emission than excitation?
excitation light has lower wavelength but higher energy, then some energy is lost due to non-radiative transition/internal conversion so light is emitted at higher wavelength
57
what principle explains why excitation and emission peaks are mirror images?
FRANCK-CONDON PRINCIPLE
58
what is the franck-condon principle?
electrons can only move certain ways --> during the movement from ground to excited state, the electrons move when the waves overlap the most the way you excite electrons is the opposite the way electrons emit
59
what causes aberrations in absorption and emission spectra?
interactions with solvent molecules
60
why do we measure fluorescence in a black plate?
photons can go in all directions --> if plate was clear, would not get accurate results
61
what does FRET stand for?
Fluorescence Resonance Energy Transfer
62
what is the principle of FRET?
mix 2 specific fluorophores photons emitted from an excited fluorophore are not emitted via internal conversion/radiation, the photons excite another fluorophore within 10nm
63
what are the 2 types of fluorophores used in FRET
1. DONOR --> lower wavelength, excited first 2. ACCEPTOR --> higher wavelength, excited by donor
64
give an example of FRET used to inhibit interaction of kinase with substrate
tag kinase with CFP (donor) and substrate with YFP (acceptor) - if kinase is interacting with substrate, they are within 10nm of each other and will see YFP fluorescence - if kinase is not interacting with substrate, the drug is successful and will only see CFP fluorescence
65
what does BRET stand for?
Bioluminescence Energy Transfer
66
describe BRET
substrate undergoes reaction with O2 to be oxidized and release light --> this light undergoes FRET to excite an acceptor fluorophore that is in close proximity at 490nm
67
why don't we need an input of light for BRET?
biochemical reaction produces light (bioluminescence)
68
why is BRET more simple than FRET?
can just add the substrate to wells, don't have to add inside wells like with FRET
69
what is quenching used to study?
enzymatic activity
70
how does quenching work?
fluorophore is linked to quencher --> if fluorophore is excited, energy is not released as a photon and is instead absorbed by quencher so no light is produced
71
give an example of quenching for a cleaving molecule
- if enzyme is inactive and doesn't cleave --> no fluorescence bc quencher absorbs the energy - if enzyme is active and cleave --> there is fluorescence bc energy can be emitted as a photon
72
describe fluorophore dipole
fluorophores have DIPOLES (aka transition moment) that depend on the structure
73
describe the fluorophore dipole when light is added
if you add POLARIZED LIGHT (light with 1 plane of oscillation), only fluorophores whose dipole aligns with the polarized light will be excited
74
what happens to polarized light when a fluorophore is excited?
when the fluorophore is in its excited state, the fluorophore moves so not all emitted light is polarized anymore, causing DEPOLARIZED EMISSION
75
describe ANISOTROPY
anisotropy describes how much emitted light remains polarized which indicates how fast the protein moves which indicates what is interacting with the protein
76
what are the 3 possible movements of a fluorophore bound to protein?
1. rotational diffusion of fluorphore 2. mobility of protein segment 3. rotation of whole protein
77
what do we measure in anisotropy?
measure how much polarized light comes out
78
what happens if we see high amount of polarized light?
fluorophore is bound to protein so it is moving slowly
79
what happens if we see low amount of polarized light?
fluorophore is free floating so it can move fast and rotate less polarized light = light emitted in all directions

PHAR 503 (10 decks)

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