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lecture II: drug-target characterizations Flashcards

1
Q

What is needed to experimentally determine the affinity of a drug on a target?

A
  1. Pure drug
  2. Pure target
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2
Q

How can a pure target be obtained?

A

Protein purification

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3
Q

Protein purification methods

A
  1. Protein expression in bacteria
  2. Chromatography
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4
Q

Protein expression in bacteria steps

A
  1. cDNA encoding for protein of interest ligated into plasmid/vector for bacterial expression
  2. Transform bacteria
  3. Grow over night
  4. Harvest bacteria
  5. Cell lysis
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5
Q

Which method often uses protein expressed in bacteria?

A

Crystallography

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6
Q

Crystallography

A

Crystallography is the experimental science of determining the arrangement of atoms in crystalline solids.

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7
Q

Chromatography

A

Chromatography is a process for separating components of a mixture. To get the process started, the mixture is dissolved in a substance called the mobile phase, which carries it through a second substance called the stationary phase.

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8
Q

FPLC

A

Fast protein liquid chromatography

A typical laboratory FPLC consist of one or two high-precision pumps, a control unit, a column, a detection system and a fraction collector.

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9
Q

Types of chromatography

A
  1. Size exclusion
  2. Affinity
  3. Ion exchange
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10
Q

Radioligand competition assay

A
  • competitive binding assay
  • measures affinity
  • method: A known antagonist (or ligand) for the target receptor is labelled with radioactivity and is added to cells or tissue such that it can bind to the receptors present. Once an equilibrium has been reached, the unbound ligands are removed by washing, filtration, or centrifugation. The extent of binding can then be measured by detecting the amount of radioactivity present in the cells or tissue, and the amount of radioactivity that was removed.
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11
Q

Radioligand competition assay evaluation

A

log([competing ligand]) vs. amount of radioligand bound

→gives IC50

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12
Q

IC50

A

Inhibitory concentration 50

Concentration at which the competing ligand displaces 50% of the radioligand.

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13
Q

Identification of same or different binding sites

A

Radiolabeled drug with known binding site (drug A) and addition of other drugs with unknown binding sites.

→ if the drug competes with drug A, then it has the same binding site
→ if the drug does not compete with drug A, then it has a different binding site

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14
Q

Elution

A

Elution is the process of extracting one material from another by washing with a solvent; as in washing of loaded ion-exchange resins to remove captured ions.

→ essentially, a type of competition

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15
Q

Size exclusion chromatography

A

Size exclusion chromatography separates molecules based on their size by filtration through a gel. The gel consists of spherical beads containing pores of a specific size distribution. Separation occurs when molecules of different sizes are included or excluded from the pores within the matrix.

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16
Q

Size exclusion chromatography elution order

A
  • constant buffer flow & porous material
  1. Large molecules excluded from pores → elute first
  2. Small molecules diffuse in pores → elute later
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17
Q

Gel filtration: 205 nm absorbance

A

Peptide bond

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18
Q

Gel filtration: 280 nm absorbance

A

Tryptophan

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19
Q

Affinity chromatography

A

To separate recombinant protein from other proteins by its biophysical traits.

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20
Q

Affinity chromatography: different traits

A
  1. Hydrophobicity
  2. Ligands
    →lectins, protein A/G, collagen
  3. Metals
    →IMAC
  4. Protein tags
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21
Q

Protein tag examples

A
  1. His ∞ Ni-NTA
    →6 His in a row likes to bind to Ni
  2. GST ∞ glutathion-sepharose
  3. MBP ∞ amylose resin
    →larger protein tag, but it may help POI to remain in solution when in bacteria
  4. immunoprecipitation: myc, flag ∞ antibodies
    →from myc pro-oncogene expressed in our cells
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22
Q

SPR

A

Surface plasmon resonance

Surface plasmon resonance is a biophysical phenomenon happening when light strikes a gold layer at the interface of two media (glass and buffer) at the angle of total internal reflection

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23
Q

SPR: Total angle of internal reflection

A

Angle of light at which total reflection occurs - light does not enter other media anymore

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24
Q

SPR steps

A
  • POI is immobilized on surface
  • Drug passes over chip surface in buffer solution

→when molecules/drugs are injected with buffer and bind to chip, it results in a change of mass on chip surface, subsequent change in diffractive index between media, which is a proportional change in diffraction angle

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25
Evanescent wave
Evanescent waves are formed when sinusoidal waves are (internally) reflected off an interface at an angle greater than the critical angle so that total internal reflection occurs. →the size of the evanescent wave changes as drug binds to protein on the chip, therefore the shadow/angle changes as well (proportional to mass change)
26
SPR: light angle
Light is shone at the total reflection angle
27
SPR: gold
Gold layer absorbs energy, resulting in decreased light intensity at a certain angle
28
SPR sensorgram
Measures the resonance signal, and thus affinity → time vs. resonance signal
29
Resonance signal
Change in angle of “shadow” in reflected light →dependent on the mass found on the chip, which is then reflected in the shadow angle
30
SPR sensorgram phases
1. Buffer flow *injection of drug* 2. Association of drug with POI till equilibrium →on rate only 3. Equilibrium point *wash off bound drug (buffer flow only)* 4. Dissociation of drug →off rate only
31
KA
Equilibrium constant KA describes association tendency
32
KA equation
KA = [LR] / ([L][R]) →low KA = low affinity →unit: M^-1
33
KD
Equilibrium constant KD describes dissociation tendency →When drug is given at a concentration equal to its dissociation constant KD, 50% of the receptors will be occupied.
34
KD equation
KD = ([L][R]) / [LR] →low KD = high affinity →unit: M
35
How to test specificity of drug binding?
Use the concept of “competition” to show specificity →using SPR, inject drug+buffer, then inject buffer only as a control
36
Target identification method
Use of trifunctional capture compounds, where you modify the drug to fish for protein-interaction partners
37
Trifunctional capture compound components
1. Capture compound →drug of interest 2. Reactive azide group →forms a covalent bond upon UV light 3. Biotin →for purification
38
Avidin-Biotin
Naturally occurring strong interaction between avidin and biotin. →interaction: KD ≈ 10^-14 →synthetic avidin will be used and bind to the biotin moiety will very high affinity, almost ensuring that our protein will be pulled out
39
Biotin
Essential cofactor for a lot of enzymes.
40
Avidin
Functions as an antibiotic in the eggs of birds, reptiles and amphibians.
41
Target identification: trifunctional capture compound steps
1. Add trifunctional compound to cells/tissue/mice... 2. Crosslink compound to proteins by UV light (like a shot of the moment where it is bound) 3. Purify trifunctional compound using biotin-streptavidin 4. Protein digestion 5. Perform mass spectrometry to identify proteins that were co-purified and thus qualify as potential drug-binding partners
42
Protein digestion
Typically done trypsin (protease), which only specifically cleaves after Lysine (K) or Arginine (R)
43
Peptide mass fingerprint
Each protein has its individual unique pattern of tryptic peptides.
44
Mass spectrometry
Mass spectrometry is an analytical tool useful for measuring the mass-to-charge ratio (m/z) of one or more molecules present in a sample. → m/z vs. intensity
45
Mass spectrometry: functional compounds
1. Ion source →ionization 2. Mass analyzer →ion separation 3. Detector →ion detection
46
Ionization methods
1. Electrospray ionization 2. Matrix-assisted laser desorption ionization
47
Mass analyzer methods
1. Time-of-flight (TOF) 2. Quadrupole 3. Ion trap
48
Detector methods
1. Electron multiplier
49
Fingerprint analysis
1. Generate list with exact peptide masses measured in spectrum 2. Compare list to database →“in silico” cleavage of known proteins in a certain species
50
Peptide validation by MS
- Collision of peptides with other molecules (air) will break peptide bonds →N-terminal peptide fragment & C-terminal peptide fragment - Distance between two peaks usually mass of one amino acid
51
Florescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence.
52
Fluorophore
Molecules that absorb light, and emit light at longer wavelengths after a certain time.
53
Two features of fluorophores
1. Emission always at a higher wavelength 2. Always mirror-imaged emission/excitation peaks
54
Fluorescence measurement
Perpendicular to the light source
55
Absorption measurement
Parallel to the light source
56
Jablonski diagram
Diagram that illustrates the electronic states of a molecule and the transitions between them. →Lengths of arrows correlate with energy – emitted photon has less energy than absorbed photon
57
Short wavelength, _____ energy
high
58
Long wavelength, _____ energy
low
59
Stoke's shift
The difference (in energy, wavenumber or frequency units) between positions of the band maxima of the absorption and emission spectra of the same electronic transition. →explains why emission is always at a higher wavelength
60
Non-radiative transition / Internal conversion
Solvents take energy. →photons like to be excited before they drop, causing for a loss in energy →may occur with interaction with water
61
Fluorophore fluorescence lifetime
Time spent in the excited state →E will be lost after a while
62
Franck-Condon principle
During the transfer from the ground state to the excited state, it is most likely that transfer happens between the best overlapping vibrational wave functions. →discrete positions for excitation and emission →explains why there are always mirror-imaged excitation/emission peaks
63
What may hinder ideal fluorescence spectra shape?
- dipole moments - interactions with solvents
64
Visible light spectrum
380 (violet) - 740 (red)
65
Causes of radiative relaxation
1. Internal conversion 2. Quenching 3. Chemical reactions
66
How can you measure fluorescence?
96-well plate reader for absorption, fluorescence, (luminescence) →use of excitation/emission filters to measure fluorescence
67
When is a black 96-well plate used?
For fluorescent assays. →black plates absorb light and reduce background and crosstalk
68
When is a white 96-well plate used?
For luminescent assays. →white plates reflect light and will maximize light output signal
69
Applications for fluorescence
1. Protein-protein interactions 2. Quenching 3. Mobility of proteins 4. Processes at the plasma membrane 5. High resolution microscopy 6. Fluorescence anisotropy
70
Applications for fluorescence: Protein-protein interactions methods
1. FRET 2. BRET 3. Split-YFP system
71
FRET
Fluorescence Resonance Energy Transfer. →when emission spectrum of donor overlaps with excitation spectrum of acceptor, FRET will occur in close proximity of fluorophors (less than 10 nm) →radiation of the donor will be transferred to the acceptor →shine excitation light of donor and, if the two proteins are in close proximity, the emission of the acceptor will be the output →use as a ruler to see how close proteins in sample are
72
BRET
Bioluminescence energy transfer. →exploits the naturally occurring phenomenon of dipole-dipole energy transfer from a donor enzyme (luciferase) to an acceptor fluorophore following enzyme-mediated oxidation of a substrate →light emission through luciferases →substrate luminol undergoes reaction with oxygen to release light →donor fluorophore is not fluorophore, but rather a luminescent substrate →when emitted light is in close proximity to a fluorophor that is being excited at 490 nm
73
Quenching
Gain of fluorescence after enzymatic activity. →quencher is not a fluorophore (it accepts light, but doesn't do anything with it) →excite fluorophore and a large proportion of its E will be transferred to quencher, which will not do anything with that light, but then post-reaction you get fluorescence if the protease completed its function
74
Applications of fluorescence: mobility of proteins method
Fluorescence recovery after photobleaching (FRAP)
75
Applications of fluorescence: processes at the plasma membrane method
Total internal reflection (TIRF)
76
Applications of fluorescence: high resolution microscopy method
Stimulated emission (STED microscopy)
77
Fluorescence anisotropy
A measurement of how a molecule changes its orientation in space, with respect to the time between absorption and emission events. →describes how much emitted light is still polarized →indicator of how fast the protein moves with/without inhibitor →depends on the direction of the fluorophore's dipole
78
Photoselection
Only the fluorophores that align to the orientation of the polarized light will get (best) excited by polarized light.
79
Polarization filter with vertical passage
Allows unpolarized light (all directions) to be linear polarized light with only one plane of oscillation.
80
Explain theory behind anisotropy.
See slides 57, 59, 61
81
Possible movements of fluorophore
1. Rotational diffusion of the fluorophore 2. Mobility of protein segment 3. Rotation of the whole protein
82
Anisotropy measurement: slow fluorophore movement
- polarized light emitted (parallel) - bound to something - high affinity
83
Anisotropy measurement: fast fluorophore movement
- depolarized light emitted (in all directions, more perpendicular) - unbound - low affinity
84
Applications of anisotropy
1. Compound screening assays 2. Fluorophore label at selected positions in target – monitor mobility 3. Use of tryptophan residues (intrinsically fluorescent) →often used to measure binding affinity
85
Advantage of anisotropy
It is a ratio measurement, thus values are intrinsically normalized

PHAR 503 (6 decks)

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