ITC and SPR (biophysical methods) Flashcards
what does ITC and SPR stand for
ITC=isothermal titration calorimetry
SPR=surface plasmon resonance
docking, posing, scoring accuracy considerations
-accuracy of molecular model for protein and ligands
-completeness of conformational sampling
-accuracy of energy calculation
-can be very low hit rate ~1-5%
ITC uses in drug discovery
characterisation of biomolecular interactions
-confirms binding and activity, determines stoichiometry and thermodynamic parameters, complements structural biology and structure-activity relationships
how do pharmaceutical companies estimate values of ΔH and ΔS?
-in silico using computer graphics/molecular modelling/docking for ΔH only
-in vitro using calorimetry for both ΔH and ΔS
describe primary enthalpic contributions (ΔH)
-hydrogen bonding, VDW interactions
-negative ΔH means enthalpy change favours binding
-solvent plays a role
describe primary entropic contributions (ΔS)
-hydrophobic effect=water release=favourable
-conformational changes and reduction in degrees of freedom=unfavourable
-positive ΔS for entropically driven reactions
how does ITC work/what is DP
DP=differential power=measured power differential between reference and sample cells to maintain a zero temperature between the cells
in 1 ITC experiment you obtain…
-affinity (KD)=strength of binding
-heat of binding (ΔH) and entropy=mechanism and driving force of interaction
-stoichiometry (n)
how to calculate entropy of binding and ΔG
ΔG=-RTln(1/KD)
ΔG=ΔH-TΔS
explain all the things in these equations:
ΔG=-RTln(1/KD)
ΔG=ΔH-TΔS
ΔH=enthalpy=indication of changes in hydrogen and VDW bonding
-TΔS=indication of changes in hydrophobic interactions and/or conformational changes
N=stoichiometry=indicates ratio of ligand to macromolecule binding
ΔG=gibbs free energy
R=gas constant
T=temperature in kelvin
KD=equilibrium dissociation constant
ITC thermodynamic signatures used to gain insight on
mechanism of binding
network of interactions →intra/intermolecular →H bonds, VDW, electrostatic int, covalent int
describe the thermodynamics for a good drug
good drug which binds strongly=large and negative ΔG (bc ΔH is large and negative or ΔS is large and positive)
-these two factors work together/against each other
ITC can be used to determine what, what do those things show, what does it minimise, what can it be used to assess
-determines kd and ΔH and TΔS
-shows the chemistry behind success/failure of an optimisation strategy
-minimises number of xray structures required
-used to assess the success of medicinal chemistry strategies
what is BIAcore and how does it work
biomolecular interaction analysis
-one binding partner (ligand) immobilised on chip and analyte injected (microfluids)
-pc collects binding data in real time
what is the detection principle of BIAcore
SPR
draw the different phases of SPR
notes
how to calculate Kd using hill plot from graph of response vs concentration
Kd is ligand conc at 50% occupancy of receptor
why SPR experiments are good
-ligand immobilised on chip surface
-analyte free in solution
-analyte flowed over ligand immobilised surface
-multiple chips surface offererd and chemistries
-flow over chip surface is laminar, faster in centre of capillary
-label free technique
-ligand present on surface
-gives Kon and Koff
why are SPR experiments bad
-immobilisation might be difficult
-amine chemistry, lysine in active sight means alternative immobilisation is required
-ligand active site is prsented and not free in solution
-non specific binding
-difficult regeneration
-contamination is reoccuring problem
-cant work with non aqueous systems
-consumables are expensive
-crowding effects of matrix might have conformational effects on the ligand
compare ITC to SPR, in ITC…
molecules are free in solution, no surface immobilisation, no regeneration required, one experiment provides full picture of binding mechanism, accurate ΔH and ΔS and stoichiometry, gives info on molecular interactions without model assumptions. recoverable sample
SPR has medium throughput and ITC has low
ITC requires larger amount of sample
why is it ideal to use both ITC and SPR techniques
high confidence if both gives the same Kd
