Measuring the competitive binding of diazepam and ibuprofen tohuman serum albumin (HSA) using Chirascan™ CD Spectroscopy
Lindsay Cole and David Gregson
Applied Photophysics Ltd, Leatherhead, UK
In this study Chirascan™ CD spectroscopy was used to evaluate thebinding interactions of two well-characterised small moleculedrugs, ibuprofen and diazepam[1, 2], to the abundant plasmaprotein, human serum albumin (HSA).
Chirascan™ was used to study the interaction of these achiral drugswith HSA by following the circular dichroism induced in the drugsby the chiral environment of the protein binding sites. Induced CDis a sensitive, label-free method for observing drug-proteininteractions in free solution. The binding of diazepam needed atwo-exponential model to satisfactorily fit the data whilst itsdisplacement by ibuprofen was modelled using a single-exponentialmodel.
These studies show the value of using Chirascan™ to furtherunderstand the pharmacokinetics of drugs and drug interactions.
Many drug molecules bind reversibly to plasma proteins and oftencirculate in the body as this bound form with a small populationfree in solution. The binding of drug molecules to plasma proteinshas an impact on the pharmacokinetics of the drug and the changesin free plasma concentrations have significant bearing on thepharmacological activity. Drug and plasma protein binding valuesare also important for establishing potential drug safety marginsfor human exposure and for selecting the final dose range for humantrials.
Particular drugs, metabolites and other molecules have highaffinities for certain binding sites on plasma proteins and thesedifferent affinities for specific binding sites can result in adrug being displaced from the protein by another molecule. The*****plex interactions can significantly change the ADME profile of adrug and is also one of the mechanisms by which multi-druginteractions occur.
Monitoring changes in circular dichroism (CD) spectra of either theprotein or the drug ligand can be used to study the structuralchanges induced following interactions. Addition of a Stopped flowaccessory to the Chirascan™ instrument also enables the kinetics ofthe interaction to be studied.
Experimental conditions and instrument setup
A Chirascan™ CD-spectrometer fitted with a titration accessory wasused for all the equilibrium titration experiments. The HSA proteinwas kept at a concentration of 0.5mg/ml for all equilibriumexperiments, in a pH7 buffer (50mM Na3PO4). Protein/buffer solutionin a 1cm cuvette was titrated with a solution of protein/bufferwith a high concentration of the ligand, by automated removal andaddition of volumes. The cuvette
was stirred and allowed to equilibrate for 1 minute after additionsbefore spectra were recorded.
All stopped-flow CD kinetic data were collected on a Chirascan™fitted with the SF.3 stopped-flow accessory, and a 150Wxenon-mercury lamp as the light source. The monochromator bandpasswas 4nm. The cell pathlength was 2mm.
Diazepam binding to HSA
Diazepam was titrated into HSA in the concentration range of 0 to0.8mM in 20 concentration steps. Diazepam binding to HSA resultedin a change to the CD spectra in the near-UV that reflected theinteraction and corresponding structural change (Figure 1).
At 320nm (a region that has no contribution from protein CD) thereis significant CD induced by the interaction of the achiraldiazepam molecule with the chiral protein binding site. Themajority of the CD changes observed are due to the inducedchirality in the bound diazepam molecule.
Figure 1 Binding of diazepam to HSA monitored by changes in near-UVCD. Left: spectra of free (red) and diazepam bound (blue) HSA.Right: change in the near-UV CD with increasing concentration ofdiazepam.
Figure 2 The pre-steady state kinetics of binding of diazepam(0.5mM) to HSA (2.5mg/mL). Stopped-flow CD was measured using theSF.3 accessory for the Chirascan. Both wavelengths (320nm and260nm) could be fitted successfully with a simultaneous doubleexponential function, the fitted rate constants were k1= 19s-1 andk2 =0.72s-1.
Diazepam is known to bind to two binding sites on HSA[1, 2] andthis is supported by the complex nature of the model required todescribe the stopped-flow kinetics. A two exponential function wasneeded to satisfactorily model the reaction traces at 320nm and260nm (Figure 2).
Ibuprofen binding to HSA
Ibuprofen was titrated into HSA in the concentration range of 0 to1.4mM in 21 concentration steps. There was little observable changein the CD spectra upon binding ibuprofen suggesting no structuralchange (Figure 3).
Figure 3 Near UV CD spectra of ligand free (red) and ibuprofenbound (blue, 1.4mM ibuprofen) HSA. There is little change in CDspectra for the protein or the ligand when ibuprofen binds.
The absence of a change in the CD spectra does not mean there is nobinding of ibuprofen to the protein; it may just demonstrate thatthere is no significant change in the structure of the protein orthe ligand upon binding the molecule.
To further explore the ibuprofen binding to HSA and a possibleinteraction with diazepam, HSA with bound diazepam (0.5mM) wastitrated with ibuprofen from 0 to 1.5mM concentration. The changein CD signal (Figure 4) shows a change from the diazepam-boundspectrum, to the ibuprofen-bound spectrum shown in Figure 3, as thediazepam is replaced with ibuprofen.
Figure 4 Displacement of bound diazepam by ibuprofen on HSAmonitored by changes in near-UV CD. Left: diazepam-bound (0.5mM,blue) HAS and ibuprofen-bound (1.5mM, red) HSA after displacementof diazepam. Right: change in the near-UV CD spectrum of bounddiazepam with increasing concentration of ibuprofen.
These observations demonstrate that ibuprofen does bind to HSA andwill competitively displace the diazepam ligand from the protein.There is no induced CD change on ibuprofen binding to HSA, but thedisplacement of another ligand with an induced CD signal can beused to study the protein-ligand interaction of this molecule.
Pre-steady-state stopped-flow CD kinetics of the displacement ofdiazepam by ibuprofen were then measured and the displacement ofthe diazepam by ibuprofen could be modelled by a single-stepprocess (Figure 5).
Figure 5 The pre-steady-state kinetics of displacement of diazepam(0.5mM) bound HSA (2.5mg/mL) by ibuprofen (2mM). Stopped-flow CDmeasured using the SF.3 accessory for the Chirascan. Bothwavelengths (320 and 260nm) could be fitted with a singleexponential model, with an apparent rate constant of k= 0.5s-1.
The results show the binding of two well known achiral drugs,ibuprofen and diazepam, to HSA and demonstrate that ibuprofen willcompetitively displace diazepam in this system. The changes in CDspectra are caused by CD induced in diazepam by the chiralenvironment of the protein binding sites. Ibuprofen shows no suchinduced CD on binding and its interaction is monitored by the lossof induced CD as diazepam is displaced. Similarly, the CD inducedin diazepam on binding to HSA and lost on displacement by ibuprofenis used to follow the kinetics of the interactions.
These studies show the value of using Chirascan™ CD spectroscopy tofollow drug-protein interactions, even if the drug itself isachiral and has no native CD signal in its unbound state. In thisexample the interaction of two drugs, ibuprofen and diazepam, withHSA has been used to demonstrate the potential of the technique toimprove the understanding of drug interactions and theirpharmacokinetics.
For further reading see Zsila et al., a review paper outliningthe use of induced chirality observed by CD for protein bindingstudies.
 R. Brodersen, T. Sjodin and I Sjoholm J. Biol. Chem. 1977, 252,5067-5072.
 U. Kragh-Hansen Biolchem. J. 1983, 209, 135-142.
 F. Zsila, Z. Bikadi, I Fitos, M. Simonyi, Current drugDiscovery Technologies, 2004, 1, 133-153
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