Plasmon-waveguide resonance spectroscopy applied to three potential drug targets: cyclooxygenase-2, hepatitis C virus RNA polymerase and integrin alpha V beta 3.
Plasmon-waveguide resonance (PWR) spectroscopy has been used to study the interactions between ligands that correspond to inhibitors, activators or substrates and three integral membrane proteins representing potential drug targets; cyclooxygenases 1 and 2 (COX-1 and -2), integrin alphaVbeta3, and hepatitis C virus RNA polymerase. The proteins were incorporated into an egg phosphatidylcholine bilayer deposited onto the surface of the PWR resonator, and changes in the amplitude and position of the PWR spectra due to mass density increases and conformational transitions have been used to characterize the kinetics and binding affinities corresponding to these interactions. Although the partition of COX-2 into the bilayer was not as efficient as was the case with the other two proteins, sufficient protein could be incorporated to allow ligand binding to be observed. It was also possible to incorporate COX-1 into a lipid bilayer by adding a suspension of microsomal membrane fragments containing this enzyme to a preformed bilayer, and to observe binding of an inhibitory ligand. The interactions between integrin alphaVbeta3 and two ligands with different in vivo efficacies could be distinguished by both spectral measurements and binding kinetics. In the case of the RNA polymerase, the kinetics of PWR spectral changes upon adding a substrate solution to an enzyme-template complex were consistent with those obtained from direct measurements of enzymatic turnover. These experiments demonstrate the utility of PWR spectroscopy to provide novel information regarding drug interactions with membrane proteins in a lipid environment and to distinguish conformational changes induced by binding of various drug molecules.
Devanathan
Savitha
S
Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, AZ 85721, USA.
Walker
Mark C
MC
Salamon
Zdzislaw
Z
Tollin
Gordon
G
D051546:Cyclooxygenase 2; D012321:DNA-Directed RNA Polymerases / Q000032:analysis / Q000378:metabolism*; D016503:Drug Delivery Systems / Q000379:methods; D016174:Hepacivirus / Q000201:enzymology*; D039302:Integrin alphaVbeta3 / Q000032:analysis / Q000378:metabolism*; D011451:Prostaglandin-Endoperoxide Synthases / Q000032:analysis / Q000378:metabolism*; D020349:Surface Plasmon Resonance / Q000379:methods*
D039302:Integrin alphaVbeta3; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; D012321:DNA-Directed RNA Polymerases