Nature of interaction between basic fibroblast growth factor and the antiangiogenic drug 7,7-(Carbonyl-bis[imino-N-methyl-4, 2-pyrrolecarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino] )bis-(1, 3-naphthalene disulfonate).
PNU145156E (7,7-(carbonyl-bis[imino-N-methyl-4, 2-pyrrolecarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino]) -bis-(1, 3-naphthalene disulfonate)) is a naphthalene sulfonic distamycin A derivative that interacts with heparin-binding growth factors. Because PNU145156E inhibits tumor angiogenesis, it was selected for clinical development. Picosecond time-resolved fluorescence emission and anisotropy were used to characterize the binding of PNU145156E to the basic fibroblast growth factor (a protein associated with tumor angiogenesis). A decrease in PNU145156E fluorescence lifetime was observed as a function of human basic fibroblast growth factor (bFGF) concentration. Nonlinear least-squares fitting of the binding isotherm yielded Kd = 145 nM for a single class of binding sites. Time-resolved anisotropy gave Kd = 174 nM. Kd = 150 nM was independently verified by quantitative high-performance affinity chromatography. The displaced volume of the complex, calculated from its rotational correlation time, fitted a sphere of 1:1 stoichiometry. These results account for the formation of a tight yet reversible PNU145156E:bFGF complex. An evaluation of PNU145156E fluorescence lifetimes in various solvents has highlighted the forces involved in stabilizing the complex. These are mostly electrostatic-hydrophobic in nature, with a relatively low contribution from hydrogen bonding. Both polar and nonpolar groups are involved on the protein-binding site within a largely hydrophobic cleft. A potential binding trajectory, based on a combination of these results with site-directed chemical modification and known bFGF x-ray structure, is suggested.
Zamai
M
M
Department of Oncology, Preclinical Research, Pharmacia & Upjohn, 20014 Nerviano, Milan, Italy.
Caiolfa
V R
VR
Pines
D
D
Pines
E
E
Parola
A H
AH
D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D018360:Crystallography, X-Ray; D004214:Distamycins / Q000737:chemistry* / Q000494:pharmacology; D016222:Fibroblast Growth Factor 2 / Q000737:chemistry* / Q000378:metabolism*; D005454:Fluorescence Polarization; D006801:Humans; D007700:Kinetics; D016018:Least-Squares Analysis; D008958:Models, Molecular; D009389:Neovascularization, Pathologic / Q000517:prevention & control; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D004214:Distamycins; C085720:FCE 26644; D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2