Mechanism of binding of the new antimitotic drug MDL 27048 to the colchicine site of tubulin: Equilibrium studies

Abstract

MDL 27048 [trans-1-(2,5-dimethoxyphenyl)-3-[4-(dimethylamino)phenyl]-2- methyl-2-propen-1-one] fluoresces when bound to tubulin but not in solution. This effect has been investigated and found to be mimicked by viscous solvents. Therefore, MDL 27048 appears to be a fluorescent compound whose intramolecular rotational relaxation varies as a function of microenvironment viscosity. The binding parameters of MDL 27048 to tubulin have been firmly established by fluorescence of the ligand, quenching of the protein fluorescence, and gel equilibrium chromatography. The apparent binding equilibrium constant was (2.75 +/- 0.45) x 10(6)M-1, and the binding site number was 0.81 +/- 0.12 (10 mM sodium phosphate-0.1 mM GTP, pH 7.0, at 25 degrees C). The binding is exothermic. The binding of MDL 27048 overlaps the colchicine and podophyllotoxin binding sites. Binding of MDL 27048 to the colchicine site was also measured by competition with MTC [2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one] , a well-characterized reversibly binding probe of the colchicine site [Andreu et al. (1984) Biochemistry 23, 1742-1752; Bane et al., (1984) J. Biol. Chem. 259, 7391-7398]. In contrast with close analogues of colchicine, MDL 27048 and podophyllotoxin neither affected the far-ultraviolet circular dichroism spectrum of tubulin, within experimental error, nor induced tubulin GTPase activity. Like podophyllotoxin, an excess of MDL 27048 over tubulin induced no abnormal cooperative polymerization of tubulin, which is characteristic of colchicine binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of a Novel Nitro-Derivative of Noscapine for the Potential Treatment of Drug-Resistant Ovarian Cancer and T-Cell Lymphoma

“…ment around these amino acids in the folded protein. Agents that bind tubulin typically change the microenvironment and alter the fluorescent properties of the target protein (Peyrot et al, 1992;Panda et al, 1997;Ye et al, 1998). Measuring these fluorescent changes has become a standard method for determining the binding properties of tubulin ligands, including the classic compound colchicine (Peyrot et al, 1989;Andreu et al, 1991).…”

Synthesis of microtubule-interfering halogenated noscapine analogs that perturb mitosis in cancer cells followed by cell death

“…Tubulin, like many other proteins, contains fluorescent amino acids like tryptophans and tyrosines and the intensity of the fluorescence emission is dependent upon the micro-environment around these amino acids in the folded protein. Agents that bind tubulin typically change the micro-environment and the fluorescent properties of the target protein [18,40,41]. Measuring these fluorescent changes has become a standard method for determining the binding properties of tubulin ligands including the classical compound colchicine [42].…”

Opium alkaloid noscapine is an antitumor agent that arrests metaphase and induces apoptosis in dividing cells

“…Noscapine Binds Tubulins. Fluorescence titration for determining the binding constant was performed as described (20). In brief, at room temperature, 2 M tubulin in 100 mM Pipes, pH 6.8͞2 mM EGTA͞1 mM MgCl 2 was excited at 278 nm, and the fluorescence emission spectra were recorded with bandwidths of 2 nm.…”

“…CD spectra measurements were performed on Jasco 600 spectrometer, in cells with a 0.1-cm path at 25°C, as described (20).…”