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)

The far-ultraviolet circular dichroism spectrum of the alpha beta-tubulin dimer analyzed by six different methods indicates an average content of approximately 33% alpha helix, 21% beta sheet, and 45% other secondary structure. Deconvolution of Fourier transform infrared spectra indicates 24% sheet, 37% (maximum) helix, and 38% (minimum) other structure. Separate alignments of 75 alpha-tubulin, 106 beta-tubulin, and 14 gamma-tubulin sequences and 12 sequences of the bacterial cell division protein FtsZ have been employed to predict their secondary structures with the multiple-sequence method PHD [Rost, B., & Sander, C. (1993a) J. Mol. Biol. 232, 584-599]. The predicted secondary structures average of 33% alpha helix, 24% beta sheet, and 43% loop for the alpha beta dimer. The predictions have been compared with sites of limited proteolysis by 12 proteases at the surfaces of the heterodimer and taxol-induced microtubules [de Pereda, J. M., & Andreu, J. M. (1996) Biochemistry 35, 14184-14202]. From 24 experimentally determined nicking sites, 18 are at predicted loops or at the extremes of secondary structure elements. Proteolysis zone A (including acetylable Lys40 and probably Lys60 in alpha-tubulin and Gly93 in beta-tubulin) and proteolysis zone B (extending between residues 167 and 183 in both chains) are accessible in microtubules. Proteolysis zone C, between residues 278 and 295, becomes partially occluded in microtubules. The alpha-tubulin nicking site Arg339-Ser340 is at a loop following a predicted alpha helix in proteolysis zone D. This site is protected in taxol microtubules; however, a new tryptic site appears which is probably located at the N-terminal end of the same helix. Zone D also contains beta-tubulin Cys354, which is accessible in microtubules. Proteolysis zone E includes the C-terminal hypervariable loops (10-20 residues) of each tubulin chain. These follow the two larger predicted helical zones (residues 372-395 and 405-432 in beta-tubulin), which also are the longer conserved part of the alpha- and beta-tubulin sequences. Through combination of this with other biochemical information, a set of surface and distance constraints is proposed for the folding of beta-tubulin. The FtsZ sequences are only 10-18% identical to the tubulin sequences. However, the predicted secondary structures show two clearly similar (85-87 and 51-78%) regions, at tubulin positions 95-175 and 305-350, corresponding to FtsZ 65-135 and 255-300, respectively. The first region is flanked by tubulin proteolysis zones A and B. It consists of a predicted loop1-helix-loop2-sheet-loop3-helix-loop4-sheet fold, which contains the motif (KR)GXXXXG (loop1), and the tubulin-FtsZ signature G-box motif (SAG)GGTG(SAT)G (loop3). A simple working model envisages loop1 and loop3 together at the nucleotide binding site, while loops 2 and 4 are at the surface of the protein, in agreement with proteolytic and antigenic accessibility results in tubulin. The model is compatible with studies of tubulin and FtsZ mutants. It is proposed that this r...

Several fluorescence properties of two enantiomers, NSC 613862 (S)-(-) and NSC 613863 (R)-(+), have been compared. Even though the two isomers showed the same fluorescence behavior in solution in different solvents, drastic differences were observed after binding to purified calf brain tubulin. Binding measurements for the two compounds were performed both by fluorescence spectroscopy and by column gel permeation, a direct method of measurement. For both isomers, the binding was characterized by the presence of one high-affinity binding site with an apparent association constant of (3.2 f 0.5) X lo6 M-I and (4.1 f 0.9) X lo6 M-' for the Rand S-isomer, respectively, and by several low-affinity sites.Both isomers were also shown to induce GTPase activity in tubulin. The high-affinity binding site seems to be the same for the two isomers. Moreover, fluorescence competition experiments suggest at least a partial overlap of the colchicine and podophyllotoxin site. To explain the differences in fluorescence behavior after binding to tubulin, we hypothesize that the R-isomer is positioned differently in its binding locus as compared with the S-isomer.Initially synthesized by Elliott et al. (1968) to act as analogues of folic acid, a number of compounds that possess an aromatic nucleus with a l-deaza-7,8-dihydropteridine structure have been reported to inhibit mitosis and produce an accumulation of cells in metaphase (Wheeler et al., 1981). Among them, NSC 181928, ethyl (5-amino-1,2-dihydro-3-[ (N-methy1anilino)methyll pyrido [3,4-b]pyrazin-7-yl)carbamate has been studied in particular. Hamel and Lin (1982) demonstrated that this drug was an active antitubulin agent which inhibited both polymerization and binding of colchicine to tubulin and stimulated tubulin-dependent GTP hydrolysis.Bowdon et al. (1987) showed that another member of the same series, NSC 370147, ethyl (5-amino-1,2-dihydro-2methyl-3-phenylpyrido [ 3,4-b] pyrazin-7-yl)carbamate, was able to competitively inhibit the binding of [3H]c~lchicine and slightly enhance the binding of [3H]vincristine to purified tubulin.However, NSC 370147 is a racemic compound. Its two chiral isomers, NSC 613862 (S)-(-) and NSC 613863 (R)- (+) (see Chart I), have displayed significant differences in Abstract published in Advance ACS Abstracts, September 1, 1993. I Abbreviations: BSA, bovine serum albumin; PG buffer, 10 mM sodium phosphate and 0.1 mM GTP, pH 7.0; MDL 27048, trans-1-(2,5-dimethoxyphenyl)-3-[4-(dimethylam~o)phenyl]-2-methyl-2-pro~n-1 -one; MTC, 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6cyclohep~~en-1-one; SDS, sodium dodecyl sulfate; TME, tropolone methyl ether; AS,,, difference between theapparent sedimentation coefficient of tubulin alone and liganded.

NSC 613862 (S)-(-) and NSC 613863 (R)-(+) are the two chiral isomers of ethyl-5-amino-2-methyl-1,2-dihydro-3-phenylpyrido[3, 4-b]pyrazin-7-yl carbamate. Both compounds bind to tubulin in a region that overlaps the colchicine site. They induce formation of abnormal polymers from purified GTP-Mg-tubulin, the active assembly form of tubulin, in glycerol-free buffer with magnesium [De Ines, C., Leynadier, D., Barasoain, I., Peyrot, V., Garcia, P., Briand, C., Rener, G. A., and Temple, C., Jr. (1994) Cancer Res. 54, 75-84]. In this study, we observed that the S-isomer can promote polymerization of GDP-tubulin, the inactive assembly-incompetent form of tubulin, into nonmicrotubular structures at a critical protein concentration of 1 mg/mL (12 mM MgCl2). Neither the R-isomer nor colchicine have this ability. By electron microscopy, these tubulin polymers showed the same poorly defined filamentous structure when GDP-tubulin or GTP-Mg-tubulin were used. By HPLC measurements, we demonstrated that a dissociated GTP hydrolysis and exchange of nucleotide occurred during the isomer-induced abnormal assembly. Both isomers inhibited the Mg2+-induced tubulin self-association leading to 42 S double ring formation from GTP-Mg-tubulin or GDP-tubulin. Measurement of their binding under nonassociation conditions revealed a 3-fold decrease in the apparent equilibrium binding constant of the R-isomer to GDP-tubulin relative to GTP-Mg-tubulin. For the S-isomer, the decrease in the binding constant was less pronounced. Binding data, analyzed in terms of a system of linked conformational and association equilibria, provide evidence that the active ("straight") rather than the inactive ("curved") conformation of tubulin differentially recognizes these ligands. Whereas binding of colchicine to tubulin is well-known to induce GTP hydrolysis, this is the first case in which the interaction of a ligand with the colchicine site is shown to be sensitive to the presence of GDP or GTP at the distant nucleotide binding site.

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