Biochemistry zyxwvu 1993,32, zyxwvu 10675-10682 10675 Tubulin Binding of Two 1 zyxwv -Deaza-7,8-dihydropteridines with Different Biological Properties: Enantiomers NSC 613862 zyxw (S)-(-) and NSC 613863 zy (R)-( +)t Daniel Leynadier, Vincent Peyrot, Marcel Sarrazin, and Claudette Briand' Groupe de Recherche sur les Interactions des Proteines en Pharmacologie, FacultC de Pharmacie, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 5, France Jose Manuel Andreu Centro de Investigaciones Biologicas. Velazquez 144, Consejo Superior de Investigaciones Cientificas, 28006 Madrid, Spain Gregory A. Rener and Carol1 Temple, Jr. Kettering-Meyer Laboratory. Southern Research Institute, P.O. Box 55305, Birmingham, Alabama Received March 19, 1993; Revised Manuscript Received June 25, 1993" ABSTRACT: 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 R- and 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 podophyllotoxinsite. 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- zyxwvu [ (N-methy1anilino)methyll pyrido [3,4-b]pyrazin-7-yl)carbam- ate 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-2- methyl-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 This work was supported in part by joint FrenchSpanish grants, the Spanish DGICyt Grant PB870220, grants from ARC, FCLCC, and FEGEFLUC, and European Community Contract SCl *-CT91-0658. To whom correspondence should be addressed. e 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 dodecylsulfate;TME, tropolone methyl ether; zyxwvut AS,,, differencebetween theapparentsedimentation coefficient of tubulin alone and liganded. 0006-2960/93/0432- 10675$04.00/0 Chart I CH30 CH30 HCeCH3 C H 3 M : c H 3 CH3 CH30 0 CH 3 OCH3 COLCHICINE MTC NSC613862(S(-)) :R,=CH3; R2=H NSC 613863 ( R (+)) : R q'H ; R2 =CH 3 OH CH3O OCH3 OCH3 PODOPHYLLOTOXIN potency in several biological tests, for example, the deter- mination of their activity against cultured lymphoid leukemia L1210 cells (Temple & Rener, 1989). 0 1993 American Chemical Society