Tubulin binding of two 1-deaza-7,8-dihydropteridines with different biological properties: Enantiomers NSC 613862 (S)-(-) and NSC 613863 (R)-(+)

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 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.

Stathmin and Interfacial Microtubule Inhibitors Recognize a Naturally Curved Conformation of Tubulin Dimers

“…1C), whereas this group would be in an essentially hydrophobic environment if its location was exchanged with that of the phenyl ring. Consistent with the results of competition experiments (43), the ligands overlap with colchicine; this overlap is limited to the phenyl of R (or S) and to the trimethoxyphenyl ring of colchicine. By contrast, the electron densities of tubulin-bound R (or S) and TN-16 (21) largely colocalize (Fig.…”

“…The apparent binding constants are summarized in Table 1 for comparison. The S isomer bound to tubulin with a slightly higher affinity than the R isomer, as previously shown (43,45). The R and S isomer affinities were not modified in the stathmin complexes.…”

“…The binding of R and S isomers induced a decrease of the intrinsic tryptophan tubulin fluorescence (43,45). We used this property to compare the binding affinity of both isomers to the ␣␤-tubulin dimers and to the T 2 Stath complexes.…”

“…The two chiral isomers NSC 613862-(CI980) (S) and NSC 613863 (R) (42) have antitumor activity, bind to tubulin with a high affinity overlapping the colchicine site and powerfully inhibit microtubule assembly in vitro (43) and in cells (44). Interestingly, the R and S compounds bind to GTP-tubulin with higher affinity than to GDP-tubulin dimers, allosterically distinguishing the bound nucleotide (45).…”

Antivascular and antitumor properties of the tubulin-binding chalcone TUB091

“…The affinity of the compounds for bovine αβ-tubulin was studied by competition experiments with R-PT, whose binding to tubulin is characterized by fluorescence emission (K a of 3.2 × 10 6 M −1 ) [21, 22]. The binding constant obtained for compound 10 by R-PT displacement was (1.3 ± 0.2) × 10 7 M −1 , similar to the reported value for colchicine (1.2 × 10 7 M −1 at 37°C).…”