A fluorescent probe and a photoaffinity labeling reagent to study the binding site of maytansine and rhizoxin on tubulin.
A fluorescent probe (20-demethoxy-20-[3-[[[5-(dimethylamino)naphthalen-1-yl]sulfonyl] amino]propyl]maytansinol 3-isobutyrate, Dan-PDM-3) and a photoaffinity labeling reagent (20-demethoxy-20-[(p-azidobenzoyl)oxy]maytansinol 3-isobutyrate, DABMI) were prepared by derivatization of ansamitocin P-3 (ASMP-3), a maytansinoid. Dan-PDM-3 consists of a tethered dansyl moiety and a maytansinoid moiety. DABMI contains a p-azidobenzoyl group instead of the tethered dansyl moiety of Dan-PDM-3. These compounds were synthesized by reacting 20-demethoxy-20-hydroxymaytansinol-3 isobutyrate (PDM-3) with the corresponding alkyl halide or benzoic acid. Both inhibit tubulin polymerization as potently as ASMP-3 and compete with ASMP-3 for binding to tubulin. The inhibition constants (Ki) of DABMI for the binding to tubulin of rhizoxin and ASMP-3 were 0.54 and 0.36 microM, respectively, which were nearly equal to the dissociation constant (Kd = 0.43 microM) of DABMI measured by the use of [14C]DABMI. The results suggest that Dan-PDM-3 and DABMI interacted with tubulin at the same site as rhizoxin and maytansine. DABMI is irreversibly bound to tubulin upon irradiation. Dan-PDM-3 and DABMI should be useful probes for studying the binding site.
Sawada
T
T
Institute of Molecular and Cellular Biosciences, University of Tokyo, Japan.
Kato
Y
Y
Kobayashi
H
H
Hashimoto
Y
Y
Watanabe
T
T
Sugiyama
Y
Y
Iwasaki
S
S
D000345:Affinity Labels / Q000138:chemical synthesis*; D000818:Animals; D000935:Antifungal Agents / Q000737:chemistry*; D001386:Azides / Q000138:chemical synthesis*; D001667:Binding, Competitive; D003619:Dansyl Compounds / Q000138:chemical synthesis*; D005456:Fluorescent Dyes / Q000138:chemical synthesis*; D007700:Kinetics; D007783:Lactones / Q000737:chemistry; D018942:Macrolides; D008453:Maytansine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry*; D008870:Microtubules / Q000737:chemistry; D011485:Protein Binding; D012568:Schizosaccharomyces / Q000187:drug effects; D013552:Swine; D014404:Tubulin / Q000737:chemistry*
D000345:Affinity Labels; D000935:Antifungal Agents; D001386:Azides; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D007783:Lactones; D018942:Macrolides; D014404:Tubulin; D008453:Maytansine; C083919:20-demethoxy-20-(3-(((5-dimethylamino)naphthalen-1-yl)sulfonyl)amino)propylmaytansinol-3-isobutyrate; C083920:2 0-demethoxy-20-((4-azidobenzoyl)oxy)maytansinol-3-isobutyrate; C041443:rhizoxin