Interactions of tubulin with guanine nucleotides that have paclitaxel-like effects on tubulin assembly: 2',3'-dideoxyguanosine 5'-[alpha,beta-methylene]triphosphate, guanosine 5'-[alpha,beta-methylene]triphosphate, and 2',3'-dideoxyguanosine 5'-triphosphate.
Despite reduced affinity for the exchangeable nucleotide binding site of tubulin relative to GTP, 2',3'-dideoxyguanosine 5'-triphosphate (ddGTP) and guanosine 5'-[alpha, beta-methylene]triphosphate [pp(CH2)pG] are highly active in promoting tubulin assembly. Like the antimitotic drug paclitaxel, which interacts with the same part of the beta-tubulin molecule as exchangeable-site GTP, both analogs enhance nucleation reactions and promote formation hyperstable polymers. These observations led us to synthesize the doubly modified analog 2',3'-dideoxyguanosine 5'-[alpha, beta-methylene]triphosphate [pp(CH2)pddG]. We compared the effects of pp(CH2)pddG to those of ddGTP, pp(CH2)pG, and the three-cognate diphosphates in their interactions with tubulin. We found that pp(CH2)pddG was as active as ddGTP and pp(CH2)pG in supporting formation of polymer of increased stability, but that its affinity for the exchangeable site was lower than that of both singly modified analogs [relative affinities for the exchangeable site for pp(CH2)pddG:ddGTP:pp(CH2)-pG:GTP were 1:2.8:10:273]. There were significant differences in interactions of each of the three analogs with tubulin, and the behavior of pp(CH2)pddG was intermediate between that of ddGTP and that of pp(CH2)pG. Most importantly, under the reaction conditions studied, with heat-treated microtubule-associated proteins (MAPs) ddGTP-induced polymer consisted of short microtubules, while polymer formed with both pp(CH2)pddG and pp(CH2)pG consisted of short sheets. On the other hand, assembly without MAPs had a fivefold lower critical concentration for tubulin with ddGTP and pp(CH2)pddG (0.5 mg/ml) than with pp(CH2)pG (2.5 mg/ml). De novo assembly, which occurs readily with 2',3'-dideoxyguanosine 5'-diphosphate, was not observed with either alpha, beta-methylenediphosphate GDP analog.
Hamel
E
E
Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Vaughns
J
J
Getahun
Z
Z
Johnson
R
R
Lin
C M
CM
D000970:Antineoplastic Agents / Q000378:metabolism; D001667:Binding, Competitive; D002851:Chromatography, High Pressure Liquid; D003848:Deoxyguanine Nucleotides / Q000378:metabolism; D054306:Dideoxynucleotides; D004305:Dose-Response Relationship, Drug; D006150:Guanine Nucleotides / Q000302:isolation & purification / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D006868:Hydrolysis; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism / Q000648:ultrastructure; D017239:Paclitaxel / Q000378:metabolism; D011108:Polymers; D013696:Temperature; D014404:Tubulin / Q000378:metabolism*
C095927:2',3'-dideoxyguanosine 5'-(alpha,beta-methylene)triphosphate; D000970:Antineoplastic Agents; D003848:Deoxyguanine Nucleotides; D054306:Dideoxynucleotides; D006150:Guanine Nucleotides; D008869:Microtubule-Associated Proteins; D011108:Polymers; D014404:Tubulin; D006153:Guanosine Diphosphate; C025675:guanosine 5'-(alpha,beta-methylene)triphosphate; C031709:2',3'-dideoxyguanosine 5'-triphosphate; D006160:Guanosine Triphosphate; D017239:Paclitaxel