Interaction of pseudolaric acid B with the colchicine site of tubulin.
We purified pseudolaric acid B (PAB) from the root and stem bark of Pseudolarix kaempferi (Lindl.) Gorden. Confirming previous findings, we found that the compound had high nanomolar IC₅₀ antiproliferative effects in several cultured cell lines, causing mitotic arrest and the disappearance of intracellular microtubules. PAB strongly inhibited tubulin assembly (IC₅₀, 1.1 μM) but weakly inhibited the binding of colchicine to tubulin, as demonstrated by fluorescence and with [³H]colchicine. Kinetic analysis demonstrated that the mechanism of inhibition was competitive, with an apparent K(i) of 12-15 μM. Indirect studies demonstrated that PAB bound rapidly to tubulin and dissociated more rapidly from tubulin than the colchicine analog 2-methoxy-5-(2',3',4'-trimethoxyphenyl)tropone, whose complex with tubulin is known to have a half-life of 17s at 37 °C. We modeled PAB into the colchicine site of tubulin, using the crystal structure 1SA0 that contains two αβ-tubulin heterodimers, both bound to a colchicinoid and to a stathmin fragment. The binding model of PAB revealed common pharmacophoric features between PAB and colchicinoids, not readily apparent from their chemical structures.
Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute at Frederick, National Institutes of Health, Frederick, MD 21702 USA.
Taradas
T
Sarkar
Tam Luong
TL
Nguyen
Zhi-Wei
ZW
Su
Jun
J
Hao
Ruoli
R
Bai
Rick
R
Gussio
Samuel X
SX
Qiu
Ernest
E
Hamel
D001665:Binding Sites; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D003078:Colchicine / Q000737:chemistry / Q000378:metabolism*; D004224:Diterpenes / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D005453:Fluorescence; D006801:Humans; D007700:Kinetics; D008938:Mitosis / Q000187:drug effects; D008958:Models, Molecular; D028201:Pinaceae / Q000737:chemistry*; D011485:Protein Binding; D050777:Stathmin / Q000737:chemistry; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism*; D050257:Tubulin Modulators / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*
D004224:Diterpenes; D050777:Stathmin; D014404:Tubulin; D050257:Tubulin Modulators; C058391:pseudolaric acid B; D003078:Colchicine