Cancer Chemother Pharmacol (1997) 40: 513±520 Ó Springer-Verlag 1997

ORIGINAL ARTICLE

Yasushi Shintani á Toshimasa Tanaka

Yukimasa Nozaki

GS-164, a small synthetic compound, stimulates tubulin polymerization

by a similar mechanism to that of Taxol

Received: 20 September 1996 / Accepted: 5 March 1997

Abstract Purpose: During our search for new microtu- Although the cytotoxicity of GS-164 against human
bule e€ectors as anticancer agents, we have found that a tumor cells was 1000-fold lower than that of Taxol and
small synthetic molecule designated GS-164 interferes GS-164 was one-tenth as active as Taxol in vitro, these
with the assembly of porcine microtubule proteins and ®ndings pave the way for synthesizing clinically useful
has cytotoxic activity against a wide range of human anticancer agents using GS-164 as a lead compound.
tumor cell lines. In this study, we investigated mode of
action of the compound in comparison with Taxol and Key words GS-164 á Taxol á Mitotic agent á
colcemid. Methods: To gain an insight into the mode of Microtubule á Anticancer
action of GS-164, we used an in vitro microtubule po-
lymerization assay and a ¯ow-cytometric measurement
technique. Microtubule organization and the level of Introduction
tubulin polymerization in HeLa cells were also examined
by immuno¯uorescence microscopy and cytoskeletal Taxol (paclitaxel) [17, 21, 26, 39, 46] is an exciting new
protein analyses, respectively. Results: GS-164 stimu- therapeutic agent with antitumor activity against ovari-
lated assembly of microtubule proteins in vitro in a an, breast, and lung carcinomas. This compound is ex-
concentration-dependent and a GTP-independent man- tracted from the bark of the Paci®c yew tree Taxus
ner. Furthermore, as with Taxol, the microtubule poly- brevifolia, as well as from needles and stems of this and
merization induced by GS-164 was antagonized by other Taxus species, and its complex chemical structure
podophyllotoxin, a tubulin polymerization inhibitor, has been determined [57]. Its activity as an antitumor
and microtubules formed by GS-164 were resistant to agent was recognized in an early preclinical screening
disassembly by calcium or low temperatures. GS-164 in program at the NCI using P388 murine leukemia-bear-
the micromolar range arrested the cell cycle of HeLa ing mice [11]. Interest in this compound arises from not
cells in the mitotic phase leading to cell death. GS-164 only its clinical activity against poorly responsive solid
also increased the amounts of cellular microtubules in tumors but also from its unique mechanism of action. It
HeLa cells, resulting in the formation of microtubule promotes tubulin polymerization and stabilizes micro-
bundles. Conclusion: These results indicate that GS-164 tubules that result in the inhibition of cell migration and
stimulates microtubule assembly by a similar mechanism chromosome segregation by blocking the transit of cy-
to that of Taxol. A comparative conformational analysis cling cells in the G2/M phase [48].
of GS-164 and Taxol suggested that the structure of the A principal obstacle to the wider therapeutic use of
former mimics the minimum essential sites of Taxol re- Taxol is limited supply. Since Taxol can be obtained
quired to exert the Taxol-like activities described above. only in low yields (40±165 mg/kg bark) from the very
slow growing Paci®c yew trees which are sparsely dis-
tributed in the Northern hemisphere [17, 21, 26, 39, 46],
Y. Shintani á Y. Nozaki (&) alternative approaches for obtaining Taxol have been
Discovery Research Laboratories II, Pharmaceutical Discovery explored, such as isolation from renewable foliage and
Research Division, Takeda Chemical Industries Ltd., other tissues of Taxus species [1, 13, 25, 27, 29], pro-
Wadai 10, Tsukuba, Ibaraki 300-42, Japan
Tel. 0298-64-6343; Fax 0298-64-6308 duction in tissue culture of Taxus plant cells [12, 32],
production by culture of Taxomyces andreanae, a Taxol-
T. Tanaka
Molecular Chemistry Laboratory, Pharmaceutical Research
producing endophytic fungus isolated from a Paci®c yew
Division, Takeda Chemical Industries Ltd., 2-17-85 tree [50], and semisynthesis of the drug or its analogues
Jusohonmachi, Yodogawa-ku, Osaka 532, Japan such as Taxotere from Baccatin III or related taxoid
514

metabolites that are more readily available from re- turbidity measurement (400 nm, Beckman DU250 spectropho-
newable sources [2, 15, 19, 43, 56]. Although the total tometer) at 37 °C using 0.7 mg of microtubule protein per ml of a
reaction mixture [100 mM 4-morpholinoethanesulfonic acid
synthesis of Taxol has also been achieved by Nicolaou (MES), 1 mM ethyleneglycol bis(2-aminoethylether) tetraacetic
et al. [40] and Holton et al. [20], it is not yet commer- acid (EGTA), 0.5 mM 2-mercaptoethanol (2ME) and 0.5 mM
cially viable owing to the multiple synthetic processes MgSO4 (pH 6.5)]. Drugs dissolved in ethanol were added at the
involved. start of the reaction.
In this report, we describe a novel small synthetic
compound designated GS-164, which stimulates tubulin Analysis for cellular tubulin and actin polymerization level
polymerization and stabilizes microtubules. GS-164 has The cytoskeletal fraction containing microtubule, actin and other
activities similar to those of Taxol in vitro and in vivo. cellular network proteins was prepared from mammalian cells as
The conformations of GS-164 and Taxol were com- described by Thrower et al. [54] with slight modi®cation. Brie¯y,
pared, and the structure-activity relationships of these 2 ml cell suspension (1 ´ 106 cells) of HeLa S3 cells cultured in the
presence or absence of a drug was centrifuged at 2000 g for 5 min
drugs are discussed. at 4 °C, and the pellet was resuspended in 2 ml lysis bu€er [0.1 M
piperazine-N,N¢-bis(2-ethanesulfonic acid) (Pipes), 1 mM EGTA,
1 mM MgSO4, 30% glycerol, 5% DMSO, 5 mM GTP, 1 mM DTT,
Materials and methods 1 mM Na-p-tosyl-L-arginine methyl ester (TAME), 0.05 mg/ml
aprotinin, 0.02% sodium azide and 0.125% NP-40 (pH 6.9)] and
Materials and human cell lines incubated at 37 °C for 20 min. Lysis of cells was veri®ed by light
microscopy. Bu€er (1 ml) lacking NP-40 was added to the lysed cell
GS-164 (Fig. 1) was synthesized by Cyclan Co. (Moscow, Russia). suspension, and then the mixture was centrifuged at 200 000 g for
Taxol, colchicine, colcemid, podophyllotoxin, bovine serum albu- 90 min at 37 °C. After the supernatant was aspirated, 0.2 ml of a
min and a monoclonal antibody against b-tubulin (TUB2.1) were depolymerization bu€er (0.1 M MES, 1 mM MgSO4, 10 mM
obtained from Sigma Co. (St. Louis, Mo.). All other chemicals CaCl2, 5 mM GTP, 1 mM TAME, 0.05 mg/ml aprotinin and
were reagent grade. The following human cell lines were purchased 0.02% sodium azide, pH 6.9) was added to the pellet. The solution
from the American Type Culture Collection (Rockville, Md.): co- was homogenized with a glass pestle and allowed to stand for 1 h
lon adenocarcinomas SW48, SW620 and SW948, breast adeno- on ice to ensure depolymerization of cytoskeletal proteins. Frac-
carcinomas MCF-7, MDA-MB231, MDA-MB435S, MDA-MB453 tions thus obtained were electrophoresed on 10% polyacrylamide
and MDA-MB468, hepatocellular carcinoma HepG2, lung small- gels and transferred to nitrocellulose ®lters (Hybond-ECL; Amer-
cell carcinoma H69, pancreatic carcinoma MIA PaCa-2, ®brosar- sham) using a semidry electroblotter (Bio-rad). Beta-tubulin pro-
coma HT-1080, prostate adenocarcinoma PC-3, embryonal tein conjugated with TUB2.1 [36] was detected with the enhanced
rhabdomyosarcoma RD, and osteogenic sarcoma Saos-2. The chemiluminescence system (ECL; Amersham). Proteins on the
following human cell lines were purchased from Flow Laboratories electrophoresed gel were stained with Coomassie brilliant blue. The
(Irvine, UK): metastatic pancreas adenocarcinoma AsPC-1, colon identity of actin was con®rmed by immunoblotting with actin-
adenocarcinoma SW480, and epidermoid carcinoma A431. The speci®c antibody and sensitivity to cytochalasin B.
human epitheloid carcinoma HeLa S3, colon adenocarcinoma
WiDr, lung carcinoma A549, and melanoma G361 were obtained Immuno¯uorescence microscopy
from the Institute for Fermentation (Osaka, Japan).
The cellular microtubule organization was observed by ¯uores-
cence microscopy using the procedure of Ohta et al. [41]. Brie¯y,
Cytotoxicity assay HeLa cells (105 cells/ml) were cultured in chamber slides for 20 h in
the presence or absence of GS-164 and ®xed with 4% formaldehyde
Cytotoxicity was assayed colorimetrically by the tetrazolium salt in phosphate-bu€ered saline (PBS) for 30 min. The ®xed cells were
(MTT) method [37]. The cytotoxic activity of the drugs was de- washed with PBS twice and incubated with 0.3% Nonidet P-40 for
termined in terms of the IC50 (lM ), the concentration required to 10 min. After washing with PBS twice, the cells were incubated
inhibit 50% of the cell growth. Cell viability was determined by with TUB2.1 (200 ll/ml, 1:200 dilution) for 1 h at 37 °C, washed
trypan blue exclusion. twice, then incubated with ¯uorescein-conjugated goat antimouse
immunogloblin antibody (Cappel, Malvern, Pa.) for 30 min. The
Preparation of microtubule proteins and polymerization assay immunostained slides were then rinsed before mounting in Im-
munon (Lipshaw Pittsburgh, Pa.) and viewed with a Nikon Diap-
Microtubule protein was prepared from porcine brains according hot photomicroscope (objective, ´ 40).
to the polymerization±depolymerization procedure described by Li
et al. [30]. Polymerization of microtubule proteins was followed by
Flow-cytometric measurement

DNA contents were measured by ¯ow cytometry as described by

Garcia et al. [14]. Brie¯y, HeLa cells (70±80% con¯uence) were
trypsinized and ®xed in cold methanol. The cells were then washed,
centrifuged, resuspended in PBS containing 60 lg/ml RNase and
incubated for 30 min at 37 °C. Before analysis, the cells were
stained with 20 lg/ml of propidium iodide. DNA levels were
measured using a Becton Dickinson Immunocytometry System
2350 (San Jose, Calif.).

Comparative conformational analysis

Stable conformations of GS-164 and Taxol were generated by

molecular dynamics and molecular mechanics calculations using a
Fig. 1 Structures of GS-164 and Taxol Discover CVFF force ®eld (ver. 2.95, Biosym Technologies).
 515

Conformers of Taxol were constructed by systematically bond-ro-

tating the substituents at positions 2 and 13, ®xing the taxane
skeleton to the most stable conformation which was determined
using molecular dynamics at 2000 K. The resulting structures were
minimized. For GS-164, each isomer was energy-minimized. The
pharmacophore was predicted using Apex-3D (ver. 1.4.3, Biosym
Technologies and DCL System International) based on the stable
conformations of GS-164 and Taxol.

Results

Cytotoxicity against human tumor cell lines

Fig. 2 E€ect of GS-164 on the polymerization of microtubule
The cytotoxic activities of GS-164, Taxol and colchicine protein in vitro. Polymerization of microtubule protein was
are shown in Table 1. GS-164 suppressed the growth of measured as described in Materials and methods. After 5 lM
almost all human cell lines tested, whereas the potency Taxol (2 ), 40 lM (3), 100 lM (4), 180 lM (5) or 440 lM (6)
of the cytotoxicity of GS-164 was about one-thousandth GS-164 or 1 mM GTP (7 ) was added to a reaction mixture
containing microtubule protein (0.7 mg/ml) at 0 °C or 440 lM GS-
that of the control mitotic poisons. However, the gross 164 (8) was added to a reaction mixture containing bovine serum
cytotoxicity pro®les of the three agents for these cell albumin (1.0 mg/ml) at 0 °C, the temperature was shifted to 37 °C
lines were similar. and the turbidity of the solution was measured at an absorbance of
400 nm. (lane 1 untreated control)

Stimulation of microtubule polymerization in vitro

reached a maximum at over 400 lM (Fig. 2). Electron
The incubation of porcine microtubule protein with microscopic observation of the product induced by
various concentrations of GS-164 induced the poly- GS-164 revealed a typical cylindrical microtubule
merization of microtubules as monitored spectrophoto- structure, not merely microtubule aggregation, and there
metrically by measuring the increase in turbidity. was no increase in the turbidity of the reaction mixture
Although there was no substantial microtubule poly- when bovine serum albumin or human immunoglobulin
merization in the absence of GS-164, a concentration- was used instead of porcine microtubule protein (Fig. 2,
dependent increase in the rate and extent of microtubule and data not shown). Neither the rate nor the extent of
assembly was observed with GS-164 and stimulation GS-164-induced microtubule polymerization were in¯u-
enced by adding GTP to the reaction mixture as is found
with Taxol [47] (data not shown). Furthermore, micro-
Table 1 Growth-inhibitory activity of GS-164, Taxol and colchi- tubule polymers formed in the presence of GS-164 were
cine against various human cell lines. Each cell line was cultured in stable against the depolymerizing e€ect of 1 mM CaCl2
the presence or absence of a drug in a 96-well plate for 72 h. and exposure to cold (4 °C) as are the polymers formed
Proliferation was evaluated by the MTT assay [37]. Initial density
was 2 ´ 104 cells/ml (NT not tested) in the presence of Taxol [47] (Fig. 3). Antimitotic drugs
such as colchicine and podophyllotoxin antagonize the
Cell line IC50 (lM )

GS-164 Taxol Colchicine

HeLa 9 0.023 0.005

MIA PaCa-2 14.1 0.011 0.011
A549 18.8 0.009 0.043
G361 17.5 0.017 0.018
WiDr 20.7 0.016 0.012
SW48 12 0.007 0.01
SW480 >100 0.117 0.063
SW620 11 NT NT
SW948 20 NT NT
MCF-7 5 0.0009 0.0063
MDA-MB231 13.6 0.011 0.013
MDA-MB435S 5.6 NT NT
MDA-MB453 11 NT NT
MDA-MB468 6.1 NT NT
H69 24.6 0.009 0.012 Fig. 3 E€ect of podophyllotoxin on the polymerization of micro-
AsPC-1 38.3 0.01 0.028 tubule protein induced by GS-164 in vitro. After 40 lM GS-164 (2)
Saos-2 10.2 0.0042 0.0058 or 40 lM GS-164 and 50 lM podophyllotoxin (3) was added to a
RD 17.7 0.0021 0.0058 reaction mixture containing microtubule protein (0.7 mg/ml) at
Hep G2 22.7 0.005 0.03 0 °C, the temperature was shifted to 37 °C and the turbidity of the
HT1080 10.8 0.02 0.012 solution was measured at an absorbance of 400 nm. At the time
A431 >100 0.048 0.043 indicated by the arrow, CaCl2 was added at a ®nal concentration of
PC-3 18 NT NT 1 mM (----) or the temperature of the reaction mixture was shifted
at 4 °C (-á -) (lane 1 untreated control)
516

Fig. 5A,B E€ects of GS-164, Taxol and colcemid on the levels of

tubulin and actin polymers in HeLa S3 cells. HeLa S3 cells were
cultured in the absence (lane 1) or the presence of 15 lM (lane 2)
and 30 lM (lane 3) GS-164, 0.3 lM colcemid (lane 4) and 1 lM
Taxol (lane 5) for 4 h. Cytoskeletal proteins in each lane were
detected by staining with Coomassie brilliant blue after electro-
phoresis on a 10% polyacrylamide gel (A), and tubulin proteins
were also detected by blotting with a monoclonal antibody against
Fig. 4 Relative ability of GS-164 and Taxol to stimulate polymer- human b-tubulin (TUB2.1) after transfer of the electrophoresed gel
ization of microtubule protein in vitro. Polymerization of micro- to a nitrocellulose ®lter as described in Materials and methods (B).
tubule protein was assayed at various concentrations of GS-164 The identity of actin proteins was con®rmed by the molecular
and Taxol as described in Materials and methods. The maximal weight and by immunoblotting with an actin-speci®c monoclonal
assembly rate at each concentration of drug was determined by the antibody (data not shown). The locations of tubulin and actin
initial slope of each polymerization curve as the absorbance proteins on the gel are indicated by the arrows
increase at 400 nm per minute

E€ect on cell cycle progression

microtubule polymerization induced by Taxol at con- and cellular microtubule organization
centrations substoichiometric to that of microtubule
[28]. Under our assay conditions, podophyllotoxin Mammalian cells incubated with Taxol arrest in the
interfered with GS-164-induced microtubule polymer- G2/M phase of the cell cycle and cellular microtubules
ization, and the inhibition was almost complete at are abnormally assembled either in abundant arrays of
equimolar concentrations of GS-164 and podophyllo- disorganized microtubules in parallel ``bundles'' or in
toxin (Fig. 3). mitotic asters that do not require centrioles for forma-
Though stimulation of microtubule polymerization is tion [14]. The formation of these structures is dose- and
maximal with equivalent amounts of Taxol and tubulin time-dependent. Asters have also been observed in mi-
on a molar basis [28], excess GS-164 was required to totic cells and bundles in interphase cells [14]. As shown
induce the maximum turbidity of polymers (Fig. 2). in Fig. 6, the proportion of cells in the G2/M phase in-
Figure 4 shows a comparison of the concentration-de- creased in HeLa cells after exposure to GS-164 as well as
pendent assembly rate stimulated by Taxol and GS-164. Taxol and colcemid; this is commonly observed with
The stimulatory activity of microtubule polymerization mitotic toxins. Furthermore, the microtubule organiza-
by GS-164 was one-tenth that of Taxol. tion in HeLa cells incubated with GS-164 di€ered
markedly from that of the nontreated cells as shown by
immuno¯uorescence microscopy using an anti-b-tubulin
monoclonal antibody (Fig. 7). Microtubule bundles
Stimulation of cellular tubulin polymerization were predominant and abnormal mitotic asters were
virtually absent in HeLa cells (Fig. 7). In contrast, asters
As described above, GS-164 had microtubule polymer- were prevalent in CHO cells at high concentrations of
ization-stimulatory activity in vitro and cytotoxicity GS-164 (data not shown). Although this di€erent re-
against a wide range of human tumor cells. To gain in- sponse to GS-164 between these two cell lines might be a
sight into whether the activity of GS-164 to induce consequence of a lower sensitivity of CHO cells to the
microtubule assembly directly contributed to its cyto- cytotoxic e€ects of GS-164, farther experiments are
toxic activity, we examined the cellular tubulin poly- needed to clarify this issue.
merization level in HeLa cells exposed to GS-164.
Figure 5 shows that GS-164 apparently increased the
tubulin polymerization level dose-dependently. How- Comparative conformational analysis
ever, like Taxol and colchicine, GS-164 hardly a€ected
the actin polymerization level, indicating that it speci®- To understand the structural elements of GS-164 re-
cally a€ected microtubule formation in the treated cells. sponsible for its tubulin-polymerizing activity, we ana-
Although we did not characterize the slowest migrating lyzed the stable conformations of both GS-164 and
protein on the gel, its amount was not a€ected by Taxol, and estimated the structural similarity between
treatment with any compound tested (Fig. 5A). these compounds. By conformational search for the
 517

Fig. 6 E€ect of GS-164, Taxol

and colcemid on HeLa S3 cell
cycle progression. Exponen-
tially proliferating HeLa cells
were cultured in the absence or
presence of 50 lM GS-164,
0.5 lM Taxol or 0.2 lM col-
cemid. The cells were harvested
24 h later for analytical DNA
¯ow cytometry as described in
Materials and methods

substituents at positions 2 and 13 with ®xing the taxane of Taxol and the other group of GS-164 was homolo-
skeleton of Taxol to the most stable, 24 probable con- gous to the phenyl ring at position 3¢. Furthermore, the
formations of Taxol were obtained within 20 kcal/mol. hydroxyl group and the one ring oxygen of GS-164 may
GS-164 generated four isomers because of the presence correspond to the hydroxyl group at position 1 and the
of two asymmetric carbons at comparable energy levels. ester oxygen at position 2 of Taxol, respectively (Fig. 8).
Using these stable conformations of GS-164 and Taxol We propose that the structure of GS-164 (R/R isomer)
for the pharmacophore analysis, we determined that one mimics the part of the Taxol structure that is critical for
of two phenyl groups of GS-164 was homologous to the Taxol to exert the activity reported [4, 7, 8, 16, 18, 19,
phenyl group in the benzoyloxy side-chain at position 2 22, 42, 51, 53, 55].

Fig. 7A±C Indirect immuno-

¯uorescence staining of GS-164-
induced microtubules in HeLa
S3 cells. HeLa S3 cells were
cultured in the absence (A) or
presence of 0.1 lM Taxol (B)
and 50 lM GS-164 (C) for 20 h
at 37 °C. The cells were then
®xed and stained with an anti-
b-tubulin monoclonal antibody
as described in Materials and
methods
518

Fig. 8 Structural similarity be-

tween GS-164 and Taxol. A
stereoscopic view was obtained
by overlapping GS-164 (green)
and Taxol (magenta) with two
benzene rings (orange spheres)
and two oxygen atoms (white
spheres) in similar positions

of Taxol. Although we have shown G2/M phase arrest

Discussion by GS-164 in this study, it remains to be determined
whether GS-164 interferes with other cell-cycle transi-
We demonstrated that a novel low-molecular-weight tions. Thus, to elucidate the mode of action of GS-164 in
compound, GS-164, can stimulate microtubule assembly more detail, further experiments to compare the activi-
by a similar mechanism to that of Taxol by the following ties of GS-164 and Taxol at equipotent doses are re-
in vitro and in vivo experimental evidence. In the in vitro quired.
microtubule polymerization assay, (1) GS-164 induced Taxol may modulate either the interaction of growth
microtubule assembly in the absence of GTP in a con- factors with their receptors or the resulting intracellular
centration-dependent manner (Fig. 2), (2) the microtu- signalling [3, 10, 33, 34, 44, 49, 58]. For example, Bog-
bules formed by the drug were resistant to disassembly dan and Ding [3] and other groups [10, 33, 34, 58] have
by calcium or low temperatures and (3) podophyllotoxin shown that Taxol downregulates tumor necrosis factor-a
interfered with the microtubule polymerization induced (TNF-a) receptors on macrophages and induces TNF-a
by GS-164 (Fig. 3). In addition, in the cell-based assay, and interleukin-1 (IL-1) expression in these cells as ob-
GS-164 increased the amounts of cellular microtubules served with lipopolysaccharide. However, GS-164 did
in HeLa cells without a€ecting those of actin ®laments not stimulate TNF-a production by macrophages under
(Fig. 5). Furthermore, GS-164 induced microtubule experimental conditions whereas Taxol did (data not
bundles (Fig. 7), and arrested the cell cycle progression shown). Burkhart et al. [6] have reported that the e€ect
of HeLa cells in the G2/M phase leading to cell death of Taxol on TNF-a expression is distinct from its mic-
(Fig. 6). All of these activities exerted by GS-164 have rotubule-stabilizing activity. Thus these biological re-
also been attributed to Taxol. sponses to Taxol might be due to its side-chain e€ect,
In addition to the activities described above, Taxol which is not related to tubulin polymerization-stimula-
inhibits the transition from G0 to S phase when serum- tory activity.
starved ®broblasts are stimulated by growth factors, Recently, Bollag et al. [5] have reported that
suggesting that Taxol also a€ects the interphase cyto- epothilones A and B, 16-membered macrolides isolated
skeleton by disrupting the normal function of the cell from an extract of the myxobacterium Sorangium cell-
membrane, transmembrane signalling, intracellular ulosum, mimic the biological e€ects of Taxol and bind to
transport and/or locomotion [24, 45]. Jordan et al. [23] the same microtubule-binding site as Taxol in a dis-
and Long and Fairchild [31] have also shown that low placement competition assay. Although all the biological
concentrations of Taxol inhibit the progression of mi- activities of these compounds are almost equipotent to
totic cells to G1 phase by interfering with spindle for- those of Taxol, they di€er from Taxol in that they are
mation without a€ecting other microtubule functions, also e€ective against multidrug-resistant tumor cells
suggesting that this is the primary cytotoxic mechanism against which Taxol is ine€ective. This might be due to
 519

the failure of P-glycoprotein to recognize epothilones as GS-164 could be the mother compound from which to
substrates because the structures of epothilones are to- develop clinically useful anticancer agents by synthetic
tally di€erent from that of Taxol. approaches.
Our comparative conformational analysis of GS-164
and Taxol suggested that GS-164 mimics the minimum Acknowledgments The authors thank Drs. Y. Li and S. Iwasaki of
the Institute of Molecular and Cellular Bioscience, University of
essential structural elements of taxol responsible for its Tokyo, for their helpful advice on the preparation of microtubule
tubulin polymerization-stimulating activity (Fig. 8). protein and the in vitro polymerization assay. We thank
GueÂard et al. [19] have postulated that during the Drs. M. Fujino, H. Okazaki, K. Kitano and H. Shirafuji for their
binding of Taxol to tubulin, two structural features of continual interest and encouragement. We also thank Mrs. Y. Kato
for skilful technical assistance.
Taxol which constitute part of ``the southern hemi-
sphere'' of the molecule [51] can be assumed to be crit-
ical for the recognition: the hydrophobic area including
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