Inhibition of Microtubules and Cell Cycle Arrest by a New 1-Deaza-7,8- Dihydropteridine Antitumor Drug, CI 980, and by Its Chiral Isomer, NSC 6138631

[CANCERRESEARCH 54, 75-84, January 1, 1994] Inhibition of Microtubules and Cell Cycle Arrest by a New 1-Deaza-7,8- dihydropteridine Antitumor Drug, CI 980, and by Its Chiral Isomer, NSC 6138631

Concepcion de Ines, Daniel Leynadier, Isabel Barasoain, 2 Vincent Peyrot, Patrick Garcia, Claudette Briand, Gregory A. Rener, and Carroll Temple, Jr. Centro de Investigaciones Biologicas, Consejo Superior de Investigaciones Cientificas, Velazquez 144, 28006 Madrid, Spain [C. I., L B.]; Groupe de Recherche sur les Interactions des Proteines en Pharmacologie, Facult~ de Pharmacie, 27Bd. Jean Moulin, 13385 Marseille Cedex 5, France [D. L., V. P., P. G., C. B.]; and Organic Chemistry Research Department, Southern Research Institute, Birmingham, Alabama [G. A. R., C. T.]

ABSTRACT binding of [3H]colchicine to purified tubulin and slightly enhanced the binding of [3H]vincristine to tubulin. NSC 370147 was synergistic CI 980 (NSC 613862; [S-(-)]) and NSC 613863 [R-(+)] are the two with vincristine in killing cultured L1210 cells and increasing life chiral isomers of ethyl 5-amino 1,2-dihydro-2-methyl-3.phenylpyrido[3,4- spans of mice bearing P388 leukemia (4). Moreover, this compound b]pyrazin-7-ylcarbamate (NSC 370147), which is a mitotic inhibitor with had good antitumor activity against several tumors in culture and also in vivo and in vitro activity against murine multidrug-resistant sublines. We have characterized the inhibition of in vitro microtubule assembly by in mice as well as against murine leukemic sublines that were resistant the S (CI 980) and R (NSC 613863) enantiomers, their actions on the to most drugs used in cancer therapy (5, 6). cytoplasmic microtubule network of epithelial-like PtK2 cells, and on the NSC 370147 is a racemic compound. Its two chiral isomers, NSC cell cycle of different human and murine leukemias and PtK2 cells. As- 613862 [S-(-)] (CI 980) and NSC 613863 [R-(+)] (Fig. 1), have sembly of purified tubulin, or tubulin plus microtubule-associated pro- been prepared and present significant differences in inhibiting the teins, into microtubules was substoichiometrically inhibited by both com- proliferation of L1210 (7-10) and U937 cells (11). CI 980 is presently pounds, which also induced a slow depolymerization of preassembled undergoing Phase I clinical trials (12). Frequently, one isomer of a microtubules. Half inhibitory concentrations were 0.4-0.7/~M and 1.6-2.1 drug may produce a desired effect while another may be less active, /zM for the S and R isomers, respectively. Excess of both drugs induced inactive, or even produce some undesired effect. Microtubule inhibi- polymerization of liganded tubulin into abnormal polymers similar to tors are not an exception. Inversion of optical activity of the acetamide colchicine. The cytoplasmic microtubules of PtK2 cells were disrupted by both compounds in a concentration- and time-dependent manner, which group of colchicine abolishes its activity (13), and isomerization of was observed by indirect immunofluorescence microscopy and quantified podophyllotoxin (cis-lactone) results in a 10-fold loss of activity (14). by an enzyme-linked immunoassay of cytoskeletal tubulin. Half inhibitory It has been shown that both isomers CI 980 [S-(-)] and NSC 613863 concentrations were 6 nM S isomer, 100 nM R isomer, and 1 pM colchicine. [R-(+)] are able to bind to purified tubulin with equilibrium disso- Twenty ma S isomer or 500-700 nra R isomer gave nearly maximal effect. ciation constants of approximately 0.2 ~M and 0.3 p,M, respectively, At these concentrations, half maximal microtubule depolymerization took apparently overlapping the trimethoxybenzene locus of the colchicine place after 2 h of treatment. After drug removal, slow microtubule assem- binding site. Their different fluorescence properties upon binding to bly and nearly complete reorganization of the cytoplasmic microtubules of tubulin have suggested a different orientation for the isomers in the PtK2 cells were observed (24 h). One nM S enantiomer or 25 nM R enan- same locus (15). tiomer induced mitotic arrest in 8 h in U937, HL60, and EIA leukemias. In order to gain insight into the mechanism of action of CI 980 and PtK2 cells also stopped in mitosis after a 24-h incubation with 50 nM R isomer or 5 nM S isomer. The inhibition of cell division was irreversible in NSC 613863, we have studied their effect on in vitro microtubule the leukemic cells, while PtK2 cells partially resumed growth. Although polymerization and their action on the cytoplasmic microtubule net- the interactions of CI 980 with microtubules in vitro are not very different work and cell division by using PtK2 cells as well as human and from other drugs, it is a most potent cellular microtubule and mitotic murine leukemias. inhibitor. MATERIALS AND METHODS INTRODUCTION Chemicals, Culture Media, and Antibodies. The synthesis of NSC Several 1,2-dihydropyrido[3,4-b]pyrazines were prepared as inter- 613862 (CI 980) and NSC 613863 has been reported (8). Stock solutions were mediates for the synthesis of certain antifolates (1, 2); they did not made in DMSO 3 and stored at -20~ Their concentrations were measured function as analogues of folic acid but inhibited mitosis and caused spectrophotometrically using extinction coefficients E374 nm 15100 and 15400 cultured cells to accumulate in metaphase (3). Therefore, new com- M-1 cm-1 for the S and R compounds, respectively (8). MTC was a gift from Dr. T. J. Fitzgerald (16). Its concentration was measured spectrophotometri- pounds of this type have been synthesized and tested as inhibitors of cally using the extinction coefficient •343 nm :" 17600 M-I"cm-1 (17). GTP, cell proliferation and anticancer agents. Ethyl 5-amino-l,2-dihydro- disodium salt, and colchicine were from Fluka. Podophyllotoxin was from 2-methyl-3-phenylpyrido[3,4-b]pyrazin-7-ylcarbamate, 2-hydroxy- Aldrich Chemical Co. Dulbecco's modified Eagle's medium, RPMI-1640, ethanesulfonate, hydrate (NSC 370147) was very active in inhibiting FCS, L-glutamine, nonessential aminoacids, and antibiotics were from Flow proliferation of cultured L1210 cells and in increasing the mitotic Laboratories (Irvine, United Kingdom). Propidium iodide, DM1A anti-tubulin index. It also inhibited pig brain microtubule polymerization and the monoclonal antibody, and goat-antimouse polyvalent immunoglobulins (fluo- resceine-conjugated) were from Sigma Chemical Co. Rabbit- antimouse immunoglobulins (peroxidase-conjugated) were from Dakopatts (Copenhagen, Received 7/16/93; accepted 10/29/93. Denmark). The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact. 3 The abbreviationsused are: DMSO, dimethylsulfoxide; MTC, 2-methoxy-5-(2',3',4- 1 This work was supported by Grant PB 87-0230 from the Direccion General de trimethoxyphenyl)-2,4,6-cycloheptatrien-l-one;MTP, microtubuleprotein; FCS, fetal calf Investigacion Cientifica y Tecnica, French-Spanishgrants, and grants from Association serum; MES, 4-morpholineethanesulfonicacid; PEM, 100 mM piperazine-N,N'-bis(2- pour la Recherchesur le Cancer Federation de Centres de lutte Contre Cancer. ethanosulfonic acid), 1 mM EGTA, and 1 mM MgC12, pH 6.8; PBS, phosphate-buffered 2 To whom requests for reprints should be addressed. saline; BSA, bovine serum albumin. 75 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1994 American Association for Cancer Research. EFFECTS OF CI 980 AND NSC 613863 ON MICROTUBULES AND CELL DIVISION

buffer was 100 mM 2-(N-morpholino)ethanesulfonic acid, 0.5 mM MgCI2, 1 mM H H ethylene-bis(oxyethylenenitrilo)tetraacetic acid, and 1 mM GTP, pH 6.7. The I I R2 buffer solution used to polymerize purified tubulin consisted of 10 mM sodium phosphate, i mu ethylene-bis(oxyethylenenitrilo)tetraaceticacid, 8 mu MgCI2, CzHsOoc/NN~ / N 3.4 M glycerol, and 0.1 mM GTP, pH 6.8. The reaction was started by a "~ temperature shift from 4 to 37~ R and S enantiomers containing samples and their controls had 1% residual DMSO. Samples were fixed with 0.5% glutar- aldehyde, adsorbed to Formvar coated grids, negatively stained with 1.5% N\ uranyl acetate, and examined under a Jeol 1200 electron microscope (Labo- ratoire de Microscopie Electronique, Facult6 de M6decine, Marseille, France). The assembly reaction of tubulin into abnormal polymers induced by colchicine and both R and S enantiomers was followed turbidimetrically at 475 nm in 10 mM sodium phosphate, 16 mM MgCI2, and 0.1 mM GTP, pH 7.0, at 37~ CI980(S(-)) "R 1 =CH 3 ; Rz=H This wavelength was chosen in order to minimize light absorption by the compounds (22). Cell Lines. U937 monocytic and HL60 promyelocytic human leukemias NSC61::5865(R (+)) "R 1 = H ; R2 =CH3 and EL-4 C57B1 benzanthracene-induced murine lymphoma were grown in RPMI-1640 supplemented with 2 mM glutamine, 10% FCS, and antibiotics at Fig. 1. Chemical structures of CI 980 and NSC 613863. 37~ in a humidified atmosphere containing 5% CO2. PtK2 potoroo epithelial- like kidney cells (American Type Culture Collection, Rockville, MD) were Microtubule Protein and Purified Tubulin. MTP was purified from pig grown in Dulbecco's modified Eagle's medium supplemented with 10% FCS, brain according to the method of Shelanski et al. (18) through three cycles of 2 mu glutamine, 0.1 mu nonessential aminoacids, and antibiotics. in vitro assembly (37~ and disassembly (4~ as described previously (19). Immunofluoreseence Experiments. PtK2 cells were plated onto 9 x 9 mm The MTP solution was stored at -80~ Calf brain tubulin was purified by glass coverslips (Bellco) at a density of 100,000-200,000 cells/ml, cultured for ammonium sulfate fractionation and ion-exchange chromatography, stored in 1 day, and then treated with the drugs in culture medium for the desired liquid nitrogen, and prepared for use as described previously (17, 20, 21). concentration and time. Residual DMSO in cultures was 0.1% (v/v). The Tubulin concentrations were determined spectrometrically at 275 nm in 6 M coverslips were processed as described previously (24) with modifications. In guanidine hydrochloride (E275nm = 1.09 liter-g-l'cm-1; Ref. 22) or in 0.5% brief, the coverslips were washed with PEM containing 4% polyethylene sodium dodecyl sulfate in neutral aqueous buffer (E275nm = 1.07 liter'g -1" glycol 8000 (Sigma). The cells were lysed with 0.5% (v/v) Triton X-100 and cm-1; Ref. 17). Microtubule protein concentrations were determined using the 1 mM GTP in PEM for 90 seconds at room temperature; then the coverslips Bio-Rad Coomassie assay (23) with BSA and purified tubulin standards. were washed with PEM-polyethylene glycol-1 mu GTP. The coverslip-at- Mierotubule Assembly. The in vitro polymerization of microtubules was tached cytoskeletons were fixed with 3.7% (w/v) formaldehyde-l% DMSO in monitored by turbidity at 350 nm in a Beckman DU 70 spectrophotometer with PEM for 30 min and kept in PBS at 4~ Alternatively, the complete cells were a thermostated 1-cm light path cell at 37~ For MTP, the polymerization fixed with 3.7% formaldehyde (10 min at room temperature), methanol (10

0.6

a 0.5 A (R) b i,

o 0.4 v~ C (s) Fig. 2. Turbidity time course of in vitro microtubules assembly. Twenty /./,M tubulin (a) with added 0.3 ~M R isomer (b) or with 0.3 ~M S isomer (c). o 0.3 Inset, in a different experiment, the plateau absor- bance values of tubulin polymerization were measured without ligand 01), in presence of 0.5 ktu R isomer (~), or in the presence of 0.5 p,u S isomer I'4 5 (A). m0 0.2 / / ,a // / 03 o

0.1 I// i ~ ~ 0.00 TObullSn~m~ 0

0.0 I I I I 6 12 18 24 30 Time (min) 76 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1994 American Association for Cancer Research. EFFECTS OF CI 980 AND NSC 613863 ON MICROTUBULES AND CELL DIVISION

i , 1 I i , I I I 9 :]; 0.05% gelatin PBS) were incubated for 45 min at room temperature. The plates ...-'" were washed five times in 0.075% Tween-PBS and developped with 1,2- 0.9 A phenylendiamine and HaO2. The absence of undesirable background or cross- reaction was verified by omitting the first antibody, by the use of an irrelevant

370C 13~ C 37 ~ C monoclonal antibody (P1C3 against a neutrophil membrane protein; Ref. 26), E ,0. ,0. and by using wells without cells. c- Determination of DNA Content. Flow cytometric analysis was performed to I',- 0.6 as described (27) with modifications. Ceils were plated at 100,000- ,r ., ." ": 300,000/mL and incubated with drugs. One ml of resuspended cells was .~ centrifuged, and the cell pellet was resuspended in 350/xl of RPMI-1640. Then l-- 125 /xl of RPMI-1640 containing 0.2% Nonidet P40, followed by 25 /.d of 1 ~,. ". : mg/ml propidium iodide, were added. After 30 min at room temperature, the cells were analyzed in a Coulter Epics CS cytofluorometer. <.a 0.3

RESULTS

Effects of CI 980 and NSC 613863 on the Assembly of Purified 0.0 Tubulin and Microtubule Protein. Fig. 2 shows the effects of these 0 12 24 36 4.8 60 72 84 96 108 120 R and S enantiomers on the turbidimetric time course of assembly of Time(rain) microtubules from purified tubulin (2 mg/ml). Half inhibitory con- 2.0 centrations were 1.6/XM for the R isomer and 0.4/XM for the S isomer. 1.2 The half inhibitory concentrations for microtubule protein (1 mg/ml) B were 2.10 /~r~ and 0.75 ~u for R and S isomers, respectively (not

~l o 0.S shown). Therefore, in both cases a clear inhibition was noticed in the I:I o~ 0.4// presence of substoichiometric R or S isomer concentrations. However, ,~ a o.o the S enantiomer was 3-4-fold more active than the R isomer. The ~' 1.2 0.0051015202531 process was also examined at varying protein concentrations with a C \ Tubulin (mg/ml) fixed R or S concentration (0.5/xr~). The results, shown in Fig. 2, inset, ~ \ indicated an increase of the protein concentration required for the 9~ 0.8 \ assembly (abscissa intercept of the plot, 1.2 mg/ml in the presence of O x R isomer and 1.5 mg/ml in the presence of S isomer). Electron microscopy examination (not shown) indicated that the polymers formed <3 0.4 under these conditions were microtubules. a Fig. 3A displays the results of adding 20 /xM R or S isomer to assembled tubulin preparations after the turbidity plateau had been J L ~ ' 3"0 0 8 12 18 24 reached. A difference in the behavior of both compounds was noticed. Time (rain) While the R isomer induced a small decrease in turbidity, the same Fig. 3. A. Effect of the enantiomers on assembled microtubules. Samples of tubulin (1.9 concentration of S isomer induced a marked turbidity increase. Elec- mg/ml) were polymerized to plateau turbidity. At the time indicated by the black arrow, tron microscopic examination showed microtubular structures and the compounds were added. Control tubulin without addition ( ), with 20 p,M S isomer some aggregates for the R compound, while for the S enantiomer some (...), with 20 ~M R isomer (------), or with 20 ~M MTC (--'--), which is a rapidly binding colchicine analogue (37). B. Turbidity time course of in vitro abnormal polymer microtubules remained visible and large amorphous structures ap- formation. Control tubulin 2 mg.m1-1 (a), 50/XM Risomer with tubulin 2 mg.m1-1 (b), 50 peared. Polymers formed in the presence of S isomer could be depo- p,u S isomer with tubulin 2 mg-m1-1 (c), or 3 mg.m1-1 (d). At the time indicated by the arrow, the sample was cooled at 13~ Inset, dependence of polymer formation on total lymerized by cooling to 13~ for 15 min, and the residual turbidity tubulin concentration. Plateau turbidities were measured in the presence of 50 ~r~ R observed was due to protein aggregates. Rewarming to 37~ induced isomer (1) or S isomer (12]). microtubule assembly in the control. With the R isomer, a weak turbidity increase took place (no microtubules were observed), min at -20~ and finally acetone (30 s at -20~ The fixed cytoskeletons or whereas the S isomer induced a much larger turbidity, which was cells were incubated for 1 h with DM1A monoclonal antibody reacting with again cold reversible. ot-tubulin (diluted 1:400) in PBS containing 10 mg/ml BSA at 37~ After Formation of the tubulin-colchicine complex is known to induce washing twice with PBS and gentle agitation (10 min), the coverslips were overlaid with fluoresceinated goat-antimouse immunoglobulins (diluted 1:100 protein conformational changes which result in GTPase activity (28, in PBS-BSA-0.02% NAN3), incubated for 45 rain at 37~ washed with PBS in 29) and polymerization into abnormal polymers (22, 30). Like col- the dark, and mounted with antifading solution. The cytoskeletons or cells were chicine, the two compounds CI 980 and NSC 613863 induce GTP photographed on Kodak Tri-X film using a Zeiss Axioplan microscope hydrolysis when they bind to tubulin (15). Therefore, and in view of equipped with epiilumination, 63x Plan-Apochromat and Plan-Neofluar 40x the above results, it was of interest to examine their ability to induce objectives, and the appropriate filters for fluorescein. the formation of polymers that are different from microtubules in Determination of Assembled Tubulin in Cytoskeleton. Quantification of glycerol-free buffer. As shown in Fig. 3B, Trace b for R and Traces c tubulin in cytoskeletons was made by an enzyme-linked immunoassay modi- and d for S, both isomers, after a lag time, produced an increase of fied from the procedure of Nieto (25). Cells were plated at 100,000/ml (0.1 ml) turbidity which was reversible upon cooling to 13~ and was protein in 96-well plates, cultured for I day, and then treated in triplicate with the drugs concentration dependent. The inset displays a critical protein concen- for the desired time. They were then lysed and fixed as above. The plates were blocked with 0.4% gelatin for 1 h at 37~ After the plates were incubated with tration plot showing that the S enantiomer needs less tubulin (0.52 DM1A antibody (diluted 1:3200 in 0.05% gelatin PBS) for 1.5 h at room mg/ml) than the R isomer (1.00 mg/ml) to induce abnormal polymer temperature, they were washed three times with 0.075% Tween 20 in PBS. formation. Negatively stained, fixed preparations under the electron Then peroxidase-conjugated rabbit-antimouse immunoglobulins (1:1000 in microscope showed the absence of microtubules and the formation of 77 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1994 American Association for Cancer Research. EFFECTS OF CI 980 AND NSC 613863 ON MICROTUBULES AND CELL DIVISION

06 L 0 ...... r

E 05 o-'N .... 9...... r ~~ 9 "... II "', 0 " I... 0 "".,,. q- _ N~ 04 "'..... 0\. Fig. 4. Depolymerization of PtK2 cell microtubules by R and S isomers. Cells were incubated for IF..... \.\. 4 h at 37~ without drugs (ll), with colchicine (IS]), with R isomer (O), or with S isomer (O). Cytoskele- tal tubulin was quantified by enzyme-linked immu- 03 - ""t ",, nosorbent assay (see "Materials and Methods")...... "\ Data, mean ___ SD of four independent experiments made in triplicate. O~ o "... \\ 0.2

"0..... 9 9.. ,. 9.,...0 01

O.../01. I ! I / 001 0.01 0.1 I 10 [INHIBITOR] (IJM)

large, filamentous structures, similar to the results described for the Mitotic Arrest of Leukemic and Epithelial Cell Lines by C1980 polymerization of the tubulin-colchicine complex (22, 31). and NSC 613863. The effect of R and S isomers on different human Depolymerization of the Cytoplasmic Microtubules of PtK2 (U937 and HL6O) and murine (EL-4) leukemias and on PtK2 cells Cells by CI 980 and NSC 613863. We first determined the dose was studied. Cells were incubated for 17 h with different concentra- curve of both isomers on the cytoplasmic network of PtK2 cells by tions of drugs. It was found that 1 nMS enantiomer or 25 nM R enan- immunoassay tubulin determination in cytoskeletons and also by im- tiomer fully stopped the cell cycle in mitosis in U937 (Fig. 9A), munofluorescence microscopy. The first method permitted quantifi- HL60, and EIA cell cultures (results not shown). This inhibition of cation of microtubule inhibition (Fig. 4), while the second permitted cell proliferation occurred in a very narrow concentration range, un- visualization of the microtubule network morphology (Fig. 5). The like the depolymerization of cytoplasmic microtubules which took depolymerization by the S isomer is significant at 3 nM, while the place in a wider range of concentrations (Fig. 4). There was an accu- effect of the R isomer is significant at 50 nM. The immunoassay data mulation of cells that had probably finished their S phase but did not correspond with the observation by indirect immunofluorescence. The proceed through mitosis. The PtK2 cells were more resistant to these half inhibitory concentrations for the S and R isomers were 6 nM and drugs; nevertheless, they were also stopped in mitosis by concentra- 100 nM, respectively, while that of colchicine was 1 /~M. The effect tions of 5 nM and 50 nM S and R isomers, respectively (Fig. 9B). The increased gradually until reaching maximal depolymerization of mi- PtK2 cell line presented some polyploidies both in control and crotubules. Both drugs were more clearly active than colchicinc. We treated cells, and with longer drug incubation periods, both octoploid next studied the time course of the microtubule depolymerization by and tetraploid cells appeared. The time course of this cell arrest was 20 nMS and 700 nM R isomers. Figs. 5 and 6 show the results for both studied next. In these experiments, it was found that U937, HL60, drugs. The effect of the R isomer was significant 30 min after treat- and EL4 cells were maximally arrested after 8 h of treatment (Fig. ment, and the effect of the S isomer was noticeable 15 rain after 10). PtK2 cells were arrested after 24 h of treatment (results not incubation. Half maximal inhibition was reached after two h treatment shown). To examine if the effect was reversible, cells were incubated with both R and S isomers, and inhibition was nearly maximal at 4 h. for 24 h in the absence or presence of 5 nMS isomer or 50 nm R iso- Then both drugs slowly increased their effect with time until 24 h. In mer. Then some were washed and their recovery was studied, while reversibility experiments (Figs. 7 and 8), microtubules of cells treated other cells were not washed. As a reversible control drug, we used with R isomer began to repolymerize after 15 rain, while PtK2 cells 250 nu MTC. In PtK2 cell cultures treated with MTC, R and S iso- treated with S isomer required more than 30 min. In the reverting PtK2 mers were partially recovered on day 5 after washing (Table 1). cells, bright microtubule organizing centers in their cytoskeletons Much debris was found in unwashed cells as well as tetraploid and were observed. Half-recovery times were about 1 and 2 h, respec- octoploid cells (results not shown). On the other hand, with U937, tively. Then the reversion continued, and at 24 h of incubation, the R HL60, and EL-4, no reversibility was found with either 25 nM R or 1 isomer-treated cells had totally reverted, while those treated with the nMS isomers nor with 50--100 nM MTC (results not shown). In some S isomer had not completely recovered. This experiment shows again experiments, U937 and HL60 cultures treated with R isomer and that the S isomer is more active than the R isomer, since its reversion washed had around 10% of the control of viable cells 5 days later is slower and is still not completed 24 h later. (results not shown). In summary, the effect of R and S enantiomers 78 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1994 American Association for Cancer Research. EFFECTS OF CI 980 AND NSC 613863 ON MICROTUBULES AND CELL DIVISION

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enantiomers on preformed microtubules diverged. The S isomer was able to induce the formation of thermosensible anomalous structures under microtubule assembly conditions much more markedly than the .6 ,,~ ,~176 R isomer. This difference in the behavior of both enantiomers could be related to the critical protein concentration required to form abnormal structures in the glycerol-containing microtubule polymerization E . r- buffer. In the absence of glycerol, the critical protein concentration necessary for abnormal polymerization was two times higher for the R isomer than for the S isomer. Effects of CI 980 and NSC 613863 on Cytoplasmic Microtu- LIJ 0 bules. The S and R enantiomers induce a maximal disruption of the Z cytoplasmic microtubule network of PtK2 cells with the S enantiomer m being approximately 16 times more active than the R enantiomer. n- These drugs are much more powerful than the usual inhibitors of O (D cellular microtubules such as colchicine, MTC, and nocodazole m (Fig.4; Refs. 38 and 39). The assembly of the microtubules of PtK2 cells after the S and R enantiomer removal is considerable slower than with MTC (38), nocodazole (39, 40), MDL 27048 (35), or Colcemide .o (41) and similar to that of colchicine (41). Complete microtubule repolymerization requires 24 h of incubation for the R enantiomer and longer periods for the S enantiomer. Effects of CI 980 and NSC 613863 on Cell Division. Our results _/,~ t I I ,_1 I 0 0.4 I 4, I0 4,0 with human (U937 and HL60) and murine (EL4) leukemias indicate that as little as 1 nMS or 25 nM R enantiomers arrested these cells in TIME (h) mitosis with CI 980 being the most active proliferation inhibitor. The Fig. 6. Time course of R and S isomer-induced depolymerizationof the microtubules growth arrest was obtained after 8 h of incubation in the presence of of PtK2 cells. Cells were incubated without drugs (I), 700 nra R isomer (Q), or 20 nMS the drugs. The high degree of mitotic arrest found after a single cell isomer ((3) at 37~ during different time periods. Cytoskeletaltubulin was determined as in Fig. 4. cycle time of exposure to these drugs is remarkable. It is similar to that found in HeLa cells in response to Vinca alkaloids (42, 43), colchicine (44), and nocodazole (45). We have also found that the inhibition of on cell division was found to be essentially irreversible in the leuke- cell proliferation and mitotic arrest occur at concentrations of drugs mic cells, while they were partially reversible in PtK2 cells.

DISCUSSION Interactions of CI 980 and NSC 613863 with Tubulin and Mi- crotubules in Vitro. Both S and R isomers inhibited substoichiometrically the in vitro assembly of microtubules in a similar way to 0.6 known microtubule inhibitors such as colchicine, podophyllotoxin, vinblastine, or MDL 27048 (32-35). This inhibition was related to an :4/- l -I---.I-I ...... I increase in the critical concentration required for tubulin and MTP E 0.5 C: ...... polymerization, and the S enantiomer was approximately 2--4-fold (xl more powerful than the R enantiomer. 0") In a previous report (15), both R and S enantiomers were shown to 0.4 .."6"'"" O W ..'" O~ bind at the colchicine site and compete with podophyllotoxin, sug- 0 gesting an overlap of the ring A (trimethoxyphenyl) binding subsite, Z _ , :'0~"'"/0 which is common to podophyllotoxin and colchicine. Both enantiom- nn 0.3 ers and colchicine induced the tubulin GTPase activity, whereas podo- n- O phyllotoxin did not. Preliminary results obtained using tropolone 0') m methyl ether as a blocking agent of the ring C (tropolone) subsite 0.2 appeared to indicate that this part of the colchicine site is also involved in the binding of both compounds. In the present study, it has been shown that both ligands induced the formation of anomalous tubulin polymers, which is characteristic of 0.1 colchicine ligands such as MTC, allocolchicine, and biphenyl colchicine analogues (17, 31, 36, 37). It has been proposed that in order to induce the abnormal tubulin polymers, colchicine analogues have to _4/ I I ~ I bind with their C rings in proper orientation into the corresponding 0 0.4 I 4 I0 40 subsite of the colchicine binding site (37). The present results indicate TIME (h) that the two enantiomers act in the same manner as colchicine at stoichiometric concentrations, perturbing the normal protein-protein Fig. 7. Reversibilityof R and S isomer-induced depolymerizationof the microtubules of PtK2 ceils. Cells were incubated without drugs (l), 700 nM R isomer (O), or 20 nMS interactions involved in microtubule formation, which supports the isomer ((3) at 37~ for 4 h. Then cells were washed three times with fresh culture medium hypothesis that they also bind to the ring C locus. The action of both and incubated in drug-free medium during different periods of time. 80 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1994 American Association for Cancer Research. EFFECTS OF CI 980 AND NSC 613863 ON MICROTUBULES AND CELL DIVISION

ti~. N. FiIIl~_' C(~tlr~,u of r11i~-'Ft}WhLIIC r~_'pOl>Tl/cri/tttilH1 I~>IIL~'~',ila~ .% J',~m~.'r rcnl~)~.ttl. [~11'~-~ ~.'clJ~, \~ L'rc tr~_'tttcd \,,ill1 7(,~() i~xl R <~i 21) T/\l >,' is~mcrs Ic~r 4 h at 37~ Then the drug was rcn'~c~vc.'d ;_[Tll.I the\ were ctlltLlrccl iT1 l'rosh rllcciiunl clciring ditti.'rcilt time' p<_'rit~d,,: ~<,il~plct~' <.'ell,, w~'rc lixcct and pic~ccs<,cd For imllltlll~llitlorcsccrlcr o[ microlubulcs. Ba#; 10 #am. A, cclls :~t iil~lc li cil[t:r .$' ismnlcr re'ill()\ ~tl. Thi, is ~tlso rcl~rc'scntati~ c_' oH ~ h~I i,, ~t~',cr~ cd x~ ith ih~_' f7 i~,on?c.'r :tt 15 n]in ~illd ~.~,ith i.hu .S' i,>c~mcr ~ll 15 ~inct :ilso 3(1 nlin. /7+ cells i~t 1 h after S isomer rcnlt)\ ~ll. This is zil,,c) rc'l~lc<,cnl~Hi\ c tH \\h~l~. i~, sct_'il ~ilh tlw 17 i,,~mml .111 it~iiT tilter cliu 7 rul~l~)\ttl. ( . cells {ll lime.' 3 h tiflor .~ i-,~nlcr ic.'rll~)~,~il. This is also observed with the <7 isomer :tt 2-24 h ~ti]ct with the R i:-,t)i]~c,i ;1{ [ 4. h. 1). c{_'ll <, 24 h zdl~..r f7 i,,c~mc.'r lC..lllt~\ <11.

A that obtained with MTC (38) and not complete. The differences found in reversibility with the different human and murine leukemias and 0 Ptk2 cells might be due to differences in sensitivity to induction of apoptosis by microtubule-disrupting agents (48) in some of these cell lines. Studies are in progress to examine this possibility. Mechanisms of Action of C1980 and NSC 613863. There are two to L L puzzling observations in this study: (a) while both enantiomers bind o to tubulin and inhibit microtubule assembly in vitro not very differ-

0 ently from known microtubule inhibitors, the inhibitory concentrations in the cells examined are orders of magnitude smaller; and (b) ..Q the binding to tubulin is weakly selective for the S enantiomer, and E ZD this compound inhibits microtubule assembly in vitro 3-4 times more powerfully that the R enantiomer. However, the cellular microtubule inhibition and the mitotic arrest are strongly chiral selective by a factor of -20 in favor of the S compound. This selectivity is in qualitative agreement with previous results (7, 8,), although in our Fluorescence intensity experimental conditions, the differences in activity between the S and R enantiomers are less pronounced. B The difference between the inhibitory activity of the S and R compounds in vitro and in cells is reminiscent of results with HeLa cells and Vinca alkaloids (42, 46, 49, 50). HeLa cells incubated with vincristine and vinblastine accumulate significantly higher concentra- if} tions intracellularly, in some cases 200-fold higher than the concen- (D o tration in the external medium (42). Recent results with the S enantiomer and L1210 cells indicate that it rapidly reached intracellu- 0 lar concentrations in considerable excess of that in culture medium,

o and a model involving passive diffusion followed by significant re- E versible binding to intracellular or cell membrane components has D z been suggested (10). The in vitro affinity of tubulin for this compound does not seem to account for such a mechanism. Another possible explanation could be the metabolism of both compounds. However, a recent study has indicated that both the activity and the difference in potency between the R and S enantiomers are retained in major hy- Fluorescence intensity droxylated metabolites (9). Other possible explanations could be sought in the following hypotheses that: (a) high sensitivity to these Fig. 9. Dose effect of R and S isomers on cell division. U937 (A) and PtK2 cells (B) were incubated for 17 h in the absence (a, b, g, and h) or in the presence of the following compounds by a particular subset of cellular tubulin molecules is not drug concentrations: R isomer, 1 (c), 25 (e and i), or 50 (k) nM; and S isomer, 0.5 (d), 1 well represented in the brain preparations used; (b) there exists cel- (jr), 2.5 (j) or 5 (1) riM. The relative DNA content per cell was measured by fluorocy- lular factors which modulate the intrinsic sensitivity of microtubules tometry (see "Materials and Methods") of 5000 cells. to these compounds; (c) there is an additional, sensitive cellular target, different from tubulin, which would be chiral selective for CI 980 much lower than those needed for a complete depolymerization of more than for the R isomer, causing the inhibition of microtubules; or cytoplasmic microtubules. This could indicate that the inhibition of (d) CI 980 could have an additional target that does not inhibit proliferation by the R and S enantiomers, as well as by MTC, colchi- microtubules but inhibits cell division, i.e., C1980 could have multiple cine, and nocodazole, could be due to perturbation of the spindle and independent mechanisms of action. microtubule dynamics and not due to depolymerization of the microtubules, as demonstrated by Jordan et al. (42, 46) for HeLa cells and Table 1 PtK2 cell cycle distribution following R- and S-isomer exposure and removal Vinca alkaloids. During 17 h of exposure of the cultures to R and S Percentage of cells in specific cell cycle phases following treatment with 50 nM R- or enantiomers, cells did not multiply, and following the washing of the 5 nM S-isomer for 24 h and then removal of the drug (time 0). Results of a representative drugs, very little or no reversibility in the inhibitory effect was found. experiment out of 3 are shown here. Similar results were obtained with MTC that seem to be irreversible Time in these leukemias, although this inhibitor has been found to be Treatment (h) G1 S (G2 + M) reversible with K562 myeloblastic leukemia (unpublished results). Control 0 58 12 30 Previous results with the racemic NSC 370147 (47) indicated that the R-isomer 0 11 7 82 inhibition of L1210 proliferation after 12 h exposure was irreversible S-isomer 0 11 8 81 MTC 0 8 13 79 24 h after drug removal. On the other hand, exposure of PtK2 cells to 5 nM S or 50 nM R isomers (during the 24-h incubation period) resulted Control 48 64 17 19 in a partially reversible growth arrest. Cells arrested in mitosis for R-isomer 48 40 22 38 S-isomer 48 41 22 37 extended periods of time can die or return to interphase without the MTC 48 40 20 40 separation of sister chromatids, resulting in polyploidy (45). After a Control 120 63 11 26 long exposure of the cell culture to these drugs, a high proportion of R-isomer 120 52 23 34 polyploid cells were found. This could affect the normal reversion of S-isomer 120 43 14 43 the cell growth after removal of the drug, which was much slower than MTC 120 46 16 38 82 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1994 American Association for Cancer Research. EFFECTS OF CI 980 AND NSC 613863 ON MICROTUBULES AND CELL DIVISION

A B C

3h 3h 3h L 6h 6h 6h

Fig. 10. Time course of the effect of R and S ~ k. isomers on cell division. Control (Column A), 25 nM A A R isomer (Column B), and 1 n~ S isomer (Column 0 C) treated U937 cells were incubated for 3, 6, 8, and ~D~ 8 h 8h 8h 16 h. r Z A .A 16h 16h 16h k, Fluorescence intensity

In conclusion, the compound CI 980 was found to be a most potent 5-amino-l,2-dihydro-2-methyl-3-phenylpyrido[3,4-b]pyrazine-7-carbamate. J. Med. mitotic and microtubule inhibitor by causes not exclusively attribut- Chem., 32: 2089-2092, 1989. 9. Temple, C., Jr., and Rener, G. A. Antimitotic agents. Chiral isomers of ethyl-(5-amino- able to its intrinsic binding to tubulin. CI 980 is presently being tested 1,2-dihydro-3-(4-hydroxiphenyl)-2-methylpyrido[3,4-b]pyrazin-7-yl)-carbamate. J. in clinical trials (12) and could be of great interest in cancer therapy Med. Chem., 35: 988-993, 1992. 10. Hook, K. E., Przybrnowski, S. A., and Leopold, W. R. Cytotoxicity and uptake of due to its superior or equivalent in vitro activity to vincristine and its CI-980 and its R-enantiomer. Proc. Am. Assoc. Canc. Res., 33: 427, 1992. full activity against vincristine-resistant tumors. 11. Leynadier, D., Peyrot, V., Briand, C., Barasoain, I., de Ines, C., Andreu, J. M., Temple, C. G., Jr., and Rener, G. A. Action of a deazapteridine on microtubular system. Effect of chiralty. Proc. Am. Assoc. Cancer Res., 33: 405, 1992. 12. Leopold, W. R., Elliot, W. L., Prysbranowsky, S. A., and Wand, R. A. In vivo ACKNOWLEDGMENTS evaluation of the potential for therapeutic synergy between CI 980 and standard chemotherapeutic agents. Proc. Am. Assoc. Cancer Res., 34: 296, 1993. We thank Drs. Jos6 Manuel Andreu and Juan Miguel Nieto for discussion, 13. Yeh, H. J. C., Chrzanowska, M., and Brossi, A. The importance of the phenyl- Dr. T. J. Fitzgerald for MTC, Dr. Santiago Abarca and Tom,is Huelamo tropolone "aS" configuration in colchicine's binding to tubulin. FEBS Lett., 229: 82-86, 1988. for technical assistance, and Pedro Lastres for expert assistance with flow 14. Kelleher, J. K. Tubulin binding affinities of podophyllotoxin and colchicine analogs. cytometry. Mol. Pharmacol., 13: 232-241, 1977. 15. Leynadier, D., Peyrot, V., Sarrazin, M., Briand, C., Andreu, J. M., Rennet, G. A., and Temple, C. 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84 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1994 American Association for Cancer Research. Inhibition of Microtubules and Cell Cycle Arrest by a New 1-Deaza-7,8-dihydropteridine Antitumor Drug, CI 980, and by Its Chiral Isomer, NSC 613863

Concepcion de Ines, Daniel Leynadier, Isabel Barasoain, et al.

Cancer Res 1994;54:75-84.

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