Taxane Anticancer Agents - American Chemical Society

Chapter 11

Photoincorporation of a Paclitaxel Photoaffinity Analogue into the N-Terminal 31 Amino Acids of ß-Tubulin 1

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Susan Band Horwitz , Srinivasa Rao , Nancy E. Krauss , Julie M . Heerding , Charles S. Swindell , Israel Ringel , and George A. Orr 2

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Downloaded by CORNELL UNIV on May 17, 2017 | http://pubs.acs.org Publication Date: December 7, 1994 | doi: 10.1021/bk-1995-0583.ch011

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Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461 Department of Chemistry, Bryn Mawr College, Bryn Mawr, PA 19010 Department of Pharmacology, The Hebrew University, Jerusalem 91120, Israel

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3'-(p-azidobenzamido)taxol, a photoaffinity analogue of taxol, covalently binds to the N-terminal domain of ß-tubulin after irradiation of the microtubule-drug complex. The azido analogue has biological properties that are similar to those of taxol, such as the ability to polymerize tubulin into stable microtubules in the absence of GTP. Taxol competes with [ H]3'-(p-azidobenzamido)taxol binding, indicating that both compounds interact with the same site on the microtubule. Cleavage of [ H]3'-(p-azidobenzamido)taxol -photolabeled β-tubulin by formic acid and subsequent mass analysis and protein sequencing have identified the N-terminal 31 amino acids as the major site for [ H]3'-(p-azidobenzamido)taxol photoincorporation. 3

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Taxol is a compound of considerable interest due to its clinical activity as an antitumor drug in humans, its unusual chemical structure, and its unique mechanism of action. The drug was isolated originally from the bark of the tree Taxus brevifolia and the elucidation of its structure was accomplished in 1971 (1). Taxol displayed cytotoxicity in a number of rodent tumor model systems and in tumor cells growing in tissue culture. Studies reported in the late seventies and early eighties clearly indicated that taxol blocked cells in the late G /M phase of the cell cycle, thereby acting as an antimitotic agent (2). Taxol is unusual in its ability to enhance the assembly of microtubules in vitro, even in the absence of GTP that is normally required to achieve assembly. The drug also stabilizes microtubules against depolymerization by cold and C a , treatments that depolymerize normal microtubules to their α-and β- tubulin dimers (3,4). Taxol binds to the microtubule with a stoichiometry, relative to the constituent tubulin heterodimers, approaching 2

+ +

N O T E : Paclitaxel is the generic name for Taxol, which is now a registered trademark. 0097-6156/95/0583-0154$08.00/0 © 1995 American Chemical Society

Georg et al.; Taxane Anticancer Agents ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

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HORWITZ E T A L .

Photoincorporation of Paclitaxel Analogue

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1 (5,(5). When cells are treated with taxol, the microtubule cytoskeleton is reorganized and stable bundles of microtubules that are diagnostic of taxol treatment are present in the cell (2). Although it has been known for the past fifteen years that taxol interacts with the tubulin/microtubule system, there has been no definitive information available on the taxol binding site on the microtubule. An excellent method for decipherinjg the binding site is photoaffinity labeling. In the absence of effective radiolabeledphotoaffinity analogues of taxol, [ H]taxol was used directly to photolabel tubulin. When a complex of microtubule protein and [ H]taxol was irradiated at 254nm and analyzed by denaturing gel electrophoresis, it was observed that the drug bound covalently to the 0-subunit of tubulin (7). However, the extent of photoincorporation was too low to pursue these studies further. The ability to prepare 3'-(p-azidobenzamido)taxol (Figure 1) and to radiolabel it with tritium to approximately 2.0 Ci/mmol has allowed the further delineation of the taxol binding site (8). A comparison of the effects of GTP, taxol and the azido analogue on microtubule assembly in vitro is seen in Figure 2. Both taxol and the azido analogue induced the assembly of C a stable microtubules in the absence of GTP, a unique characteristic of taxol activity. In a series of related experiments, it was observed that the binding of H-taxol to microtubules was specifically inhibited by both unlabeled taxol and the azido analogue, indicating that both are occupying the same binding site (Figure 3). Although the azido analogue was slightly less active than taxol in vitro, its effects in cells were similar to those of taxol (9). Like taxol, it induced the formation of normal microtubules plus hoops and ribbons as determined by electron microscopy, and immunofluorescence studies revealed the presence of stable microtubule bundles in Chinese hamster ovary cells (CHO). In terms of cytotoxicity, the azido analogue was 3-fold less active than taxol when tested in the murine cell line, J774.2 (9). jS-Tubulin was specifically labeled with H-(p-azidobenzamido)taxol (Figure 4), and after 30 min of irradiation the efficiency of photoincorporation of the azido analogue ranged from 2 to 6%. A ten-fold excess of taxol or unlabeled 3'-(pazidobenzamido)taxol reduced photoincorporation. In contrast, baccatin III, that is known not to have taxol-like activity (10), had no effect on photoincorporation. Such experiments indicated that 3'-(p-azidobenzamido)taxol specifically labeled βtubulin under the conditions of our experiments. It is known that formic acid cleaves Asp-Pro bonds (11-13) and since there are two such bonds in β-tubulin, as indicated below, formic acid digestion was carried out with purified β-tubulin to determine into which fragment the azido analogue had been incorporated (8). Since the molecular weight of each fragment differs, they can be readily resolved on denaturing gels. 3


3

++

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3

304 305

31 32

NH2

A 1

*

A

2

*

A3

COOH

A time course of formic acid treatment demonstrated that after complete digestion of 0-tubulin all of the radiolabel was associated with the lowest molecular weight fragment. High performance electrophoresis chromatography (HPEC) was used to analyze further the photoaffinity labeled ^-tubulin and its formic acid digestion products (Figure 5). As can be seen, the radiolabeled 0-tubulin migrated as a Georg et al.; Taxane Anticancer Agents ACS Symposium Series; American Chemical Society: Washington, DC, 1994.


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TAXANE ANTICANCER AGENTS

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H-3'-(p-Azidobenzamldo)taxol 3

Figure 1. Molecular structure of [ H]3'-(p-azidobenzamido)taxol.

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Ca + Taxol

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TIME (min) Figure 2. Assembly of microtubule protein (MTP) in the presence of GTP. MTP (1.5 mg/ml) was incubated at 35°C with either ImM GTP or 20μΜ taxol or 20μΜ 3-(p-azidobenzamido)taxol [3'-(P-ABA)T]. The assembly reaction was followed turbidimetrically. At the time designated, 4mM CaCl was added to each sample. (Reproduced with permission from ref. #9). /

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HORWITZ E T A L .

I

I 10

I 20 Drug (μΜ)

I 30

Figure 3. Competition between taxol and 3'-(p-azidobenzamido)taxol for the binding site(s) on microtubules. MTP (1.5 mg/ml) was assembled in the presence of ΙΟμΜ H-taxol and various concentrations of unlabeled taxol ( · ) , or 3-(p-azidobenzamido)taxol (•). H-taxol and its competitor were added simultaneously. The microtubule polymer was centrifuged through a 50% sucrose layer and the pellet was analyzed for H-taxol. Binding obtained in the presence of H-taxol alone represents the 100% value (counts/min//*g protein). (Reproduced with permission from ref. #9) 3

/

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Figure 4. [ H]3 -(p-Azidobenzamido)taxol specifically photoincorporates into β-tubulin. [ H]3'-(p-Azidobenzamido)taxol (5μΜ, 1.7 Ci/mmol) was added to MTP (5 μΜ tubulin) and incubated at 37°C for 30 min. After the incubation, the sample was irradiated at 254nm for 30 min and analyzed by SDS-PAGE and by fluorography. A, SDS-PAGE followed by Coomassie staining. B, fluorography of A. Lane 1, no additions; lane 2, addition of 50 μΜ taxol; lane 3, 50 μΜ unlabeled 3-(p-azidobenzamido)taxol; lane 4, 50 μΜ baccatin III. The gel was exposed for 10 days. (Reproduced with permission from ref. #8) 3

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11.

FRACTION NUMBER

Figure 5. High performance electrophoresis chromatographic analysis of [ H]3'-(p-azidobenzamido)taxol-photolabeled 0-tubulin (A) and its formic acid digestion products (B). Intact 0-tubulin was resolved on a 7% polyacrylamide gel (A) and digested β-tubulin on a 12% polyacrylamide gel containing 6 M urea (B). In each case, 75,000 dpm in a total volume of 25 μ\ was loaded. Electrophoresis was conducted either at 0.3 mA for 30 min followed by 1.5 mA for 5 h (A) or at 0.4 mA for 30 min followed by 1.3 mA for 5 h (Β). Fractions were counted by liquid scintillation spectrometry. Insulin (3 kDa, A/B chains), aprotinin (6.5 kDa), lysozyme (14.3 kDa), carbonic anhydrase (29 kDa), and bovine serum albumin (67 kDa) were used as markers. (Reproduced with permission from ref. #8) 3


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single peak of the appropriate size. The major radiolabeled peptide after a 120 hr formic acid digest migrated with an apparent molecular weight of 6500 daltons. Amino acid sequencing of this radiolabeled fragment revealed that the first 20 amino acids were identical to those that have been reported for the N-terminal sequence of human 0 -tubulin (14). Due to the apparent discrepancy between the predicted molecular weight of the Ai fragment and the apparent molecular weight determined by HPEC, it was necessary to prove rigorously that formic acid was cleaving β-tubulin at Asp Pro . The mass of the N-terminal peptide of β-tubulin generated by formic acid cleavage was determined by electrospray mass spectroscopy after modification of Cys by either pyridylethylation or carboxymethylation. The calculated and experimentally determined monoisotopic mass of the NH -terminal domain of βtubulin corresponded very closely, thereby proving that formic acid cleaves at Asp -Pro in β-tubulin. 2

31

32

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2

31

32

Modification pyridylethylation carboxymethylation

Experimental Mass 3584.97 3537.85

Theoretical Mass 3584.73 3537.68

The sequence of the N-terminal 32 amino acids of β-tubulin, with an arrow indicating the site of formic acid cleavage, is shown below. 10 MET-ARG-GLU-ILE-VAL-HIS-ILE-GLN-ALA-GLY-GLN-CYS-GLY-ASN-GLN20 30 ILE-GLY-ALA-LYS-PHE-TRP-GLU-VAL-ILE-SER-ASP-GLU-HIS-GLY-ILEASP-PRO

t The N-terminal region of β-tubulin isoforms is highly conserved throughout nature (15). Since taxol is known to interact with microtubules from a variety of cell lines and sources, it is reasonable that its binding site be in a conserved domain of βtubulin. It is interesting to note that recent experiments have implicated Cys in β-tubulin as being part of the binding site for the exchangeable GTP that is hydrolyzed during microtubule polymerization (16). The only compound that can substitute for GTP in the in vitro polymerization of tubulin is taxol, a drug that is now known to bind to β-tubulin in the same region as the exchangeable GTP. A recent study that examined the binding site in tubulin for the antimitotic agent colchicine, indicated that there are two distinct domains of β-tubulin that form part of the binding site for colchicine (17). One of these regions maps to the N-terminal region of β-tubulin. Although both taxol and colchicine are antimitotic agents, colchicine, in contrast to taxol, interacts with the tubulin dimer and inhibits the 12


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polymerization of tubulin in vitro. All of these studies implicate the N-terminal domain of /8-tubulin as an important regulatory site for microtubule assembly. The interaction of taxol or colchicine with this domain of jS-tubulin could result in distinct conformational changes that would regulate tubulin polymerization in different ways. To understand the interaction of taxol with its cellular target, the microtubule, which is crucial to comprehending the basis of the antitumor activity of this drug, it is necessary to determine the binding site for the drug. This information, in addition to structure-activity data for taxol, will provide knowledge that could lead to the design and synthesis of new and improved taxol analogues. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

Wani, M.C.; Taylor, H.L.; Wall, M.E.; Coggon, P.; McPhail, A.T. J. Am. Chem. Soc. 1971, 93, 2325-2327. Schiff, P.B.; Horwitz, S.B. Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 15611565. Schiff, P.B.; Fant, J.; Horwitz, S.B. Nature 1979, 277, 665-667. Schiff, P.B.; Horwitz, S.B. Biochemistry, 1981, 20, 3247-3252. Parness, J.; Horwitz, S.B. J. Cell Biol., 1981, 91, 479-487. Diaz, J.F.; Andreu, J.M. Biochemistry, 1993, 32, 2747-2755. Rao, S.; Horwitz, S.B.; Ringel, I. J. Natl. Cancer Inst., 1992, 84, 785-788. Rao, S; Krauss, N.E.; Heerding, J.M.; Swindell, C.S.; Ringel, I.; Orr, G.A.; Horwitz, S.B. J. Biol. Chem., 1994, 269, 3132-3134. Swindell, C.S.; Heerding, J.M.; Krauss, N.E.; Horwitz, S.B.; Rao, S.; Ringel, I. J. Med. Chem., 1994, 38, 1446-1449. Parness, J.; Kingston, D.G.I.; Powell, R.G.;, Harracksingh, C.; Horwitz, S.B. Biochem. Biophys. Res. Commun., 1982, 105, 1082-1089. Sonderegger, P.; Jaussi, R.; Gehring, H . ; Brunschweiler, K.; Christen, P. Anal Biochem., 1982, 122, 298-301. Hall, J.L.; Dudley, L.; Dobner, P.R.; Lewis, S.A.; Cowan, N.J. Mol. Cell. Biol., 1983, 3, 854-862. Lee, M.G.-S.; Loomis, C.; Cowan, N.J. Nucleic Acids Res., 1984, 12, 58235836. Lewis, S.A.; Gilmartin, M.E.; Hall, J.L.; Cowan, N.J. J. Mol. Biol., 1985, 182, 11-20. Kimmel, B.E.; Samson, S.; Wu, J.; Hirschberg, R.; Yarbrough, L.R. Gene (Amst)., 1985, 35, 237-248. Shivanna, B.D.; Majillano, M.R.; Williams, T.D.; Himes, R.H. J. Biol. Chem., 1993, 268, 127-132. Uppuluri, S.; Knipling, L.; Sackett, D.L.; Wolff, J. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 11598-11602.

R E C E I V E D September 20, 1994


Taxane Anticancer Agents - American Chemical Society

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