Vol.
184,
No.
2, 1992
April
30,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
MARINE
SPONGE POLYKETIDE PROTEiN TYROSINE
INHIBITORS KINASE
765-772
OF
Rita t-l. Leel, Doris L. SlateI*, Robert Moretti2, Khisal A. Alvi3, and Phillip Crews3# ‘Institute of Cancer and Developmental Biology and *Institute of Bio-organic Chemistry, Syntex Research, Palo Alto, CA 94304 3Department
Received
March
of Chemistry and Biochemistry and institute of Marine Sciences, University of California, Santa Cruz, CA 96064 10,
1992
Summary: The marine polyketide natural product, halenaquinone, was shown to be an irreversible inhibitor of pp60 v-src, the oncogenic protein tyrosine kinase encoded by the Aous sarcoma virus. This compound had an IC50 of approximately 1.5 $vI against pp6Ov-src and also inhibited the ligand-stimulated kinase activity of the human epidermal growth factor receptor with an l&o of approximately 19 PM. Haienaquinone blocked the proliferation of a number of cuttured cell lines, including several transformed by oncogenic protein tyrosine kinases. Halenaquinol, xestoquinone, halenaquinol sulfate, and several simple synthetic quinone analogs were also shown 0 1992Academic Press,Inc. to inhibit pp6Ov-src.
Protein tyrosine kinases comprise a class of enzymes involved in the regulation of cellular growth and signalling (l-3). A number of natural products of microbial, plant, and marine origin have been reported to inhibit protein tyrosine kinases. Among these are erbstatin (4). herbimycin A (5). genistein (6) lavendustin A (7), staurosporine (8) piceatannol (9) adriamycin (10). and aeroplysinin (11). Synthetic inhibitors of tyrosine kinases, especially the tyrphostins, have also been described; certain tyrphostins can block cell proliferation induced by growth factors which bind to receptors with protein tyrosine kinase activity (12-14). The structural diversity represented among the known natural product inhibitors of tyrosine kinases prompted the examination of tropical marine sponges as potential sources of new inhibitors. These sponges are known to contain diverse, bioactive secondary metabolites (15, 16). Indo-Pacific marine sponges of the Xestospongia and * To whom correspondence addressed. # To whom correspondence be addressed.
on biological assays and synthetic compounds on marine natural products isolation should
should be
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Adocia genera
are a source of polyketide quinones and hydroquinones, including halenaquinone (17-l 9) and xestoquinone (18-20), which possess antibiotic (17) cardiotonic (18), and antiproliferative (19) activity. Although the molecular basis of their antiproliferative activity is unknown, some of these structures could accommodate a nucleophilic Michael addition from reactive thiols in important cellular proteins, as postulated for cytotoxic naphthoquinones (21). We isolated halenaquinone from Fijian collections of Xestospongia ?cafbonaria and examined its effect on the protein tyrosine kinase activity of pp60v-src, the transforming gene product of the Rous sarcoma virus. It proved to be a potent, irreversible inhibitor of this enzyme. Five other marine quinones and related compounds were also studied, along with approximately 20 simple synthetic quinones. The antiproliferative activity of halenaquinone on a number of cell lines, including several transformed by oncogenes encoding protein tyrosine kinases, was also evaluated.
Materials
and Methods
Compounds: Halenaquinone, xestoquinone, halenaquinol, halenaquinol sulfate, and tetrahydrohalenaquinone A were isolated from the marine sponge Xestospongia ?carbonaria (22) gathered from the Benga Lagoon, Fiji Islands (Collection number 91007, identification made by M.C. Diaz). Their spectroscopic properties were consistent with those previously published (17-20). Tetrahydrohalenaquinone B was also isolated from this sponge, and its structure proof will be published elsewhere (Alvi and Crews, in preparation). Synthetic compounds were prepared and structures confirmed at Syntex Research. The reference protein kinase inhibitors, PI, P4di(adenosine-5’)tetraphosphate (AppppA) and S-fluorosulfonylbenzoyladenosine (FSBA), were obtained from Sigma Chemical Company, St. Louis, MO. rat Enzyme Assays: pp60 v-src was purified from Rous sarcoma virus-transformed cells and its protein kinase activity assayed essentially as described by Wong and Goldberg (23), except that 2-mercaptoethanol was omitted from the reaction buffer. Test compounds were solubilized in DMSO or water and incubated with the enzyme for 15 min prior to the addition of [val+angiotensin II (4 mg/ml final concentration, Sigma) and ATP (30 PM final concentration, Sigma) to start the kinase reaction. Approximately l-2 PCi of y[gP]-ATP (New England Nuclear, Boston, MA) was used per reaction. The l&o is defined as the concentration of compound which inhibits 50% of enzyme activity compared to the appropriate vehicle control. Epidermal growth factor receptor tyrosine kinase activity was assayed using detergent lysates of human A431 epidermoid carcinoma cells (24). The crude receptor preparation was incubated in a volume of 15 ~1 with ligand (333 nM epidermal growth factor, Sigma) in 41 mM HEPES, pH 7.4, 8.3 mM MnClz, 20 mM MgCh. 0.17% Triton X-100, 16 PM ATP, and 66.7 PM NasV04 for 15 minutes to permit ligand-stimulated autophosporylation to occur. When inhibitors were tested, they were added during this step. [Vals]-angiotensin II phosphorylation was assayed by adding 10 ~1 of substrate solution (10 mg/ml [val5]-angiotensin II, 275 mM ATP, 3 mg/ml bovine serum albumin [Sigma]) to the autophosphorylation reaction. Approximately 2-3 &i of y[zP]-ATP was used per reaction. After incubation at 35O C for 30 min, s2P-labeled peptide was quantitated by phosphocellulose filter paper binding as described by Wong and Goldberg (23). 766
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Inhibitor Reversibility: Compounds were tested at concentrations giving 60-80% inhibition in the standard pp60 v-src assay. After a 15 min incubation of compounds with pp60v’src, the incubation solution was diluted 2 or 5-fold prior to the addition of fvalsJ-angiotensin 11 and ATP. The standards employed were AppppA, a reversible competitive inhibitor binding at the ATP site (25) and FSBA, an irreversible inhibitor which reacts with a lysine residue in the ATP binding site (26). Cell Proliferation: CHRC5 multidrug-resistant Chinese hamster ovary ceils were generously provided by Dr. Victor Ling (University of Toronto, Toronto, ON). Mouse NlH3T3 cells transformed by wild-type v-src or v-erbB as well as NY684, a rat cell line transformed by a temperature-sensitive variant of v-src, were provided by Dr. Randall Schatzman, Syntex Research. RR1022 rat cells transformed by Rous sarcoma virus were obtained from the American Type Culture Collection (Rockville, MD). Cells were maintained in minimal essential medium (Irvine Scientific, Santa Ana, CA) supplemented with 10% fetal bovine serum (JR Scientific, Woodfand, CA). Cell proliferation was assessed using the method of Mosmann (27) as modified by Bruno and Slate (28).
Results Halenaquinone and five related marine pentacyclic polyketides were tested against pp6Ov-src, and IGo values determined as shown in Table 1. Hatenaquinol proved to be approximately as potent as halenaquinone, while the closely related xestoquinone was less active. Interestingly, halenaquinol sulfate was much less active than halenaquinol, and the tetrahydrohalenaquinones were both inactive at the doses tested. Halenaquinone also inhibited the ligand-stimulated tyrosine kinase activity of the human epidermal growth factor receptor with an 00 of about 19 PM. The inhibition by halenaquinone was shown to be irreversible (Table 2) in that dilution of the enzyme after pre-incubation with this compound did not appreciably alter the observed inhibition. AppppA and FSBA behaved as reversible and irreversible inhibitors, respectively. Based on the results above, approximately 20 simple quinones (Figure 1) were synthesized in order to examine the effect of adding substituents to the naphthoquinone moiety and removing or reducing various keto functionalities. As can be seen in Table 3, the most potent inhibitors had lC50 values in the 9-50 HM range, making them less potent than halenaquinone. Replacing the naphthoquinone oxygens with chlorine destroyed inhibitory activity. Replacing the furan ring of halenaquinone with a benzene ring also decreased activity. Halenaquinone was tested for its effect on cell proliferation on several cell lines. It was found to have an E&o of approximately l-10 PM on the lines studied (Table 4); these figures are in good agreement with the 2-3 pM EDso values reported by Schmitz and Bloor for P388 leukemia (19) and data provided on our samples by the National Cancer Institute (not shown). 767
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BIOCHEMICAL
Table Compound
1.
Inhibition 1C50
o$
AND
of ppbOv-src
BIOPHYSICAL
tyrosine
RESEARCH
kinase
COMMUNICATIONS
activity
Compound
(PM)
IC50
WV
0
0
11;
1;
0
0
Halenaquinone
0 6 11; Cl
Xestoquinone
1.5
Halenaquinol
sulfate
0.55
Tetrahydrohalenaquinone
A
26
>>200
.P OH
OH
H
1;
I>
&x9 0
0
I 0
0
60
Tetrahydro halenaquinone
>>200 B
Compounds were solubiliged in ,DMSO and pre-incubated with enzyme for 15 min. before the addition of [val ]-angrotensin II and ATP. The IC5s is defmed as the concentration of compound reducing enzyme activity by 50% compared to vehicle alone; final DMSO concentration was 3%.
Discussion As part of an ongoing drug screening effort, a number of biosynthetically diverse marine natural products have been tested for their ability to inhibit various protein tyrosine kinases. Halenaquinone and halenaquinol are among the most active kinase inhibitors we have evaluated to date. Halenaquinone is a potent, irreversible inhibitor
Table 2. Reversibility of pp68v-src inhibition Percent Inhibition Concentration Diluted 1:2 Diluted 1:5 after substrates added Undiluted 44.8 0 1 mM 71.8 $P~A 68.2 57.0 1 mM 74.8 51.7 Halenaguinone 5uM 61.2 683 Compounds were solubilized in water (AppppA) or DMSO (FSBA and halenaquinone) and pre-incubated with enzyme for 15 minutes. The incubation mix was then diluted as indicated and [val5]-angiotensin II and ATP added to start the kinase reaction. Percent inhibition was calculated based on appropriately diluted control reactions; results shown are the average for duplicate samples. Compound
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0
RESEARCH
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0
I
I
1;
CQlf
ca
0
1; 0
lo 0
0
OH 12
0 H
I I
I H
0
0
11
0
H
w
BIOPHYSICAL
m
0
13
I
I H
0
0%
0
1; 0
I 0
15
14
+$J oyp 19 0
0
20
0
Fiaure 1. Structures of quinone analogs tested for kinase inhibitory activity.
of pp6Ov-src protein tyrosine kinase; it also inhibits the protein tyrosine kinase activity of the epidermal growth factor receptor (IC50 = 19 $4). A comparison of the relative potency of xestoquinone and halenaquinone shows that removal of one of the three possible Michael acceptor sites in halenaquinone results in a loss of inhibitory activity. 769
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Table 3. Inhibition of pp60v’src by synthetic quinones and analogs
Comocund
00 (uM1 159.5 30 52 40
: 3 2 6 7
14 X200
Ccmoound
0.0 (uM)
Comocund
IGo l&t)
98 10 11 12
>200 >200 56 18
16 15 17 19 18
2200 =200 50 9.5
13 14
:4’
20
297
Compounds depicted in Figure 1 were tested as described in Materials and Methods. The IGo is defined as the concentration of compound reducing enzyme activity by 500/bcompared to vehicle alone; final DMSO concentration was 3Oh.
Halenaquinone shares the quinone moiety common to many of the known tyrosine kinase inhibitors; in addition, Q- or Q-catechols found in erbstatin and some tyrphostins may become oxidized to quinones during the course of testing. The activity of halenaquinol could be due to its conversion (partial or full) to halenaquinone; previous work has shown that halenaquinol can be air oxidized upon exposure to light or heat (20).
A number of simple quinones were synthesized to further define the requirements for inhibitory activity. Any modification that served to decrease the possibility for Michael addition decreased activity. Although it is not known if a free sulfhydryl is required for pp6Wsrc enzymatic activity, it has been reported that sulfhydryl reagents can negate the inhibitory activity of herbimycin A (29, 30). The apparent ICso of halenaquinone and many of the simple quinones was found to increase several-fold if 2-mercaptoethanol was added to the enzyme reaction mix (data not shown). It is not clear whether the antiproliferative effect of halenaquinone is due to its inhibition of protein tyrosine kinases. Inhibition of growth occurs at about the same concentrations in the non-transformed Chinese hamster ovary cell line as it does in cells transformed by protein tyrosine kinases. Since halenaquinone is potentially a very active Michael acceptor,
attack by any nucleophile
in a crucial position
important cellular protein might contribute to its anti-proliferative
activity.
Table 4. Inhibition of cell proliferation by halenaquinone Cell line RR1022
Oncoaene oresent v-src
NY684 NIH src NIH erbB-HX CHnCS
Temperature-sensitive v-src v-src v-erbB mm--
EC& (uM) 4.8 1.6 5.6 2.0 10.0
Cell proliferation was assessed as described in Materials and Methods,
and the concentration of halenaquinone required to reduce proliferation by 50% of control (EC50) determined for each cell line. 770
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Acknowledgments This research was partially supported by NIH grant CA52955 We express our appreciation to Dr. Robert Wilhelm for providing us with valuable chemical advice, and to Dr. Marshall Wallach and Ms. Amie Chuong for support in processing incoming marine extracts andcompounds. Drs. John Young and Richard Roth provided critical feedback on the manuscript. We wish to thank Ms. Lisa Hunter for her assistance with sponge collection and Ms. Cristina Diaz for sponge taxonomy data. We also thank the government of Fiji for their cooperation. The Fijian vessel MVIMollie-Dean was used to make the sponge collections; sadly, she was iost at sea on August 13, 1991.
References 1. Brickell, P.M. (1991) Int. J. Exp. Pathol. 72, 97-108. 2. White, M.F. J. Bioenerg. Biomemb. (1991) 23,63-82. 3. Cantfey, L.C., Auger, K.R., Carpenter, C., Duckworth, B., Graziani, A., Kapeller, R., and Soltoff, S. (1991) Cell 64, 281-302. 4. Umezawa, H., Imoto, M., Sawa, T., Isshiki, K., Matsuda, N., Uchida, T., linuma, H., Hamada, M., and Takeuchi, T. (1986) J. Antibiot. 39, 170-l 73. 5. Uehara, Y., Hori, M., Takeuchi, T., and Umezawa, H. Jpn. J. Cancer Res. (1985) 76, 672-675. 6. Akiyama, T., Ishida, J., Nakagawa, S., Ogawara, H., Watanabe, S., Itoh, N., Shibuya, M., and Fukami, Y. (1987) J. Biol. Chem. 262, 55925595. 7. Onoda, T., Isshiki, K., Takeuchi, T., Tatsuta, K., and Umezawa, K. (1990) Drugs Exptl. Clin. Res. 16, 249-253. 8. Nakano, H., Kobayashi, E., Takahashi, I., Tamaoki, T., Kuzuu, Y., and Iba, H. (1987) J. Antibiot. 40, 706708. 9. Geahlen, R.L. and McLaughlin, J.L. (1989) Biochem. Biophys. Res. Commun. 165, 241-245. 10. Donella-Deana, A., Monti, E., and Pinna, L.A. (1989) Biochem. Biophys. Res. Commun. 160, 13091315. 11. Kreuter, M.-H., Leake, R.E., Rinaldi, F., Muller-Klieser, W., Maidhof, A., Muller, W.E.G., and Schroder, H-C. (1990) Comp. Biochem. Physiol. 978, 151-158. 12. Gazit, A., Yaish, P., Gilon, C., and Levitzki, A. (I 989) J. Med. Chem. 32,2344-2352. 13. Levitzki, A. (1990) Biochem. Pharmacol. 40, 913-l 918. 14. Yaish, P., Gazit, A., Gilon, C., and Levitzki, A. (1988) Science 242, 933-935. 15. Crews, P. and Hunter, 1-M. In: Marine Biotechnology (OR. Zaborsky and D. Attaway, Eds.), Vol. 1, in press. 16. Alvi, K.A., Tenenbaum, L., and Crews, P. (1991) J. Nat. Prod. 54, 71-78. 17. Roll, D.M., Scheuer, PJ., Matsumoto, G.K., and Clardy, J. (1983) J. Am Chem. Sot. 105, 6177-6178. 18. Nakamura, H., Kobayashi, J., Kobayashi, M., Ohizumi, Y., and Hirata, Y. (1985) Chem. Lett. , 713-7: 6. 19. Schmitz, F.J. and Eloor, S.J. (1988) J. Org. Chem. 53,3922-3925. 20. Kobayashi, M., Shimizu, N., Kyogoku, Y., and Kitagawa, I. (1985) Chem. Pharmacol. Bullet. 33, 1305-l 308. 21. Hayashi, T., Smith, F.T., and Lee, K.-H. J. Med. Chem. (1987’) 30,2005-2008. 22. Van Soesi, R.W.M. (1989) Netherlands J. Sea Res. 23.223-230. 23.Wong, T.W. and Goldberg, A.R. (S83) J. Biol. Chem. 258, 1022-1025. 24. Yarden, Y. and Schlessinger, J. (1987) Biochem. 26, 1434-1442. 25. Manes% P-F., Perry, M.E., and Levy, B.T. (1983) J. Biol. Chem. 258,40554058. 771
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26. Russo, M-W., Lukas, TJ., Cohen, S., and Staros, J.V. (1985) J. Biol. Chem. 260, 5205-5208. 27. Mosmann, T. (1983) J. Immunol. Meth. 65, 56-63. 28. Bruno, N-A. and Slate, D.L. (1990) J. Natl. Cancer Inst. 82,419-424. 29. Uehara, Y., Kukazawa, H., Murakami, Y., and Miruno, S. (1990) Biochem. Biophys. Res. Commun. 163,8033-8039. 30. Fukuzawa, H., Mizuno, S., and Uehara, Y. (1990) Biochem. Biophys. Res. Commun. 173 276-282.
772
184,
No.
2, 1992
April
30,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
MARINE
SPONGE POLYKETIDE PROTEiN TYROSINE
INHIBITORS KINASE
765-772
OF
Rita t-l. Leel, Doris L. SlateI*, Robert Moretti2, Khisal A. Alvi3, and Phillip Crews3# ‘Institute of Cancer and Developmental Biology and *Institute of Bio-organic Chemistry, Syntex Research, Palo Alto, CA 94304 3Department
Received
March
of Chemistry and Biochemistry and institute of Marine Sciences, University of California, Santa Cruz, CA 96064 10,
1992
Summary: The marine polyketide natural product, halenaquinone, was shown to be an irreversible inhibitor of pp60 v-src, the oncogenic protein tyrosine kinase encoded by the Aous sarcoma virus. This compound had an IC50 of approximately 1.5 $vI against pp6Ov-src and also inhibited the ligand-stimulated kinase activity of the human epidermal growth factor receptor with an l&o of approximately 19 PM. Haienaquinone blocked the proliferation of a number of cuttured cell lines, including several transformed by oncogenic protein tyrosine kinases. Halenaquinol, xestoquinone, halenaquinol sulfate, and several simple synthetic quinone analogs were also shown 0 1992Academic Press,Inc. to inhibit pp6Ov-src.
Protein tyrosine kinases comprise a class of enzymes involved in the regulation of cellular growth and signalling (l-3). A number of natural products of microbial, plant, and marine origin have been reported to inhibit protein tyrosine kinases. Among these are erbstatin (4). herbimycin A (5). genistein (6) lavendustin A (7), staurosporine (8) piceatannol (9) adriamycin (10). and aeroplysinin (11). Synthetic inhibitors of tyrosine kinases, especially the tyrphostins, have also been described; certain tyrphostins can block cell proliferation induced by growth factors which bind to receptors with protein tyrosine kinase activity (12-14). The structural diversity represented among the known natural product inhibitors of tyrosine kinases prompted the examination of tropical marine sponges as potential sources of new inhibitors. These sponges are known to contain diverse, bioactive secondary metabolites (15, 16). Indo-Pacific marine sponges of the Xestospongia and * To whom correspondence addressed. # To whom correspondence be addressed.
on biological assays and synthetic compounds on marine natural products isolation should
should be
Vol.
184,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Adocia genera
are a source of polyketide quinones and hydroquinones, including halenaquinone (17-l 9) and xestoquinone (18-20), which possess antibiotic (17) cardiotonic (18), and antiproliferative (19) activity. Although the molecular basis of their antiproliferative activity is unknown, some of these structures could accommodate a nucleophilic Michael addition from reactive thiols in important cellular proteins, as postulated for cytotoxic naphthoquinones (21). We isolated halenaquinone from Fijian collections of Xestospongia ?cafbonaria and examined its effect on the protein tyrosine kinase activity of pp60v-src, the transforming gene product of the Rous sarcoma virus. It proved to be a potent, irreversible inhibitor of this enzyme. Five other marine quinones and related compounds were also studied, along with approximately 20 simple synthetic quinones. The antiproliferative activity of halenaquinone on a number of cell lines, including several transformed by oncogenes encoding protein tyrosine kinases, was also evaluated.
Materials
and Methods
Compounds: Halenaquinone, xestoquinone, halenaquinol, halenaquinol sulfate, and tetrahydrohalenaquinone A were isolated from the marine sponge Xestospongia ?carbonaria (22) gathered from the Benga Lagoon, Fiji Islands (Collection number 91007, identification made by M.C. Diaz). Their spectroscopic properties were consistent with those previously published (17-20). Tetrahydrohalenaquinone B was also isolated from this sponge, and its structure proof will be published elsewhere (Alvi and Crews, in preparation). Synthetic compounds were prepared and structures confirmed at Syntex Research. The reference protein kinase inhibitors, PI, P4di(adenosine-5’)tetraphosphate (AppppA) and S-fluorosulfonylbenzoyladenosine (FSBA), were obtained from Sigma Chemical Company, St. Louis, MO. rat Enzyme Assays: pp60 v-src was purified from Rous sarcoma virus-transformed cells and its protein kinase activity assayed essentially as described by Wong and Goldberg (23), except that 2-mercaptoethanol was omitted from the reaction buffer. Test compounds were solubilized in DMSO or water and incubated with the enzyme for 15 min prior to the addition of [val+angiotensin II (4 mg/ml final concentration, Sigma) and ATP (30 PM final concentration, Sigma) to start the kinase reaction. Approximately l-2 PCi of y[gP]-ATP (New England Nuclear, Boston, MA) was used per reaction. The l&o is defined as the concentration of compound which inhibits 50% of enzyme activity compared to the appropriate vehicle control. Epidermal growth factor receptor tyrosine kinase activity was assayed using detergent lysates of human A431 epidermoid carcinoma cells (24). The crude receptor preparation was incubated in a volume of 15 ~1 with ligand (333 nM epidermal growth factor, Sigma) in 41 mM HEPES, pH 7.4, 8.3 mM MnClz, 20 mM MgCh. 0.17% Triton X-100, 16 PM ATP, and 66.7 PM NasV04 for 15 minutes to permit ligand-stimulated autophosporylation to occur. When inhibitors were tested, they were added during this step. [Vals]-angiotensin II phosphorylation was assayed by adding 10 ~1 of substrate solution (10 mg/ml [val5]-angiotensin II, 275 mM ATP, 3 mg/ml bovine serum albumin [Sigma]) to the autophosphorylation reaction. Approximately 2-3 &i of y[zP]-ATP was used per reaction. After incubation at 35O C for 30 min, s2P-labeled peptide was quantitated by phosphocellulose filter paper binding as described by Wong and Goldberg (23). 766
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Inhibitor Reversibility: Compounds were tested at concentrations giving 60-80% inhibition in the standard pp60 v-src assay. After a 15 min incubation of compounds with pp60v’src, the incubation solution was diluted 2 or 5-fold prior to the addition of fvalsJ-angiotensin 11 and ATP. The standards employed were AppppA, a reversible competitive inhibitor binding at the ATP site (25) and FSBA, an irreversible inhibitor which reacts with a lysine residue in the ATP binding site (26). Cell Proliferation: CHRC5 multidrug-resistant Chinese hamster ovary ceils were generously provided by Dr. Victor Ling (University of Toronto, Toronto, ON). Mouse NlH3T3 cells transformed by wild-type v-src or v-erbB as well as NY684, a rat cell line transformed by a temperature-sensitive variant of v-src, were provided by Dr. Randall Schatzman, Syntex Research. RR1022 rat cells transformed by Rous sarcoma virus were obtained from the American Type Culture Collection (Rockville, MD). Cells were maintained in minimal essential medium (Irvine Scientific, Santa Ana, CA) supplemented with 10% fetal bovine serum (JR Scientific, Woodfand, CA). Cell proliferation was assessed using the method of Mosmann (27) as modified by Bruno and Slate (28).
Results Halenaquinone and five related marine pentacyclic polyketides were tested against pp6Ov-src, and IGo values determined as shown in Table 1. Hatenaquinol proved to be approximately as potent as halenaquinone, while the closely related xestoquinone was less active. Interestingly, halenaquinol sulfate was much less active than halenaquinol, and the tetrahydrohalenaquinones were both inactive at the doses tested. Halenaquinone also inhibited the ligand-stimulated tyrosine kinase activity of the human epidermal growth factor receptor with an 00 of about 19 PM. The inhibition by halenaquinone was shown to be irreversible (Table 2) in that dilution of the enzyme after pre-incubation with this compound did not appreciably alter the observed inhibition. AppppA and FSBA behaved as reversible and irreversible inhibitors, respectively. Based on the results above, approximately 20 simple quinones (Figure 1) were synthesized in order to examine the effect of adding substituents to the naphthoquinone moiety and removing or reducing various keto functionalities. As can be seen in Table 3, the most potent inhibitors had lC50 values in the 9-50 HM range, making them less potent than halenaquinone. Replacing the naphthoquinone oxygens with chlorine destroyed inhibitory activity. Replacing the furan ring of halenaquinone with a benzene ring also decreased activity. Halenaquinone was tested for its effect on cell proliferation on several cell lines. It was found to have an E&o of approximately l-10 PM on the lines studied (Table 4); these figures are in good agreement with the 2-3 pM EDso values reported by Schmitz and Bloor for P388 leukemia (19) and data provided on our samples by the National Cancer Institute (not shown). 767
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BIOCHEMICAL
Table Compound
1.
Inhibition 1C50
o$
AND
of ppbOv-src
BIOPHYSICAL
tyrosine
RESEARCH
kinase
COMMUNICATIONS
activity
Compound
(PM)
IC50
WV
0
0
11;
1;
0
0
Halenaquinone
0 6 11; Cl
Xestoquinone
1.5
Halenaquinol
sulfate
0.55
Tetrahydrohalenaquinone
A
26
>>200
.P OH
OH
H
1;
I>
&x9 0
0
I 0
0
60
Tetrahydro halenaquinone
>>200 B
Compounds were solubiliged in ,DMSO and pre-incubated with enzyme for 15 min. before the addition of [val ]-angrotensin II and ATP. The IC5s is defmed as the concentration of compound reducing enzyme activity by 50% compared to vehicle alone; final DMSO concentration was 3%.
Discussion As part of an ongoing drug screening effort, a number of biosynthetically diverse marine natural products have been tested for their ability to inhibit various protein tyrosine kinases. Halenaquinone and halenaquinol are among the most active kinase inhibitors we have evaluated to date. Halenaquinone is a potent, irreversible inhibitor
Table 2. Reversibility of pp68v-src inhibition Percent Inhibition Concentration Diluted 1:2 Diluted 1:5 after substrates added Undiluted 44.8 0 1 mM 71.8 $P~A 68.2 57.0 1 mM 74.8 51.7 Halenaguinone 5uM 61.2 683 Compounds were solubilized in water (AppppA) or DMSO (FSBA and halenaquinone) and pre-incubated with enzyme for 15 minutes. The incubation mix was then diluted as indicated and [val5]-angiotensin II and ATP added to start the kinase reaction. Percent inhibition was calculated based on appropriately diluted control reactions; results shown are the average for duplicate samples. Compound
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0
I
I
1;
CQlf
ca
0
1; 0
lo 0
0
OH 12
0 H
I I
I H
0
0
11
0
H
w
BIOPHYSICAL
m
0
13
I
I H
0
0%
0
1; 0
I 0
15
14
+$J oyp 19 0
0
20
0
Fiaure 1. Structures of quinone analogs tested for kinase inhibitory activity.
of pp6Ov-src protein tyrosine kinase; it also inhibits the protein tyrosine kinase activity of the epidermal growth factor receptor (IC50 = 19 $4). A comparison of the relative potency of xestoquinone and halenaquinone shows that removal of one of the three possible Michael acceptor sites in halenaquinone results in a loss of inhibitory activity. 769
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Table 3. Inhibition of pp60v’src by synthetic quinones and analogs
Comocund
00 (uM1 159.5 30 52 40
: 3 2 6 7
14 X200
Ccmoound
0.0 (uM)
Comocund
IGo l&t)
98 10 11 12
>200 >200 56 18
16 15 17 19 18
2200 =200 50 9.5
13 14
:4’
20
297
Compounds depicted in Figure 1 were tested as described in Materials and Methods. The IGo is defined as the concentration of compound reducing enzyme activity by 500/bcompared to vehicle alone; final DMSO concentration was 3Oh.
Halenaquinone shares the quinone moiety common to many of the known tyrosine kinase inhibitors; in addition, Q- or Q-catechols found in erbstatin and some tyrphostins may become oxidized to quinones during the course of testing. The activity of halenaquinol could be due to its conversion (partial or full) to halenaquinone; previous work has shown that halenaquinol can be air oxidized upon exposure to light or heat (20).
A number of simple quinones were synthesized to further define the requirements for inhibitory activity. Any modification that served to decrease the possibility for Michael addition decreased activity. Although it is not known if a free sulfhydryl is required for pp6Wsrc enzymatic activity, it has been reported that sulfhydryl reagents can negate the inhibitory activity of herbimycin A (29, 30). The apparent ICso of halenaquinone and many of the simple quinones was found to increase several-fold if 2-mercaptoethanol was added to the enzyme reaction mix (data not shown). It is not clear whether the antiproliferative effect of halenaquinone is due to its inhibition of protein tyrosine kinases. Inhibition of growth occurs at about the same concentrations in the non-transformed Chinese hamster ovary cell line as it does in cells transformed by protein tyrosine kinases. Since halenaquinone is potentially a very active Michael acceptor,
attack by any nucleophile
in a crucial position
important cellular protein might contribute to its anti-proliferative
activity.
Table 4. Inhibition of cell proliferation by halenaquinone Cell line RR1022
Oncoaene oresent v-src
NY684 NIH src NIH erbB-HX CHnCS
Temperature-sensitive v-src v-src v-erbB mm--
EC& (uM) 4.8 1.6 5.6 2.0 10.0
Cell proliferation was assessed as described in Materials and Methods,
and the concentration of halenaquinone required to reduce proliferation by 50% of control (EC50) determined for each cell line. 770
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Acknowledgments This research was partially supported by NIH grant CA52955 We express our appreciation to Dr. Robert Wilhelm for providing us with valuable chemical advice, and to Dr. Marshall Wallach and Ms. Amie Chuong for support in processing incoming marine extracts andcompounds. Drs. John Young and Richard Roth provided critical feedback on the manuscript. We wish to thank Ms. Lisa Hunter for her assistance with sponge collection and Ms. Cristina Diaz for sponge taxonomy data. We also thank the government of Fiji for their cooperation. The Fijian vessel MVIMollie-Dean was used to make the sponge collections; sadly, she was iost at sea on August 13, 1991.
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