Calphostin C (UCN-1028C), a novel microbial compound, is a highly potent and specific inhibitor of protein kinase C

Vol. 159, No. 2, 1989 March 15, 1989

BIOCHEMICAL

CALPHOSTIN

c (DCN-1028c),

A NOVEL MICROBIAL

HIGHLY POTENT AND SPECIFIC

Eiji Kobayashi’,

l

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 540-553

INHIBITOR

COMPOUND, IS A

OF PROTEIN KINASE C

Hirofumi Nakano*, Makoto and Tatsuya Tamaoki*

Morimoto#

Tokyo Research Laboratories, Kyowa Hakko Kogyo Co. Ltd., 3-6-6 Asahimachi, Machida, Tokyo 194, Japan

#Pharmaceutical

Received

January

18,

Research Laboratory, Kyowa Hakko Kogyo Co. Ltd., Nagaizumi, Shizuoka, Japan 1989

Calphostin C (UCN-1028C), a newly isolated compound from . . onum c@&8ponor& is a potent and specific inhibitor of protein kinase C, because it was 1000’times more inhibitory to protein kinase C (IC50, 0.05 PM) than other protein kinases such as CAMP-dependent protein kinase and tyrosine-specific protein kinase (IC50, >50pM). Calphostin C did not inhibit calcium activated neutral protease (calpain)-digested protein kinase C, indicating that it interacts with the regulatory domain of protein kinase C. In addition this compound showed inhibitory effects on the binding of [3H]PDBu to protein kinase C. The potent cytotoxic activity and antitumor activity of calphostin C might be due to the inhibition of protein kinase C, and thus it may be Q 1989 Academic Press, Inc. potentially useful for the therapeutic application.

Protein kinase C plays crucial roles in signal transduction, cellular proliferation and differentiation (1). Further interest in protein kinase C was generated

by the discovery that tumor promoting

phorbol esters activate the

enzyme (2). Therefore, the specific inhibitor of protein kinase C is very useful for studying the role of protein kinase C in the regulation of various cellular functions and may be value as therapeutic

agents.

Many compounds

has been

reported to inhibit protein kinase C, but the specificity of most of them for protein kinase C is questionable. In the course of screening inhibitors of protein kinase C partially purified from rat brain, we have found staurosporine (3), the most potent inhibitor of protein kinases and UCN-01 (4), a selective inhibitor of protein kinase C. These findings prompted

4BBREVIATION: 0006-291X/89 Copyright All rights

[3H]PDBu,

us to continue further screening for specific

[3H]phorbol-12,13-dibutyrate.

$1.50

Q 1989 by Academic Press, Inc. of reproduction in any form reserved.

548

Vol. 159, No. 2, 1989

inhibitors

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of protein kinase C, and now we have found a specific inhibitor of

protein kinase C. We now report that the microbial compound,

designated

calphostin

C

(originally called UCN-1028C), produced by Cladosporium cladosoorioides novel and specific inhibitor of protein kinase C with high potency.

is a

Materials: Protein kinase C was partially purified from rat brain with DE-52 and phenylsepharose column chromatography, as described by Kikkawa u.(5). CAMP-dependent protein kinase was partially purified from bovine heart by the method of Kuo ti. (6). Histone type IfIS, histone type ff and PDBu were purchased from Sigma Chemical Co. Phosphatidylserine and diolein were purchased from Serdary Res. Lab. Inc., and Nakarai Chemicals, respectively. [Y-~~P]ATP (3000 Ci/mmol) and [3H]PDBu (13.4 Ci/mmol) were purchased from Amersham Co., England. Fnzvme assay and f3HlPDRu biw: Protein kinase C activity was assayed in a reaction mixture (0.25 pl) containing 20 mM Tris/HCI, pH 7.5, 10 mM magnesium acetate, 50 pg histone Ills, 20 pg phosphatidylserine, 0.88 pg diolein, 5 x 1O-5 M CaC12, 5 pM [y-32P]ATP (105 cpmlnmol) and 1 pg partially purified enzyme, according to the method of Kikkawa et. al. (5). Calpaindigested protein kinase C activity was assayed in the reaction mixture (250 ~1) containing 20 mM Tris/HCI, pH 7.5, 10 mM magnesium acetate, 50 pg histone Ills, 30 mM 2-mercaptoethanol, 1 mM EGTA and 5 pM [y-32P]ATP. CAMPdependent protein kinase activity was assayed by the method of Kuo u. (6). Tyrosine-specific protein kinase of p60 transforming protein of Rous sarcoma virus was assayed as described previously (7). The binding of [3H]PDBu to protein kinase C was analysed by the method of Miyake ti. (8). The reaction mixture (200 p.1)contained 4 pmol Tris/malate, pH 6.8, 20 p.mol KCI, 30 nmol CaCl2, 20 pg phosphatidylserine, 2 pg partially purified protein kinase C, 0.5 % (final concentration) DMSO, 10 pmol [3H]PDBu (l-3 x 104 cpm / pmol) and 10 pl of various concentrations of drugs. After incubation of the reaction mixture at 300C for 20 min, the reaction was stopped by the addition of 3 ml ice-cold DMSO (0.5%). The mixture was collected on a polyethyleneimine-treated Whatman GF/B glass filter and washed with 6 ml of 0.5% ice-cold DMSO. The radioactivity on the filter was quantitated using a liquid scintillation spectrometer. lian cell culu: HeLa S3 cells and human breast cancer MCF-7 cells were cultured in modified Eagle’s minimum essential medium (Nissui Co. Ltd.,), and RPM1 1640 (GIBCO Lab.), respectively, supplemented with 10 % fetal bovine serum.

RFsUf TS AND ~fSW5iSlQN Calphostins

produced by Cladosoorium

cladospgrioides

have a unique

structure which includes perylene quinone, and is consisted of many components, among which calphostin C shows the most potent biological activity (Fig. 1). The production, calphostins

isolation and structural elucidation

will be reported in a separate paper (9). We investigated

549

of the

Vol. 159, No. 2, 1989

BIOCHEMICAL

AND BIOPHYSICALRESEARCH COMMUNiCATlONS

Fig. 1 Chemicalstructure of calphostinC (UCN-1028C). Fig. 2 Inhibition of protein kinase C and calpain-digestedprotein kinase C by calphostin C. The definition of % activity was as follows : (protein kinaseC activity in the presenceof calphostin C / protein kinase C activity in the absence of calphostin C) x 100. Inhibition of protein kinase C ( l ), calpain-digestedprotein kinase C ( o ), CAMP-dependentprotein kinase ( A ) and tyrosone specific-proteinkinase by calphostin C ( n ).

inhibitory effects of calphostin C on protein kinase C activity, and it inhibited protein kinase C activity with an IC50 value of 0.05 uM (Fig. 2). On the contrary, even as high as 50 pM of calphostin C only slightly inhibited CAMP-dependent protein kinase which was partially purified from bovine heart, and tyrosine specific-protein kinase of p60 transforming protein of Rous sarcoma virus (Fig. 2). Protein kinase C is known to have two separate functional domains, the catalytic and the regulatory domain, by proteolysis (10). This phenomenon was confirmed by cloning cDNAs of protein kinase C (1 l-l 4). As shown in Fig. 2, the calpain-digested protein kinase C activity was independent of Ca2+ and phospholipid, and was not inhibited by calphostin C. This result indicates that calphostin C interacts with the regulatory domain of protein kinase C.

In

addition the inhibition of protein kinase C was not overcome by adding an excess amount of Ca2+ or phospholipid, both of which interact with the regulatory domain (Fig. 3).

Local anesthetic compounds such as

chlorpromazine (15) and tamoxifen (16) have been reported to interact with the regulatory domain and compete with phospholipids, but the IC50 value is uM order and so protein kinase C must not be the most sensitive target of these compounds ~JIIvivq.

Unlike these compounds, calphostin C is a potent inhibitor

of protein kinase C without competing with phospholipids. Furthermore, calphostin C inhibited [ali]PDBu binding to protein kinase C, indicating that it may be interacting with the binding site of phorbol esters in the regulatory domain (Table 1). 550

BIOCHEMICAL

Vol. 159, No. 2, 1989

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

loo+

100 - (@

(b)

D

g

@/Y---Y 250

-

_

i50-

g

01 10

1 Cont.

of C&l2

102

0’ 103

’ 2.5 Cont.

( FM )

’ 5.0

’ 10

’ 20

’ 40

of phosphatidylserine

80

. 160

( pg/ml )

Fig. 3 Inhibition of protein kinase C by calphostinC in the various -concentrations of Ca2+ and phospholipid. Protein kinase C activity was assayed as describedin Fig. 2, and the concentrations of CaCl2 (a), and phosphatidylserine(b) were varied. The definition of % inhibitionwas as follows: [l .’ (protein kinaseC activity in the presenceof calphostin C 0.03 pM / protein kinaseC activity in the absence of calphostinC) ] x 100.

Protein kinase C is a complex family of related molecular

forms.

To

determine the sensitivity of calphostin C to the isozymes of protein kinase C, we isolated three isozymes, a, J3and ‘y, from rat brain with hydroxylapatite HPLC according to the method of Ono U.

(17). Calphostin C inhibited all isozymes

of protein kinase C to the same degree (data not shown). These results suggest that calphostin C interacts with the common regulatory region in all isozymes of

protein kinase C. We examined the effects of calphostin C on tumor cell growth to determine whether a specific inhibitor of protein kinase C causes inhibition of cellular proliferation.

Calphostin showed potent cytotoxic effects against HeLa S3 cells

and human breast cancer cell MCF-7 with IQ,0 values of 0.18 pg/ml and 0.14 pg/ml, respectively (Table 2). Interestingly, calphostin C showed antitumor activity against murine lymphocytic leukemia P388 in vivo (data not shown). The antitumor activity of this compound is now being investigated in detail and will

Table 1. Inhibition of [3H]PDBu bindingto protein kinase C by calphostin C CalphostinC (PM)

Inhibitionof [3H]PDBu binding to proteinkinaseC (%)

0.03 4.6 0.1 47.1 69.5 0.5 1.0 91.3 % inhibition = [l-( 3H-PDBu binding in the presence of calphostin C / 3H-PDBu binding in the absence of calphostin C )] x 100. 551

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 159, No. 2, 1989

Table 2. Effects of calphostin C on the growth of HeLaS3 and MCF-7 cells Cytotoxic activity, IC5o @g/ml) HeLaS3 MCF-7

Compound Calphoatin C

0.14

0.18

Logarithmically growing HeLaS3 and MCF-7 cells (5 x 104 cells) were incubated with calphostinfor 72 hr. Numberof cells were counted at 72 hr after addition of calphostin C.

be reported in a separate paper. Calphostin

C did not interact with DNA io vitro

(data not shown). Therefore, it is conceivable that the cytotoxic and antitumor activity of this compound is due to the specific inhibition of protein kinase C. Further studies are underway to confirm that calphostin

C specifically

inhibits

protein kinase C&&Q. The protein kinase C inhibitors acting on the catalytic domain with a sequence

homologous

to other protein kinases, such as staurosporine

and H-7

(18), can not have high specificity for protein kinase C. In contrast the protein kinase C inhibitors

acting on the regulatory domain which is distinct from other

protein kinases, may be specific for protein kinase C. However most of the known inhibitors acting on the regulatory domain compete with phospholipids

and are only effective at high doses, usually greater than uM concentrations. Thus, calphostin C is the first specific inhibitor of protein kinase C with high

potency, and will excellently contribute to the study of the role of protein kinase C in various cellular functions.

ACKNOWLFDGMFNTS We wish to thank Dr. Koichi Suzuki, Tokyo Metropolitan

Institute of Medical

Science for the calpain donation; Ms. Kiyoe Mokudai for technical assistance;

Dr. Hideo Iba of the University of Tokyo for inhibition

data of tyrosine-specific

protein kinase.

REFERENCES (1) Nishizuka, Y. (1986) Science m, 305-312 (2) Castagna, M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa, U. and Nishizuka, Y. (1982) J. Biol. Chem. &Z, 7847-7851 (3) Tamaoki, T., Nomoto, H., Takahashi, I., Kato, Y., Morimoto, M. and Tomita, F., (1986) Biochem. Biophys. Res. Commun. m, 397-402 (4) Takahashi, I., Kobayashi, E., Asano, K., Yoshida, M. and Nakano, H. (1987) J. Antibiotics a, 1782-l 784 (5) Kikkawa, U., Takai, Y., Minamikuchi, R., Inohara, S. and Nishizuka, Y. (1982) J. Biol. Chem. a, 13341-13348 552

Vol. 159, No. 2, 1989

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(6) Kuo, J. F. and Greengard,

P. (1969) Proc. Natl. Acad. Sci. USA 64, 13491355 (7) Nakano, H., Kobayashi, E., Takahashi, I., Tamaoki, T., Kuzuu, Y. and Iba, H. (1987) J. Antibiotics 4p, 706-708 (8) Miyake, R., Tanaka, Y., Tsuda, T., Kaibuchi, K., Kikkawa, U. and Nishizuka, Y. (1984) Biochem. Biophys. Res. Commun. 121, 649-656 (9) Kobayashi, E., Ando, K., Nakano, H. and Tamaoki, T. (1988) in press (10) Inoue, M., Kishimoto, A., Takai, Y. and Nishizuka, Y. (1977) J. Biol. Chem. a, 761 O-761 6 (11) Parker, P. J., Coussens, L., Totty, N., Rhee, L., Young, S., Chen, E., Stabel, S., Waterfield, M. D. and Ullrich, A. (1986) Science m, 853-858 M. and Bell, R. M. (1986) cell a, 491-502 1::; Ono, Y., Kurokawa, T., Fujii, T., Kawahara, K., Igarashi, K., Kikkawa U., Ogita, K. and Nishizuka, Y. (1986) FEBS Lett. a, 347-352 (14) Ohno, S., Kawasaki, H., Imajoh, S., Suzuki, K., lnagaki, M., Yokokura, H., Sakoh, T. and Hidaka, H. (1987) Nature m, 161-166 (15) Mori, T., Takai, Y., Minamikuchi, R., Yu, B. and Nishizuka, Y. (1980) J. Biol. Chem. %, 8378-8380 (16) O’Brian, C. A., Liskamp, R., Solomon, P. H. and Weinstein, I. B. (1985) Cancer Res. &j, 2462-2465 (17) Ono, Y., Kikkawa, U., Ogita, K., Tomoko, F., Kurokawa, T., Asaoka, Y., Sekiguchi, K., Ase, K., Igarashi, K. and Nishizuka, Y. (1987) Science m, 1116-1120 (18) Hidaka, H., Inagaki, M., Kawamoto, S. and Sasaki, Y. (1984) Biochemistry a,50365041

553