Rational design, synthesis, and evaluation of a new type of PKC inhibitor

Full text of DOI:10.1021/ja971270t

J. Am. Chem. Soc. 1998, 120, 457-458
457
Rational Design, Synthesis, and Evaluation of a New
Type of PKC Inhibitor
Mikiko Sodeoka,1 Midori A. Arai, Koji Adachi,
Koichiro Uotsu, and Masakatsu Shibasaki*
Graduate School of Pharmaceutical Sciences
The UniVersity of Tokyo, Hongo, Bunkyo-ku
Tokyo 113, Japan
ReceiVed April 22, 1997
Protein kinase C (PKC), a serine- and threonine-phosphory-
lating enzyme, is thought to play central roles in cellular signal
transductions.2 To clarify its physiological roles and to serve,
potentially, as an important compound in anticancer therapies, a
specific PKC inhibitor would be of great use. PKC has a catalytic
site that phosphorylates substrate proteins and a regulatory site
that controls its kinase activity. Since the catalytic site shares
many features with the catalytic sites of other kinases, such as
protein kinase A (PKA), it appears that a specific PKC inhibitor
targeting the regulatory site would be advantageous in terms of
kinase selectivity and/or PKC subtype selectivities. Most of the
potent PKC inhibitors, such as H73 and staurosporine,4 bind to
the catalytic site in competition with ATP, and thus these
inhibitors have the potential to inhibit other kinases. Although
some PKC inhibitors have been reported to act at the regulatory
site,5-7 their mode of interaction with PKC is not fully understood.
The following description of the rational design, synthesis, and
evaluation of a regulatory site-inhibitor of PKC is therefore
presented as a novel approach.
In the inactive state, PKC resides in the cytosol, and the
regulatory site of PKC interacts with the catalytic site through
the pseudosubstrate sequences, preventing the access of substrates
to the catalytic site. PKC is believed to be activated by its
translocation to the membrane and the subsequent conformational
change caused by the binding of phosphatidyl-L-serine (L-PS) and
sn-1,2-diacylglycerol (DAG), an endogenous PKC activator, to
the cysteine rich domain (CRD)8,9 in its regulatory site. Many
structurally diverse agonists have been reported, such as phorbol
12-myristate 13-acetate (PMA), ingenol, aplysiatoxin, bryostatin,
Figure 1. Strategy for the development of novel PKC inhibitor.
Figure 2. Phorbol ester-phosphatidyl-L-serine hybrid molecule (PEPS).
teleocidin, and their derivatives. However, to our knowledge,
there is no mention in the literature of a DAG antagonist. This
is probably because the binding of these agonists is highly
synergetic to the interaction of PKC with a membrane containing
L-PS (Figure 1).
We focused on this unique property of PKC activation. If a
molecule capable of blocking the binding sites of both DAG
(phorbol ester) and L-PS in CRD were made available, it would
likely prevent the interaction with the membrane and keep PKC
inactive in the cytosol (Figure 1). To this end, we designed the
phorbol ester-phosphatidyl-L-serine hybrid molecule (PEPS) 1
(Figure 2). Although the exact L-PS binding sites have not been
identified, the design of compound 1 was based on the results of
the reported photoaffinity labeling experiments using [20-3H]-
phorbol 12-azidobenzoate derivatives.10,11
Synthesis of PEPS (1) was achieved as shown in Scheme 1.
1,2:5,6-O-Diisopropylidene-D-mannitol 2 was converted to 3 in
five steps.12 Removal of an allyl group, reduction, and protection
of the resulting primary alcohol gave 4. The triphenylmethyl
group of 4 was converted to an acetyl group, and debenzylation
gave alcohol 5. The compound 5 and phosphoramidite 613 were
treated with tetrazole in anhydrous THF, and oxidation of the
resulting phosphine gave the phosphotriester 7. Deprotection of
the silyl ether gave the L-PS portion 8. The coupling of the L-PS
portion and the phorbol ester 9, which was synthesized from
phorbol in four steps,14 was achieved using Yamaguchi’s method.15
(1) Present address: Sagami Chemical Research Center, Nishi-Ohnuma,
Sagamihara, Kanagawa 229, Japan.
(2) (a) Protein Kinase C Current Concepts and Future PerspectiVes; Lester,
D. S., Epand, R. M., Eds.; Ellis Horwood Ltd.: West Sussex, 1992. (b)
Nishizuka, Y. Science 1992, 258, 607 and references therein.
(3) Hidaka, H.; Inagaki, M.; Kawamoto, S.; Sasaki, Y. Biochemistry 1984,
23, 5036.
(4) (a) Tamaoki, T.; Nomoto, H.; Takahashi, I.; Kato, Y.; Morimoto, M.;
Tomita, F. Biochem. Biophys. Res. Commun. 1986, 135, 397. (b) Bit, R. A.;
Davis, P. D.; Elliott, L. H.; Harris, W.; Hill, C. H.; Keech, E.; Kumar, H.;
Lawton, G.; Maw, A.; Nixon, J. S.; Vesey, D. R.; Wadsworth, J.; Wilkinson,
S. E. J. Med. Chem. 1993, 36, 21 and references therein.
(5) Calphostin C is reported to inhibit PKC light dependently, although
the mechanism is unknown: Bruns, R. F.; Miller, F. D.; Merriman, R. L.;
Howbert, J. J.; Heath, W. F.; Kobayashi, E.; Takahashi, I.; Tamaoki, T.;
Nakano, H. Biochem. Biophys. Res. Commun. 1991, 176, 288 and references
therein.
(6) (a) Hannun, Y. A.; Loomis, C. R.; Merill, A. H., Jr.; Bell, R. M. J.
Biol. Chem. 1986, 261, 12604. (b) Sozzani, S.; Agwu, D. E.; McCall, C. E.;
O’Flaherty, J. T.; Schmitt, J. D.; Kent, J. D.; McPhail, L. C. J. Biol. Chem.
1992, 267, 20481. (c) Benna, J. E.; Hakim, J.; Labro, M.-T. Biochem.
Pharmacol. 1992, 43, 527. It is possible that these cationic amphipathic
compounds inhibit PKC activities by neutralizing the negative phospholipids;
see chapter 3.6 in ref 2a.
(10) (a) Delclos, K. B.; Yeh, E.; Blumberg, P. M. Proc. Natl. Acad. Sci.
U.S.A. 1983, 80, 3054. (b) Schmidt, R.; Heck, K.; Sorg, B.; Hecker, E. Cancer
Lett. 1985, 26, 97.
(11) We have designed the linker of PEPS (1) based on the assumption
that the nitrene generated by the photolysis reacts with the double bond in
the oleoyl group in L-PS as shown below.
(7) Translocation inhibitor peptides were reported to interact with the
regulatory domain of PKC: Yedovitzky, M.; Mochly-Rosen, D.; Johnson, J.
A.; Gray, M. O.; Ron, D.; Abramovitch, E.; Cerasi, E.; Nesher, R. J. Biol.
Chem. 1997, 272, 1417.
(8) Binding of phorbol esters to CRD, which consists of about 50 amino
acid residues, is L-PS dependent, see: Wender, P. A.; Irie, K.; Miller, B. L.
Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 239 and references therein.
(9) The NMR and X-ray analyses of the structure of CRD in the absence
of phospholipids have been reported, see: Zhang, G.; Kazanietz, M. G.;
Blumberg, P. M.; Hurley, J. H. Cell 1995, 81, 917 and references therein.
(12) He´bert, N.; Beck, A.; Lennox, R. B.; Just, G. J. Org. Chem. 1992,
57, 1777.
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458 J. Am. Chem. Soc., Vol. 120, No. 2, 1998
Communications to the Editor
Scheme 1. Synthesis of the Phorbol Ester-Phosphatidyl-L-serine Hybrid Molecule (PEPS)
a (a) IO4- resin, BH4- resin; (b) BnBr, NaH; (c) 1 N - HCl; 73% (three steps); (d) TrCl, Et3N, DMAP; 94%; (e) 7-allyloxycarbonylheptanoic acid,
DCC, DMAP; 97%; (f) Pd(PPh3)4, morpholine; 92%; (g) BH3‚THF; 92%; (h) TBDPSCl, imidazole; 98%; (i) HCO2H; 72%; (j) Ac2O, Py; 95%; (k)
Pd-C, H2; 93%; (l) 6, tetrazole; 89%; (m) mCPBA, NaHCO3; 76%; (n) HF-Py; 41%; (o) 4-allyloxycarbonylbenzoic acid, DCC, DMAP; 95%; (p)
Pd(PPh3)4, morpholine; 93%; (q) 2,4,6-trichlorobenzoyl chloride, Et3N; then 8, DMAP; 69%; (r) anisole in TFA; 76%.
Finally, deprotection under acidic conditions gave the target
molecule PEPS (1).
The PKC-binding affinity of PEPS (1) was first examined by
its competitive inhibition of the binding of tritium-labeled phorbol
12,13-dibutyrate ([3H]PDBu) to PKC (mixture of R, â, and γ
isozymes from rat brain).16 PEPS (1) exhibits significant binding
to PKC, although it is about 10-fold weaker than PMA under
these conditions. Evaluation of the inhibiting effect of PEPS (1)
on PKC activity was then carried out by comparison of the
catalytic activities of PKC stimulated by PMA (10 nM) in both
the absence and presence of PEPS (1) according to the method
of Bell et al. with minor modifications.17 As shown in Figure 3
(lane 1), PEPS (1) significantly inhibited PKC activity. A phorbol
ester derivative 10, which lacked the L-PS portion, did not inhibit
PKC activation (lane 2), indicating that the L-PS portion of PEPS
is essential to the inhibition. The inhibition of pure PKC isozymes
(human, recombinant) was also evaluated. PEPS (1) showed
significant inhibition of the PKC R and âI (lanes 3, 4, 5, 7, and
8). To further clarify the importance of specific interaction of
the L-PS portion, the inhibition of PKC R and âI by compound
11,18 which had a D-PS component in place of L-PS, was
examined.19 The compound 11 was found to be a significantly
less potent inhibitor than PEPS (1) (lanes 6 and 9). This result
suggests that recognition of PS region of PEPS by PKC is
stereospecific and that the inhibition of PKC by PEPS is
dependent upon the interaction of PKC at both the phorbol and
L-PS sites of PEPS.
PEPS (1) displayed an ability to act alone as a partial agonist
for PKC, probably because its hydrophobic element still had some
affinity for the membrane.20 Attempts to suppress this undesired
Figure 3. Inhibition of PKC activities by PEPS (1). PKC activities in
the presence of 10 nM PMA and indicated concentration of inhibitors
dispersed in Triton X-100-L-PS mixed micelles were measured.
interaction and increase the binding affinity of PEPS (1) for PKC
are now in progress.
In summary, this is the first report on a rationally designed
phorbol ester derivative showing PKC inhibition. Although PEPS
(1) would require significant structural modification for therapeutic
applications, the concepts demonstrated herein should pave the
way for further design of novel PKC inhibitors.
(13) Kitas, E. A.; Knorr, R.; Trzeciak, A.; Bannwarth, W. HelV. Chim.
Acta 1991, 74, 1314 and references therein.
(14) Sorg, B.; Fu¨rstenberger, G.; Berry, D. L.; Hecker, E.; Marks, F. J.
Lipid. Res. 1982, 23, 443.
Acknowledgment. This study was financed with Special Coordination
Funds of the Science and Technology Agency of the Japanese Govern-
ment. We thank Dr. S. Kidokoro (Sagami Chemical Research Center)
for helpful discussion on the molecular modeling of the PEPS complex.
(15) Inanaga, J.; Hirata, K.; Saeki, H.; Katsuki, T.; Yamaguchi, M. Bull.
Chem. Soc. Jpn. 1979, 52, 1989.
(16) Tanaka, Y.; Miyake, R.; Kikkawa, U.; Nishizuka, Y. J. Biochem. 1986,
99, 257.
(17) Hannun, Y. A.; Loomis, C. R.; Bell, R. M. J. Biol. Chem. 1985, 260,
10039.
1
Supporting Information Available: Experimental procedures, H,
(18) Compound 11 was synthesized using the same techniques as for PEPS
(1) except that D-serine was used instead of L-serine.
(19) Studies using phospholipid analogues have revealed that only L-PS
can fully support PKC activity with a high degree of stereospecificity, whereas
D-PS only partially supports the activity. See: Lee, M.-H.; Bell, R. M. J.
Biol. Chem. 1989, 264, 14797.
13C NMR, IR, mass spectral data for the products, experimental procedures
for biological evaluation of the products, and modeling of the CRD-PEPS
(1) complex (46 pages). See any current masthead page for ordering
and Internet access instructions.
(20) See Supporting Information. Presence of added PS is necessary for
activation of PKC by PEPS (1), as is the case with other phorbol esters.
JA971270T