Dramatic switching of protein kinase C agonist/antagonist activity by modifying the 12-ester side chain of phorbol esters

Article abstract of DOI:10.1021/ja027048s

A dramatic switching of PKC agonist and antagonist activity was observed by modification of the hydrophilicity of the 12-ester side chain of phorbol. Thus, phorbol ester 4 that contains a glycol at the 12-ester chain demonstrated a pure and significant antagonist ability of PKC; however, 3 that contains an alkanol at the 12-ester chain demonstrated a potent PKC agonist activity. On the basis of the structural difference between 3 and 4 and results of the partition assay in the Hela cell/PBS buffer system, we propose that 4 acts as a translocation poison of the PKC-phorbol ester complex. The approach of controlling the agonist/antagonist activity of phorbol esters by the nature (i.e., hydrophilicity, charge, and rigidity, etc.) of the 12-ester chain may be very useful for developing selective PKC inhibitors and a potential pharmaceutical compound for anticancer therapies. Copyright

Full text of DOI:10.1021/ja027048s

Published on Web 08/14/2002
Dramatic Switching of Protein Kinase C Agonist/Antagonist Activity by
Modifying the 12-Ester Side Chain of Phorbol Esters
Reiko Wada, Yutaka Suto, Motomu Kanai, and Masakatsu Shibasaki*
Graduate School of Pharmaceutical Sciences, The UniVersity of Tokyo, Tokyo 113-0033, Japan
Received May 25, 2002
Protein kinase C (PKC) is a family of core enzymes (serine/
threonine kinases) in intracellular signal transduction cascades and
is intimately involved in the regulation of a variety of cellular
functions such as gene expression, cellular growth, and differentia-
1
tion. Decades of intensive studies revealed a sophisticated picture
2
2+
for PKC activation: increase of intracellular Ca concentration
and/or generation of the lipid second messenger, diacylglycerol
(
DAG), results in the recruitment of PKC from the cytosol, where
Figure 1. Phorbol derivatives.
the PKC is maintained in an inactive conformation, to the
membrane, where the PKC becomes allosterically activated by
interactions with DAG and phosphatidyl-L-serine (PS) in the
hydrophobic environment. Phorbol esters, such as phorbol 12-
myristate 13-acetate (PMA: 1) and phorbol 12,13-dibutyrate
hydrophilicity of the 12-ester chain of phorbol esters would modify
the stability of the phorbol ester-PKC-membrane complex, which
is required for PKC activation. A hydrophilic 12-ester chain would
greatly decrease the affinity of the phorbol ester-PKC complex
and the membrane, while maintaining an affinity between the
phorbol ester and PKC. Because PKC activation does not occur in
the absence of an interaction with the membrane, a phorbol ester
containing a hydrophilic ester chain would be a competitive inhibitor
of PKC activation by an agonist such as PMA. Phorbol esters
derived from aliphatic carboxylic acids of various carbon lengths
at the 12-position (R in Figure 1) are known; however, there are
no previous studies on the structure-activity relationship concerning
the nature (e.g., hydrophilicity, charge, rigidity, etc.) of the 12-
(PDBu: 2), are naturally occurring extremely potent PKC agonists
that strongly activate PKC (at least 3 orders of magnitude higher
than DAG) through the same mechanism as that of the endogenous
activator, DAG.3 In addition, although DAG is metabolized
immediately through phosphorylation or hydrolysis, phorbol esters
persistently activate PKC because there is no natural mechanism
to terminate their action in an animal cell. Because of this ability,
phorbol esters can produce malignant cell growth and are the most
potent tumor promoters known to date. In this communication, we
report that the strong PKC agonist activity of phorbol esters is
completely suppressed by a slight modification of the ester moiety
at the 12-position, but still retains the ability to bind to PKC.
Moreover, the compound demonstrated significant PKC inhibitory
activity.
ester chain. Thus, we synthesized 3 and 4, containing an alkanol
and tetra(ethylene glycol) at the 12-ester chain.9
The PKC-binding affinity of PMA (1), 3, and 4 was first assessed
by competitive inhibition of the binding of tritium-labeled 2 to
human recombinant PKCR in the presence of a PS vesicle as a
Our ongoing focus is to develop a PKC specific inhibitor as a
4
potential pharmaceutical compound for anticancer therapies.
10
membrane mimic (Figure 2a). Although 1 and 3 had a similar
Generally, inhibitors that bind to the regulatory domain of PKC
are thought to be more advantageous than catalytic domain binders
with respect to kinase selectivity, because the catalytic domain of
PKC shares many features with the catalytic sites of other kinases,
strong binding affinity to PKC (K
d
≈ 0.01 µM), the affinity of 4
was ca. 2 orders of magnitude lower (K
d
> 1 µM) than that of 1
11
and 3. As compared with the physiologic ligand DAG, however,
the binding affinity of 4 was still higher, reflecting the high binding
5
such as protein kinase A (PKA). The strong and selective binding
affinity of the phorbol moiety.
We then investigated the agonist activity of 3 and 4.12 As shown
in Figure 2b, 3 had agonist activity comparable to that of PMA
affinity of phorbol esters to the regulatory domain of PKC led us
to design a PKC inhibitor containing the phorbol moiety. Crystal-
6
7
lographic and molecular modeling studies of phorbol ester-PKC
complexes suggested that the molecular basis of action of phorbol
esters is ascribed to their bifunctional nature: on one hand, with
their hydrophilic part, phorbol esters bind PKC by a hydrogen bond
network, mainly through oxygen atoms at the 20- and 3-positions.
On the other hand, the hydrophobic ester chain at the 12-position
helps to retain the phorbol ester-PKC complex in the membrane
where PKC is activated. Thus, both sets of oxygen atoms at the
hydrophilic part and the hydrophobic ester chain are essential for
(EC50 < 0.01 µM). In sharp contrast, the agonist activity was
completely eliminated in the glycol ester 4 (EC50 . 10 µM). The
large difference from the expected binding affinity to PKC in the
agonist activity of 4 suggested that activation of PKC is a multistep
process and that 4 does not promote any of these steps other than
the binding to PKC. One possible explanation for the sharp contrast
in agonist activity between 3 and 4 might be the difference in the
affinity of the agonist-PKC complex to the membrane (Figure 3).
Thus, the 4-PKC complex might be stable in solution due to the
hydrophilicity of the poly(ethylene glycol) ester chain, and thus
translocation to the membrane where PKC is activated might be
prevented. Results of the following partition experiments appear
to support this explanation. The distribution of 3 and 4 in the water
8
phorbol esters to activate PKC. We designed a PKC inhibitor on
the basis of this mechanism of action of phorbol esters. The
*
To whom correspondence should be addressed. E-mail: mshibasa@mol.f.u-
tokyo.ac.jp.
1
0658 J. AM. CHEM. SOC. 2002, 124, 10658-10659
9
10.1021/ja027048s CCC: $22.00 © 2002 American Chemical Society

C O MMU N I C A T I O N S
Figure 2. Evaluation of new phorbol derivatives containing the 12-ester chain of different hydrophilicity. (a) The PKCR binding profile of PMA (1), 3, and
3
4
evaluated by competitive experiments with [ H]PDBu. 9, 1; b, 3; 2, 4. (b) The activation profile of PKCR evaluated by the phosphorylation of EGF-R
32
in the presence of [γ- P]ATP. 9, 1; b, 3; 2, 4. (c) The inhibitory activity of 3 and 4 against PKCR activation by 10 nM PMA. b, 3; 2, 4.
Acknowledgment. Financial support was provided by RFTF
of Japan Society for the Promotion of Science. We thank Dr.
Nishina, Ms. Tsujita, and Professor Katada of the University of
Tokyo for the partition assay.
Supporting Information Available: Experimental procedures and
characterization of the products (PDF). This material is available free
of charge via the Internet at http://pubs.acs.org.
References
(
1) (a) Protein Kinase C Current Concepts and Future PerspectiVes; Lester,
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Figure 3. Cartoon for PKC activation mechanism by phorbol derivatives.
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(
(
layer was measured in the Hela cell/PBS buffer system using HPLC
analysis (detection with UV).13 Whereas 4 mainly existed in the
water layer (water layer:cell ) 11:1), 3 was efficiently concentrated
in the Hela cell membrane (water layer:cell ) 1:3). This contrasting
distribution is probably due to the hydrophilicity difference of the
4) Our initial concept for the design of a PKC inhibitor was to prevent the
PKC conformational change to the active form by a chelate binding of
the inhibitor (PEPS): Sodeoka, M.; Arai, M. A.; Adachi, K.; Uotsu, K.;
Shibasaki, M. J. Am. Chem. Soc. 1998, 120, 457-458. Although PEPS
inhibited PKC activation by PMA, PEPS also demonstrated a partial
agonist activity.
(
5) PKC has a regulatory domain that binds to DAG (or PKC) and PS and a
catalytic domain that phosphorylates the substrate proteins. Most of the
potent PKC inhibitors, such as H7 and staurosporine, bind to the catalytic
domain in competition with ATP. There are several selective kinase
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inhibitors, see: Bridges, A. J. Chem. ReV. 2001, 101, 2541-2571.
6) Zhang, G.; Kazanietz, M. G.; Blumberg, P. M.; Hurley, J. H. Cell 1995,
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12-ester chain, and the difference might be an important factor in
determining the PKC agonist activity.
As expected from the binding ability and completely eliminated
agonist activity of 4, the inhibitory assays demonstrated that 4 is a
significant inhibitor of PMA activation of PKCR. PKC activity was
suppressed up to 30%, depending on the inhibitor concentration
(
(
7) Wang, S.; Kazanietz, M. G.; Blumberg, P. M.; Marquez, V. E.; Milane,
G. W. A. J. Med. Chem. 1996, 39, 2541-2553.
(
Figure 2c). In the case of 3, however, PKCR activity increased in
(8) For pharmacophore models of phorbol esters-PKC complexes, see: (a)
Wender, P. A.; Koehler, K. F.; Sharkey, N. A.; Dell’Aquila, M. L.;
Blumberg, P. M. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 4214-4218. (b)
Itai, A.; Kato, Y.; Tomioka, N.; Iitaka, Y.; Endo, Y.; Hasegawa, M.; Shudo,
K.; Fujiki, H.; Sakai, S.-I. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 3688-
a concentration-dependent manner, reflecting the agonistic character
of 3. Therefore, the function of the phorbol ester derivatives could
be dramatically switched from a potent agonist to a pure antagonist
with no partial agonist activity by tuning the hydrophilicity of the
3692. (c) Kishi, Y.; Rando, R. R. Acc. Chem. Res. 1998, 31, 163-172.
(
9) For synthesis of 3 and 4, see Supporting Information (SI) for details. The
phorbol can be easily isolated from commercially available croton oil in
a large scale: Crombie, L.; Games, M. L.; Pointer, D. J. J. Chem. Soc. C
1
2-ester chain.
The described results demonstrated a possibility for a rational
1968, 1347-1362.
design of a phorbol-based PKC inhibitor by modification of the
2-ester side-chain hydrophilicity, thus affecting the membrane
(10) Tanaka, Y.; Miyake, R.; Kikkawa, U.; Nishizuka, Y. J. Biochem. 1986,
99, 257-261.
1
(
11) One possible reason for the observed higher affinity of 1 and 3 to PKC
as compared to 4 might be a concentration effect on the PS vesicle surface.
Because of their hydrophobic ester chain, 1 and 3 are more efficiently
affinity of the inhibitor-PKC complex. Because the phorbol is
easily available from commercial sources and esterification of the
1
inhibitor candidates can be synthesized using this approach.
Systematically changing the nature (hydrophilicity, charge, and
rigidity) of the ester moiety to develop a more potent and selective
PKC inhibitor is currently under investigation.
3
concentrated on the PS vesicles where the [ H]PDBu exists than is 4.
2-hydroxyl group is synthetically trivial, a wide range of diverse
This situation might allow for 1 and 3 to be more effective in competing
for the binding site on PKC.
(
12) Hannun, Y. A.; Loomis, C. R.; Bell, R. M. J. Biol. Chem. 1985, 260,
10039-10043.
13) See Supporting Information for details.
(
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J. AM. CHEM. SOC.
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VOL. 124, NO. 36, 2002 10659