Indolactam-V is involved in the CH/π interaction with Pro-11 of the PKCδ C1B domain: Application for the structural optimization of the PKCδ ligand

Article abstract of DOI:10.1021/ja050447d

The CH/π interaction between the indole ring of indolactam-V (IL-V) and the hydrogen atom at position 4 of Pro-11 of the PKCδ C1B domain was evaluated using the mutant peptide of the PKCδ C1B domain, in which the CH/π interaction was inhibited by substitu

Full text of DOI:10.1021/ja050447d

Published on Web 04/02/2005
Indolactam-V Is Involved in the CH/π Interaction with Pro-11 of the PKCδ C1B
Domain: Application for the Structural Optimization of the PKCδ Ligand
Yu Nakagawa,† Kazuhiro Irie,*,† Ryo C. Yanagita,† Hajime Ohigashi,† and Ken-ichiro Tsuda‡
DiVision of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto UniVersity,
Kyoto 606-8502, Japan, and Bio-IT Business Promotion Center, NEC Corporation, Tokyo 108-8001, Japan
Received January 23, 2005; E-mail: irie@kais.kyoto-u.ac.jp
Protein kinase C (PKC) isozymes are the main receptors of tumor
promoters such as phorbol esters and teleocidins.1 PKC isozymes
are subdivided into three classes: conventional PKCs (R, âI, âII,
γ), novel PKCs (δ, ꢀ, η, θ), and atypical PKCs (λ/ι, ú).2,3 Tumor
promoters bind to the C1A and/or C1B domains of conventional
and novel PKCs. Since tumor promoters are structurally quite
different from each other, the binding mode of each tumor promoter
with each C1 domain of the PKC isozymes has attracted much
attention from medicinal chemists. X-ray analysis of the crystal
structure of the isolated PKCδ C1B domain in complex with
phorbol 13-acetate revealed how the phorbol ester binds to the
PKCδ C1B domain.4 A docking simulation of indolactam-V (IL-
V, Figure 1),5,6 the core structure of teleocidins, with the PKCδ
C1B domain suggested that the hydroxyl group, amide carbonyl
group, and amide hydrogen of IL-V are involved in hydrogen
bonding with Thr-12, Leu-21, and Gly-23 of the PKCδ C1B
domain.7,8 A similar hydrogen bond network was observed in the
docking model of the simplified analogue of IL-V, benzolactam-
V8 (BL-V8),9 in which the indole ring of IL-V is replaced with a
benzene ring.7 Several mutational studies supported these models
and revealed that Pro-11, which is conserved among PKC isozymes,
plays important roles in IL-V binding.8 However, it remains unclear
whether Pro-11 mutation causes a conformational change in the
binding site or loss of the hydrophobic interaction with IL-V.
Moreover, these models cannot clearly explain the fact that the
binding affinity of IL-V for PKCδ is more than 20 times higher
than that of BL-V8.
To interpret the large difference in the binding affinity between
IL-V and BL-V8, we wanted to provide evidence that IL-V may
be involved in the CH/π interaction10 with Pro-11 of the PKCδ
C1B domain, whereas BL-V8 may not. The results suggested that
lack of this CH/π interaction might be the main reason for the low
binding affinity of BL-V8. The structural optimization of BL-V8
is also described as an application of this concept to the design of
a potent PKCδ ligand.
To predict the amino acid residues arising from PKCδ interacting
with the aromatic rings of IL-V and BL-V8, docking simulations
of IL-V and BL-V8 with the crystal structure of the PKCδ C1B
domain4 were performed using the FlexX program11 (Figure 2).
The resultant docking models were quite similar to those reported
by Endo et al.7 and Wang et al.8 The aromatic rings of both ligands
were close to the hydrogen atom at position 5 of Pro-11 of the
PKCδ C1B domain (2.7 Å). On the other hand, the distance between
the aromatic ring of each ligand and the hydrogen atom at position
4 was quite different (2.5 Å for IL-V and 3.3 Å for BL-V8). Since
a hydrogen atom and an aromatic ring are involved in the CH/π
interaction only on the condition that their distance is within 3.05
Å,10 these results suggest that IL-V may be involved in the CH/π
Figure 1. Structures of IL-V, BL-V8, and NL-V8.
Figure 2. Docking models of IL-V (left) and BL-V8 (right) with the PKCδ
C1B domain. Yellow dotted lines represent hydrogen bonds.
interaction with the hydrogen atoms at positions 4 and 5 of Pro-11
of the PKCδ C1B domain, but BL-V8 may interact only with the
hydrogen atom at position 5.
Substitution of the hydrogen atom involved in the CH/π
interaction with a fluorine atom is one of the reliable methods to
evaluate the CH/π interaction.12 Since a fluorine atom has an
extremely higher electronegativity but a similar van der Waals
radius compared to that of a hydrogen atom, substitution with a
fluorine atom can inhibit the CH/π interaction of the target hydrogen
atom without causing a significant conformational change of the
whole protein. To examine the existence and importance of the
CH/π interaction in the binding of IL-V and BL-V8 to the PKCδ
C1B domain, a mutant peptide of the PKCδ C1B domain, in which
Pro-11 was replaced by 4,4-difluoro-Pro,13 was prepared by solid-
phase Fmoc synthesis, as reported previously.14 The binding
affinities of IL-V and BL-V8 for the wild-type and the mutant
peptides of the PKCδ C1B domain, designated as δ-C1B and
δ-C1B(P11dfP), respectively, were evaluated by the inhibition of
the specific binding of [3H]phorbol 12,13-dibutyrate (PDBu) to these
peptides by the method reported previously.15 Table 1 shows the
inhibition constants (Ki) of IL-V and BL-V8 for δ-C1B and δ-C1B-
(P11dfP).
IL-V showed about a 10 times lower affinity for δ-C1B(P11dfP)
compared to that for δ-C1B. In contrast, the binding affinity of
BL-V8 for δ-C1B(P11dfP) was almost equal to that for δ-C1B.
These results strongly suggest that IL-V could be involved in the
CH/π interaction with the hydrogen atom at position 4 of Pro-11,
and the low binding affinity of BL-V8 could be mainly ascribable
to the absence of this CH/π interaction.
On the basis of these results, the structural optimization of BL-
V8 was carried out. The benzene part of the indole ring of IL-V
† Kyoto University.
‡ NEC Corporation.
9
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J. AM. CHEM. SOC. 2005, 127, 5746-5747
10.1021/ja050447d CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
Table 1. Ki Values (nM) of the Inhibition of [3H]PDBu Binding by
for whole PKCδ; the Ki value of NL-V8 for PKCδ was 147 ( 18
nM, which was about 12 times smaller than that of BL-V8 (1700
nM), reported by Endo et al.7 On the other hand, NL-V8 showed
an affinity to δ-C1B(P11dfP) about 4 times lower than that for
δ-C1B. These results indicate that the higher affinity of NL-V8
compared to that of BL-V8 could be partially attributed to the CH/π
interaction between the additional benzene ring and the hydrogen
atom at position 4 of Pro-11.
Our present data provide evidence that the CH/π interaction plays
a pivotal role in the binding of IL-V and its analogues to the PKCδ
C1B domain. It was also shown that the binding affinity of BL-V8
could be enhanced by the effective formation of the CH/π
interaction. The information presented in this communication is
useful for the verification of the docking model of IL-V with the
PKCδ C1B domain and for the rational design of new potent ligands
for PKCδ, which has a tumor suppresser role.18
IL-V, BL-V8, and NL-V8a
peptides
IL-V
BL-V8
NL-V8
δ-C1B
δ-C1B(P11dfP)
11.4 (1.0)b
131 (9.5)
414 (28)
436 (41)
44.1 (4.8)
139 (20)
a The Kd values for δ-C1B and δ-C1B(P11dfP) were 0.53 and 3.5 nM,
respectively. Although these values were slightly different from each other,
a significant conformational change by the mutation would not possibly
occur since the Ki values of BL-V8 for both peptides were almost similar.
b Standard deviation of at least two separate experiments.
Scheme 1. Synthesis of NL-V8
Acknowledgment. This research was partly supported by a
grant-in-aid for Scientific Research (A) (No. 15208012 for H.O.
and K.I.) and a grant-in-aid for the promotion of Science for young
scientists (Y.N.) from the Ministry of Education, Science, Culture,
Sports, and Technology of the Japanese Government.
Supporting Information Available: Modeling methods and de-
tailed experimental procedures with spectroscopic data. This material
is available free of charge via the Internet at http://pubs.acs.org.
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The binding affinity of NL-V8 for δ-C1B was about 10 times
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