Diacylglycerol lactones targeting the structural features that distinguish the atypical C1 domains of protein kinase C Ζ and ι from typical C1 domains

Article abstract of DOI:10.1021/jm500165n

To explore the feasibility of developing ligands targeted to the atypical C1 domains of protein kinase C Ζ and ι, we have prepared diacylglycerol lactones substituted with hydrophilic groups on their side chains, which potentially could interact with the

Full text of DOI:10.1021/jm500165n

Article
pubs.acs.org/jmc
Diacylglycerol Lactones Targeting the Structural Features That
Distinguish the Atypical C1 Domains of Protein Kinase C ζ and ι from
Typical C1 Domains
Yongmei Pu,† Ji-Hye Kang,‡ Dina M. Sigano,‡ Megan L. Peach,§ Nancy E. Lewin,† Victor E. Marquez,‡
and Peter M. Blumberg*,†
†Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, National Institutes of Health,
Bethesda, Maryland 20892, United States
‡Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute at Frederick, National Institutes of Health,
Frederick, Maryland 21702, United States
§Chemical Biology Laboratory, Basic Science Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical, Inc.,
Frederick, Maryland 21702, United States
ABSTRACT: To explore the feasibility of developing ligands
targeted to the atypical C1 domains of protein kinase C ζ and
ι, we have prepared diacylglycerol lactones substituted with
hydrophilic groups on their side chains, which potentially
could interact with the arginine residues that distinguish the
atypical C1 domains of PKCζ and PKCι from typical C1
domains, and we have measured their binding to mutated versions of the C1b domain of PKCδ that incorporate one or more of
these arginine residues. The most selective of the diacylglycerol lactones showed only a 10-fold reduction in binding affinity with
the triple arginine mutant (N7R/S10R/L20R) compared to the wild-type, whereas phorbol 12,13-dibutyrate showed a 6000-fold
loss of affinity. Molecular modeling confirms that these ligands are indeed able to interact with the arginine residues. Our results
show that dramatic changes in selectivity can be obtained through appropriate substitution of diacylglycerol lactones.
bound ligand promotes membrane insertion of the C1 domain
both by capping the hydrophilic cleft and by contributing
additional hydrophobicity provided by its acyl side chains. The
membrane lipids, in turn, provide further stabilization of the
complex. Functional consequences of the membrane insertion
of the C1 domain for its protein host include translocation of
the protein to membranes, bringing it into proximity with
substrates or interacting partners, and conformational change.
In the cases of both β2-chimaerin15 and PKCβII,16 X-ray
crystallography reveals that the C1 domain in its unliganded
state is involved in intramolecular interactions which are lost
when the C1 domain shifts to the membrane, providing a
further mechanistic component for the conformational change.
Analysis of the C1 domains in PKC family members
indicated that whereas the C1 domains of the so-called classical
PKC isoforms α, βI, βII, and γ and those of the so-called novel
PKC isoforms δ, ε, η, and θ were DAG-responsive, those of the
atypical PKC isoforms ζ and ι/λ failed to recognize DAG. Such
DAG-unresponsive C1 domains have been termed “atypical”.
Structural analysis indicates, moreover, that the atypical C1
domains can be further subdivided.
INTRODUCTION
■
The C1 domain has emerged as a critical structural motif
functioning as a recognition module for the lipophilic second
messenger sn-1,2-diacylglycerol (DAG).1−3 Initially identified
as the regulatory site on the classic and novel isoforms of
protein kinase C (PKC) for DAG and their ultrapotent
analogues the phorbol esters, C1 domains serve a similar
function while coupled to distinct effector domains on six other
families of proteins.4−6 These families comprise isoforms of
protein kinase D, chimaerin, RasGRP, unc13, MRCK, and
DAG kinase. DAG, the endogenous ligand for C1 domains,
plays a central role in cellular signaling. It is generated through
breakdown of phosphoinositol 4,5-bisphosphate by phospho-
lipase Cβ, activated by G-protein-coupled receptors, as well as
by phospholipase Cγ, activated by receptor tyrosine kinases.7 It
is also generated indirectly through the activity of phospholi-
pase D on phosphatidylcholine.8 Given the critical role played
by the many pathways signaling through DAG, it is not
surprising that PKC and the other family members have been
identified as attractive therapeutic targets for cancer,
Alzheimer’s disease, and a variety of other conditions.9,10
Drugs in clinical trials targeting the C1 domain of PKC include
bryostatin 1 and PEP005 (ingenol 3-angelate).11−13
The atypical C1 domains of PKCζ17 and Vav118 retain a
conformation of the binding cleft very similar to that of the
typical C1 domains but possess residues that interfere with
The structure of the complex between phorbol ester and the
C1b domain of PKCδ was solved by X-ray crystallography.14
The ligand binds to the C1 domain by inserting into a
hydrophilic cleft in an otherwise hydrophobic surface. The
Received: January 29, 2014
Published: March 31, 2014
© 2014 American Chemical Society
3835
dx.doi.org/10.1021/jm500165n | J. Med. Chem. 2014, 57, 3835−3844

Journal of Medicinal Chemistry
Article
formation of the C1 domain−ligand−phospholipid complex
(Figure 1). In the case of PKCζ/ι, we have identified four
Vav1 C1 domain, Kd for PDBu = 1.05 0.14 nM; PKCδ C1b
domain, Kd = 0.193 0.005 nM).19
The second class of atypical C1 domains differs conceptually
from the above in that they involve deletions of residues in the
loops constituting the DAG/phorbol ester binding cleft.
Examples include the atypical C1 domains of Raf,20 of KSR
(kinase suppressor of Ras),21 and of Nore1.22 In each instance,
these deletions disrupt the structure of the binding cleft and
destroy binding.
The atypical PKC isoforms ζ and ι play critical roles in
cellular signaling. Unique among PKC isoforms, PKCι is the
only isoform for which knockout is embryonic lethal.23 PKCι
functions as an oncogene in cancer in multiple tissue types
including lung and colon.24,25 PKCζ plays a critical role in
NFκB signaling and in IL-4 response, and the role of PKCι in
cell polarity has been highlighted.26
Figure 1. Amino acid sequence alignment of the C1b domain of
PKCδ, the C1 domains of PKCζ, PKCι, and hVav1. The residues in
PKCζ/ι largely responsible for the lack of phorbol ester binding are
shown in red.17 The residues in Vav1 largely responsible for the lack of
phorbol ester binding are shown in blue.18
arginine residues along the rim of the the binding cleft of the
C1 domain that are primarily responsible for its failure to bind
phorbol ester.17 Replacement of these four arginine residues
with the residues in the corresponding positions in the typical
C1b domain of PKCδ confers high affinity phorbol ester
binding on this atypical C1 domain, as evidenced by its
membrane translocation in response to PMA.17 Conversely,
replacement of the corresponding residues on the typical C1b
domain of PKCδ with these arginines led to loss of phorbol
ester binding activity.17 In the case of Vav1, we similarly
identified five residues responsible for the loss of binding
activity.19 Four residues increase the hydrophilicity along the
rim of the binding cleft, disfavoring membrane insertion of the
C1 domain. The fifth confers hydrophobicity in what is a
hydrophilic collar in typical C1 domains that stabilizes
interaction of the membrane-inserted C1 domain with lipid
head groups. Once again, substitution of these five identified
residues from Vav1 into the corresponding positions in the C1b
domain of PKCδ abolished ligand binding activity and
replacement of these five residues in the atypical C1 domain
of Vav1 with the corresponding residues in the C1b domain of
PKCδ generated potent binding activity (quintuple mutant of
In PKCζ, the role of the atypical C1 domain, like that of the
typical C1 domains of the novel and classical PKCs, has been
suggested to be regulatory, acting as an allosteric inhibitor of
enzyme activity by binding to the PIF (PDK-1 interacting
fragment) pocket in the small lobe of the kinase domain,
stabilizing interactions of the inhibited kinase.27 Ligand binding
to the PKCζ C1 domain might therefore be stimulatory, by
relieving this inhibition. A possible precedent is provided by the
λ-interacting protein (LIP), which binds to the C1 domain of
PKCλ/ι and activates the enzyme.28 On the other hand, a
prominent feature of the atypical PKCs is their coupling to
other proteins mediated through their N-terminal PB1 (Phox
Bem 1) domain.29 Ligand binding to the PKCζ C1 domain
might lead to functional inhibition if it caused the
mislocalization or sequestration of the PKC. A possible
precedent is provided by the par-4 gene product, which binds
to the C1 domain and leads to inhibition.30
Diacylglycerol lactones have proven to provide a powerful
scaffold for the design of novel, potent ligands targeting C1
domains.31 They combine relative structural simplicity with
potencies approaching those of the phorbol esters for
Table 1. Structures of DAG-lactones 1−13 including log P Values
a
R1
R2
log P
1-E
[(CH3)2CH]2CHCH2-
[(CH3)2CH]2CHCH2-
[(CH3)2CH]2CHCH2-
[(CH3)2CH]2CHCH2-
[(CH3)2CH]2CHCH2-
[(CH3)2CH]2CHCH2-
(CH3)3C-
o-C6H4OH
m-C6H4OH
p-C6H4OH
3.60
3.60
3.60
3.54
3.54
3.54
2.58
2.58
4.38
4.38
6.24
6.24
6.24
3.20
2-E
3-E
4-E
m-C6H4CO2H
5-E
p-C6H4CO2H
5-Z
6-E
p-C6H4CO2H
m-C6H4CO2H
7-E
(CH3)3C-
p-C6H4CO2H
8-E
[(CH3)2CHCH2]2CHCH2-
[(CH3)2CHCH2]2CHCH2-
[(CH3)2CHCH2]2CHCH2-
[(CH3)2CHCH2]2CHCH2-
[(CH3)2CHCH2]2CHCH2-
(HOCH2)2(CH3)C-
m-C6H4CO2H
9-E
p-C6H4CO2H
10-E
11-E
12-E
13-E
o-C6H4CH[CO2C(CH3)3]2
m-C6H4CH[CO2C(CH3)3]2
p-C6H4CH[CO2C(CH3)3]2
-CH2CH[CH2CH(CH3)2]2
a
The log P values were calculated using ChemBioDraw Ultra, version 12.0.2.
3836
dx.doi.org/10.1021/jm500165n | J. Med. Chem. 2014, 57, 3835−3844

Journal of Medicinal Chemistry
Article
optimized structures. Manipulation of the side chains using
combinatorial chemistry has yielded compounds with dramatic
differences in functional consequences at the whole cell level,
plausibly reflecting the diversity for interactions afforded by the
C1 domain−ligand−membrane ternary complex for different
cellular membrane subdomains.32 Since the atypical C1
domains of PKCζ/ι retain a binding cleft conformation similar
to that of the typical C1 domains, we postulated that DAG
lactones appropriately substituted to interact with the positively
charged residues that distinguish the C1 domains of PKCζ/ι
from the typical C1 domains might provide ligands for these
atypical C1 domains. We describe here a first step for exploring
this strategy. We prepared a series of DAG lactones
incorporating polar side chains that might interact with one
or more of the critical arginine residues that distinguish the
PKCζ/ι C1 domains. We compared their binding to the typical
C1b domain of PKCδ and to mutated versions of this C1
domain incorporating one or more of these arginine residues.
Up to a 600-fold gain in relative affinity was obtained, and
computer modeling predicted that ligand−arginine interactions
should be occurring. Our findings provide encouragement that
our approach is promising.
Our initial objective was to examine the influence of various
hydrophilic substitutions of the DAG-lactone acyl side chains
on binding affinities to variants of the PKCδ C1b domain
substituted with one of the four critical arginine residues found
in PKCζ/ι. All of the single arginine substitutions led to only a
modest decrease in PDBu binding (Table 2, first row),
permitting the measurement of DAG-lactone binding affinities
by competition for [3H]PDBu binding. Table 3 shows the ratio
of the Kd or Ki values for mutant binding to wild-type binding,
giving an indication of the relative change in binding affinity
due to the arginine mutation(s). Thus, in the first row of Table
3 it can be seen that (as described previously17) binding of
[3H]PDBu to the N7R and P11R mutants was about 5-fold
weaker than to the wild-type δC1B domain, binding to L20R
was about 35-fold weaker, and binding to S10R was essentially
the same. A value of less than 1 in Table 3, as seen for some of
the DAG-lactones, indicates that binding to the mutant was
stronger than to the wild-type domain.
The hydrophilic DAG-lactones fell into two classes. The R2
phenol (1−3) and aryl diester (10−12) derivatives retained
good potency on the wild-type C1b domain. They also showed
good retention of activity on the single variants N7R, S10R, and
P11R and in some cases even improved potency over the wild-
type domain (Table 2). The R2 phenol compounds (1−3) also
averaged only a 5-fold decrease in binding affinity to L20R
versus the wild-type C1 domain, compared to a 35-fold
reduction in binding with [3H]PDBu. Compound 3 showed as
much as a 10-fold improvement in selectivity for L20R, the
mutant with the greatest effect on [3H]PDBu binding (Table
3). There was little improvement in the selectivity of the aryl
diester class (10−12) for L20R, and this series was not further
characterized.
RESULTS
■
Diacylglycerol lactones 1−13 (Table 1) were prepared and
characterized as described in Scheme 1.
Scheme 1
The second class of DAG-lactones, the R2 benzoic acid
derivatives 4−9 and the R1 methylpropane diol derivative 13,
showed large reductions in affinity for the wild-type C1b
domain, ranging from 96- to 2100-fold (Table 2). In the case of
the benzoic acid derivatives, this was no doubt due in large part
to the formal charge on these molecules and the solvation
energy penalty for membrane interaction. Accordingly, the
reduction in affinity was generally inversely proportional to the
size and hydrophobicity of the R1 side chain; i.e., compounds 8
and 9 with the largest branched acyl groups had the highest
affinities. This correlation of Ki with log P is believed to be a
function of nonspecific membrane binding.37 Interestingly,
however, the activity of this second class of DAG-lactones
toward the single mutants did not decrease substantially relative
to the wild-type domain (Table 3). Particularly strikingly,
compounds 4 and 13 both bound almost as strongly to the
L20R mutant as to the wild-type domain, in contrast to the 35-
fold decrease in potency of [3H]PDBu for L20R. Compounds 4
and 8 also bound the P11R mutant 5 times and 3 times more
strongly, respectively, than they bound the wild-type domain.
The syntheses of DAG-lactone analogues 1−13 were
completed utilizing a well-established methodology developed
in our laboratory.33−35 From commercially available starting
reagents the doubly protected lactone I [PMP = p-
methoxyphenyl and PG = either benzyl (Bn) or tert-
butyldiphenylsilyl (TBDPS)] was synthesized and reacted
with different aldehydes (RCHO) to form the corresponding
aldol intermediates, which then underwent elimination of the
resultant β-hydroxylactone to give the corresponding olefin
(II). In some cases, this generated mixtures of E- and Z-
isomers. Consistent with previously synthesized DAG-lactones,
the E/Z geometry around the double bond was assigned by 1H
NMR: the vinyl proton for the E-isomers displayed a
characteristic multiplet that was farther downfield than that of
the corresponding Z-isomers. After separation of the geometric
E/Z-isomers and removal of the PMP group with ceric
ammonium nitrate (CAN), the compounds were individually
converted to the corresponding DAG-lactones with different R′
acyl groups by conventional methods. The target compounds
(1−13) were obtained by deprotection of intermediate IV.
When the protecting group was the benzyl ether (PG = Bn),
debenzylation was performed with BCl3, (CH2Cl2, −78 °C),
whereas in the case of the silyl ether (PG = TBDPS),
deprotection was accomplished with tetra-n-butylammonium
fluoride (TBAF) under standard conditions. The decision to
prepare racemic DAG-lactones was mainly practical, as these
compounds are easier to synthesize and we have found the
differences in binding affinity between the enantiomers to be
minimal.36
DISCUSSION AND CONCLUSIONS
■
We had observed previously that the decrease in affinity of
[3H]PDBu for variant C1b domains incorporating more than
one arginine substitution was greater than the product of the
decreases induced by the individual substitutions.17 We next
examined how the hydrophilic substituted DAG-lactones
interacted with the N7R/P11R and N7R/L20R double mutants
as well as with the triple mutants derived from these double
mutants by addition of the S10R. As previously described, the
S10R mutation had only a modest effect in combination,
3837
dx.doi.org/10.1021/jm500165n | J. Med. Chem. 2014, 57, 3835−3844

Journal of Medicinal Chemistry
Article
3838
dx.doi.org/10.1021/jm500165n | J. Med. Chem. 2014, 57, 3835−3844

Journal of Medicinal Chemistry
Article
Table 3. Relative Binding Affinities of Ligands to the Wild-Type C1b Domain of PKCδ and to Mutated Versions Incorporating
One or More of the Residues of PKCζ/ι Responsible for Its Loss of Ligand Binding Activity
a
Kd or Ki relative to that for PDBu binding to the wtδC1b
a
compd
Kd, nM, wtδC1B
N7R
S10R
P11R
L20R
N7R/P11R
N7R/L20R
N7R/S10R/P11R
N7R/S10R/L20R
PDBu
1-E
(0.23)
(2.95)
(2.46)
(2.95)
(2102)
(560)
(865)
(1206)
(1070)
(300)
(96)
5.13
1.12
1.01
0.67
0.30
1.41
0.55
1.44
0.82
0.94
1.25
0.90
1.20
1.17
1.04
1.39
0.79
0.85
0.40
0.40
1.45
0.57
1.46
0.62
1.56
0.98
1.50
1.20
1.00
1.44
5.17
3.53
0.94
0.50
0.20
1.27
1.09
2.79
0.93
0.33
1.25
1.70
2.00
2.00
2.61
34.7
4.75
7.87
3.20
1.34
12.4
3.25
4.57
5.74
5.33
7.70
17.3
17.6
17.5
1.04
147
2090
81.0
85.2
21.3
5900
224
12.5
b
b
b
b
2-E
NT
NT
NT
NT
b
b
b
b
3-E
NT
NT
NT
NT
b
b
4-E
2.47
6.79
3.25
13.2
3.49
21.8
209
NT
NT
b
b
5-E
NT
NT
b
b
5-Z
60.0
87.0
78.0
NT
NT
b
b
6-E
NT
NT
b
b
7-E
NT
NT
b
b
b
b
8-E
NT
NT
NT
NT
b
b
b
b
9-E
NT
NT
NT
NT
b
b
b
b
10-E
11-E
12-E
13-E
(1)
NT
NT
NT
NT
b
b
b
b
(0.5)
NT
NT
NT
NT
b
b
b
b
(0.6)
NT
NT
NT
NT
(777)
30.7
9.83
60.5
10.1
a
b
Data are derived from Table 2. NT, not tested.
Figure 2. Modeled complexes of DAG-lactone 4 with the N7R/P11R (A) and L20R (B) mutant C1 domains. The arginine residues are colored
magenta. The ligand is colored green, and hydrogen bond interactions are indicated with dashed orange lines.
consistent with its modest effect when alone.17 For the N7R/
L20R double C1b domain mutant, PDBu bound with a 2090-
fold decrease in affinity. In dramatic contrast, 13 showed only a
9.8-fold loss in affinity, corresponding to a 212-fold increase in
selectivity. The triple mutant N7R/S10R/L20R showed a
further modest decrease in affinity for PDBu, with a total
reduction in affinity of 5900-fold; compound 13 displayed no
further loss of affinity for an increase in selectivity relative to
PDBu of 585-fold. For the N7R/P11R double mutant, which
displayed a 147-fold loss in PDBu binding affinity, compound 4
showed only a 2.5-fold loss in affinity, corresponding to a 59-
fold increase in selectivity, whereas compound 13 was much
less tolerant, with only a 4.8-fold increase in selectivity.
Modeling provides some insight into the interactions of the
compounds with the various mutants. We docked the DAG-
lactones 4 and 13 into modeled structures of the C1 domain
mutants and explored the conformational space available to the
arginine residues and hydrophilic R1 and R2 lactone side chains
to identify low-energy salt-bridged or hydrogen-bonded
complex structures.
DAG-lactone 4, a meta-substituted R2 benzoate, is shown in
Figure 2. As seen in Figure 2A and in Table 3, the meta-isomer
seems to be favorable for strong interactions with arginine at
3839
dx.doi.org/10.1021/jm500165n | J. Med. Chem. 2014, 57, 3835−3844

Journal of Medicinal Chemistry
Article
Figure 3. Modeled complexes of DAG-lactone 13 with the N7R (A) and L20R (B) mutant C1 domains. The arginine residues are colored magenta.
The ligand is colored green, and hydrogen bond interactions are indicated with dashed orange lines.
position 11. The carboxylate group is positioned to form strong
hydrogen bonds with one of the Nη atoms and the Nε atom in
the arginine side chain (R11), as well as with the backbone
amide nitrogen. In Figure 2A compound 4 is shown
simultaneously binding the arginine at position 7 (R7). We
found that arginine at this position can bind any of the DAG-
lactones, regardless of their R1 and R2 side chains, through
interactions with the carbonyl oxygen of the ester adjacent to
R1 along with the protein backbone carbonyl at either tyrosine
Y8 (as shown) or L24. Figure 2B shows the docked complex of
compound 4 with the L20R mutant. Here the charged
carboxylate group on the ligand and the guanidinium group
on the arginine (R20) are pulled down from the binding site,
toward the location in the membrane-bound complex of the
more hydrated interfacial region. The high selectivity of
compound 4 to the L20R mutant may be an example of the
previously observed nonadditivity of mutant ligand-binding
behavior,17 as neither the other meta-benzoate compounds (6
and 8) nor the other compounds with the same R1 acyl group
(1−3, 5) are as selective.
DAG-lactone 13 differs from the other compounds under
discussion here in that its polar side chain is in the R1 position
rather than the R2. We have shown previously that there is a
strong solvation penalty for placing polar side chains at R1,
because in binding the C1 domain, this polar group would be
driven into the hydrophobic part of the membrane. Polar side
chains at R2 are located in the membrane interfacial region and
thus can remain hydrated.33 However, DAG-lactones are
capable of adopting an alternative binding mode, albeit with
somewhat weaker hydrogen bonding, in which the positions of
the side chains are essentially flipped.38 This suggests that
DAG-lactone 13 may be binding the C1 domain arginine
mutants with a mixture of the two binding modes, one with
worse solvation but better hydrogen bonding and one vice
versa. Figure 3A shows the docked structure of compound 13,
in the “standard” DAG-lactone binding mode, interacting with
arginine at position 7 (R7). The arginine hydrogen-bonds to
the hydroxyl groups in the diol but also, as with compound 4,
to the ester carbonyl and the protein backbone. Figure 3B
shows compound 13 in its alternative “reversed” orientation,
with the diol side chain interacting with arginine at position 20
(R20). With the DAG-lactone in this orientation, the arginine
can also hydrogen-bond weakly to the ether oxygen in the
lactone ring. Compound 13 does not form particularly strong
interactions with arginine at position 11 in either of its binding
modes (not shown), which may explain its reduced selectivity
for the P11R and the N7R/P11R mutants. However, this
compound could bind the N7R/L20R double mutant with a
mixture of its two binding modes which would add a favorable
amount of configurational entropy to the binding free energy.
The core conclusion is that indeed marked improvements in
selectivity are achievable for DAG-lactones incorporating
moieties that may interact with arginines corresponding to
those found in the atypical C1 domains of PKCζ/ι. Among
compounds 1−3, the ortho derivative was clearly the least
selective; for the other pairs of meta versus para there were in
general relatively small differences, depending on the specific
pair of compounds. Although the series of compounds
examined is not extensive, it is noteworthy that compounds 4
and 13 showed different relative recognition for N7R/P11R
versus N7R/L20R. The high selectivity of compound 13 in
particular suggests that further exploration of this class of DAG-
lactones may be of interest and that the exploitation of the
DAG-lactones’ ability to bind in multiple binding orientations
could be used to develop compounds targeted to multiple
mutations simultaneously.
EXPERIMENTAL SECTION
■
General Procedures. All chemical reagents were commercially
available. Melting points were determined on an MPA 100 OptiMelt
automated melting point system (Stanford Research Systems, USA) or
a MelTemp II apparatus, (Laboratory Devices, USA) and are
uncorrected. Column chromatography was performed on a Teledyne
Isco CombiFlash Companion instrument under gradient elution
3840
dx.doi.org/10.1021/jm500165n | J. Med. Chem. 2014, 57, 3835−3844

Journal of Medicinal Chemistry
Article
conditions with RediSep disposable flash columns. Analytical TLC was
performed on Analtech Uniplates silica gel GF. All of the intermediates
described in this section were oils purified by column chromatography
and confirmed by TLC to be a single spot. These compounds were
Table 4. Protecting Groups for Compound IV in Scheme 1
1
used directly for the ensuing step. H and 13C NMR spectra were
recorded on a Varian Unity Inova instrument at 400 and 100 MHz,
respectively. Spectra are referenced to the solvent in which they were
run (7.24 ppm for CDCl3). Positive-ion fast atom bombardment mass
spectra (FABMS) were obtained on a VG 7070E-HF double-focusing
mass spectrometer operated at an accelerating voltage of 6 kV under
the control of a MASPEC-II data system for Windows (Mass
Spectrometry Services, Ltd.). Either glycerol or 3-nitrobenzyl alcohol
was used as the sample matrix, and ionization was effected by a beam
of xenon atoms generated in a saddle-field ion gun at 8.0 0.5 kV.
Nominal mass spectra were obtained at a resolution of 1200, and
matrix-derived ions were background-subtracted during data system
processing. Elemental analyses were performed by Atlantic Microlab,
Inc., Norcross, GA.
Aldol Condensation Followed by Olefination (II). Under
argon, a solution of I (prepared as described previously34,35) (1 equiv)
in THF (5 mL/mmol) at −78 °C was treated dropwise with
[(CH3)3Si]2N-Li (LiHMDS, 1.5 equiv) and stirred at the same
temperature for 1 h. A solution of ArCHO (1.5 equiv dissolved in
THF) was then added dropwise, and the mixture was stirred at −78
°C until determined complete by TLC analysis. The reaction mixture
was then quenched with a saturated aqueous solution of NH4Cl and
allowed to warm to room temperature. The layers were separated, and
the aqueous layer was extracted with Et2O (3×). The combined
organics were washed with H2O (2×) and brine (1×), dried (MgSO4),
and concentrated in vacuo to give the condensation product as a
mixture of diastereomers, which was then directly dissolved in a
solution of Et3N (4 equiv) in CH2Cl2 (10 mL/mmol), cooled to 0 °C,
treated dropwise with CH3SO2Cl (MsCl, 2 equiv), and then stirred at
room temperature for 1 h. The reaction mixture was then cooled again
to 0 °C and treated dropwise with 1,8-diazabicyclo[5.4.0]non-5-ene
(DBU, 5 equiv). When the addition of DBU was completed, the
reaction mixture was allowed to reach room temperature overnight.
The volatiles were removed in vacuo, and the residue was treated with
EtOAc followed by 1 N HCl. The layers were separated, and the
aqueous layer was extracted with EtOAc (1×). The combined organics
were washed with H2O (2×) and brine (1×), dried (MgSO4), and
concentrated in vacuo. Purification by silica gel flash column
chromatography (gradient EtOAc/hexanes) gave II in 52−91% yield.
PMP Deprotection (III). Ceric ammonium nitrate (CAN, 3 equiv)
was added to a stirring solution of II (1 equiv) in acetonitrile (8 mL/
mmol) and water (2 mL/mmol) at 0 °C. The reaction was monitored
by TLC, quenched upon completion with a saturated aqueous
Na2S2O3 solution, and warmed to room temperature. The resulting
aqueous solution was extracted with CH2Cl2 (2×), dried (MgSO4),
and concentrated in vacuo. Purification by silica gel flash column
chromatography gave III in 29−97% yield.
Acylation (IV). A solution of III (1 equiv) in CH2Cl2 (12 mL/
mmol) was treated with Et3N (3 equiv) and dimethylaminopyridine
(DMAP, 2.5 equiv) and reacted with the corresponding acid chloride
(RCOCl, 1.2−1.5 equiv). The reaction was stirred at room
temperature and monitored by TLC. Upon completion, the reaction
was concentrated in vacuo and purified by silica gel flash column
chromatography to give IV in 42−100% yield.
Benzyl Deprotection (1−9, 13) (Table 4). BCl3 (3 equiv) was
added slowly to a stirring solution of IV (1 equiv) in CH2Cl2 (20 mL/
mmol) at −78 °C. The reaction was monitored by TLC and quenched
upon completion by MeOH. Immediate concentration in vacuo and
purification by silica gel column chromatography (gradient elution)
gave 1−9 (19−82% yield) and 13 (88% yield).
TBDPS Deprotection (10−12). TBAF (2 equiv) was added
slowly to a stirring solution of IV (1 equiv) in THF (20 mL/mmol) at
room temperature. The reaction was monitored by TLC and directly
concentrated in vacuo upon completion. Purification by silica gel
column chromatography (gradient elution) gave 10−12 in 27−53%
yield.
1-E: R1 = [(CH3)2CH]2CHCH2-; R2 = o-C6H4OH. This compound
has been previously published.39
2-E: R1 = [(CH3)2CH]2CHCH2-; R2 = m-C6H4OH. This compound
has been previously published.39
3-E: R1 = [(CH3)2CH]2CHCH2-; R2 = p-C6H4OH. This compound
has been previously published.39
4-E: R1 = [(CH3)2CH]2CHCH2-; R2 = m-C6H4CO2H. 1H NMR (400
MHz, CDCl3) δ 0.73, 0.76, 0.82, 0.83 (4 × d, J = 6.8 Hz, 12 H,
CH2CH(CH(CH3)2)2), 1.53 (pentet, J ≈ 5.8 Hz, 1 H, CH2CH(CH-
(CH3)2)2), 1.69 (septuplet, J ≈ 6.2 Hz, 2 H, CH2CH(CH(CH3)2)2),
2.18 (dd, J = 5.8, 1.0 Hz, 2 H, CH2CH(CH(CH3)2)2), 3.08 (dd, J =
17.9, 2.8 Hz, 1 H, H-3a), 3.33 (dd, J = 17.9, 3.0 Hz, 1 H, H-3b), 3.81
(AB q, J = 12.2 Hz, 2 H, CCH2OH), 4.29 (AB q, J = 12.0 Hz, 2 H,
CCH2OC(O)CH2CH(CH(CH3)2)2), 7.55 (t, J = 7.8 Hz, 1H, Ar),
7.60 (t, J = 2.8 Hz, 1H, CCHC6H4COOH), 7.71 (d, J = 7.9 Hz, 1H,
Ar), 8.11 (dm, 1 H, J = 7.8 Hz, Ar), 8.22 (br s, 1 H, Ar); 13C NMR
(100 MHz, CDCl3) δ 174.83, 171.14, 170.55, 135.87, 135.30, 134.94,
131.49, 131.22, 130.28, 129.44, 126.12, 83.93, 65.65, 64.85, 47.08,
32.94, 32.24, 29.47, 21.42, 21.40, 18.84, 18.77; FAB-MS (m/z, relative
intensity) 417 (M − H, 100). Anal. (C23H30O7) calcd C 66.01, H 7.73;
found C 65.85, H 7.22.
5-E: R1 = [(CH3)2CH]2CHCH2-; R2 = p-C6H4CO2H. 1H NMR (400
MHz, CDCl3) δ 0.74, 0.77, 0.84, 0.85 (4 × d, J = 6.8 Hz, 12 H,
CH2CH(CH(CH3)2)2, 1.54 (pentet, J ≈ 5.8 Hz, 1 H, CH2CH(CH-
(CH3)2)2), 1.70 (septuplet, J ≈ 6.4 Hz, 2 H, CH2CH(CH(CH3)2)2),
2.18 (dd, J = 5.8, 1.3 Hz, 2 H, CH2CH(CH(CH3)2)2), 3.07 (dd, J =
18.0, 2.8 Hz, 1 H, H-3a), 3.29 (dd, J = 18.0, 2.8 Hz, 1 H, H-3b), 3.79
(AB q, J = 12.2 Hz, 2 H, CCH2OH), 4.29 (AB q, J = 12.0 Hz, 2 H,
CCH2OC(O)CH2CH(CH(CH3)2)2), 7.59 (d, J = 8.3 Hz, 2 H, Ar),
7.60 (t, J = 2.8 (t, J = 2.8 Hz, 1H, CCHC6H4COOH), 8.15 (d, J =
8.3 Hz, 2 H, Ar); 13C NMR (100 MHz, CD3OD) δ 175.74, 172.92,
164.91, 140.12, 135.56, 131.21, 131.10, 129.61, 113.95, 85.79, 67.14,
65.19, 33.80, 33.25, 30.58, 21.68, 19.07, 19.00; FAB-MS (m/z, relative
intensity) 417 (M − H, 100). Anal. (C23H30O7·0.1H2O) calcd C
65.73, H 7.24; found C 65.56, H 7.28.
5-Z: R1 = [(CH3)2CH]2CHCH2-; R2 = p-C6H4CO2H. 1H NMR (400
MHz, CDCl3) δ 0.76, 0.77, 0.86, 0.87 (4 × d, J = 6.7 Hz, 12 H,
CH2CH(CH(CH3)2)2, 1.53 (pentet, J ≈ 5.7 Hz, 1 H, CH2CH(CH-
(CH3)2)2), 1.70 (septuplet, J ≈ 6.5 Hz, 2 H, CH2CH(CH(CH3)2)2),
3841
dx.doi.org/10.1021/jm500165n | J. Med. Chem. 2014, 57, 3835−3844

Journal of Medicinal Chemistry
Article
2.12 (d, J = 5.7 Hz, 2 H, CH2CH(CH(CH3)2)2), 3.68 (s, 2 H, H-3a,b),
3.79 (AB s, 2 H, CCH2OH), 4.31 (AB q, J = 12 Hz, 2 H,
CHC6H4CH[CO2C(CH3)3]2); 13C NMR (100 MHz, CDCl3) δ
173.17, 170.13, 167.15, 166.91, 134.72, 134.13, 133.30, 129.80,
129.52, 128.15, 128.02, 127.70, 83.26, 82.67, 82.53, 64.93, 64.40,
56.42, 44.00, 43.97, 39.20, 31.61, 30.58, 27.83, 27.81, 25.09, 25.07,
22.83, 22.78, 22.56, 22.52; FAB-MS (m/z, relative intensity) 655 (M +
K+, 25), 57 (100). Anal. (C35H52O9) calcd C 68.16, H 8.50; found C
67.96, H 8.53.
CCH2OC(O)CH2CH(CH(CH3)2)2), 6.91 (s,
1
H, C
CHC6H4COOH), 7.33 (d, J = 8.0 Hz, 2 H, Ar), 8.02 (d, J = 8.0
Hz, 2 H, Ar); 13C NMR (100 MHz, CDCl3) δ 174.70, 172.20, 171.02,
148.06, 143.17, 135.77, 130.85, 129.15, 87.93, 63.60, 63.29, 47.02,
32.81, 32.00, 29.49, 29.45, 21.44, 18.85, 18.77; FAB-MS (m/z, relative
intensity) 417 (M − H, 99), 229 (100). Anal. (C23H30O7) calcd C
66.01, H 7.73; found C 65.84, H 7.19.
11-E: R1 = [(CH3)2CHCH2]2CHCH2-; R2 = m-C6H4CH[CO2C-
(CH3)3]2H. 1H NMR (400 MHz, CDCl3) δ 0.78, 0.80, 0.82
(overlapping doublets, J = 6.3 Hz, 12 H, CH2CH[CH2CH(CH3)2]2),
0.98−1.13 (m, 4 H, 2 × CH2CH[CH2CH(CH3)2]2), 1.45 (s, 18 H,
CH[CO2C(CH3)3]2), 1.55 (septuplet, J ≈ 6.7 Hz, 2 H, CH2CH-
[CH2CH(CH3)2]2), 1.88 (pentet, J ≈ 6.7 Hz, 1 H, CH2CH[CH2CH-
(CH3)2]2), 2.20 (d, J = 6.5 Hz, 2 H, CH2CH[CH2CH(CH3)2]2), 2.89
(t, J = 6.5 Hz, 1 H, OH), 3.01 (dd, J = 17.7, 2.8 Hz, 1 H, H-3a), 3.22
(dd, J = 17.7, 2.9 Hz, 1 H, H-3b), 3.71 (AB quartet of doublets, J =
12.1, 6.4 Hz, 2 H, CCH2OH), 4.24 (AB q, J = 11.9 Hz, 2 H,
CCH2OC(O)CH2CH(CH(CH3)2)2), 4.45 (s, 1 H, CH[CO2C-
(CH3)3]2), 7.38−7.46 (m, 3 H, Ar), 7.48 (br s, 1 H, Ar), 7.52 (t, J
= 2.8 Hz, 1 H, CCHC6H4CH[CO2C(CH3)3]2); 13C NMR (100
MHz, CDCl3) δ 173.13, 171.04, 166.95, 136.80, 134.45, 134.28,
131.28, 130.96, 129.27, 128.89, 124.42, 83.28, 82.30, 82.28, 65.27,
64.60, 59.74, 43.92, 39.18, 32.06, 30.48, 27.79, 25.00, 22.74, 22.46,
22.45; FAB-MS (m/z, relative intensity) 617 (MH+, <1), 57 (100).
Anal. (C35H52O9) calcd C 68.16, H 8.50; found C 68.10, H 8.47.
1
6-E: R1 = (CH3)3C-; R2 = m-C6H4CO2H. H NMR (400 MHz,
CD3OD) δ 1.06 (s, 9 H, C(CH3)3), 3.13 (dd, J = 18.0, 2.8 Hz, 1 H, H-
3a), 2.14 (dd, J = 18.0, 3.0 Hz, 1 H, H-3b), 3.73 (AB q, J = 12.1 Hz, 2
H, CCH2OH), 4.26 (AB q, J = 11.9 Hz, 2 H, CCH2OC(O)C(CH3)3),
7.49 (t, J = 2.9 Hz, 1H, CCHC6H4COOH), 7.55 (t, J = 7.8 Hz, 1 H,
Ar), 7.76 (d, J = 7.8 Hz, 1 H, Ar), 8.03 (d, J = 7.8 Hz, 1 H, Ar), 8.18 (s,
1 H, Ar); 13C NMR (100 MHz, CD3OD) δ 179.17, 173.10, 168.98,
136.23, 135.86, 135.78, 135.41, 132.76, 131.85, 131.76, 130.28, 128.48,
85.92, 67.23, 65.08, 39.89, 33.07, 27.37, 27.33; FAB-MS (m/z, relative
intensity) 361 (M − H, 100). Anal. (C19H22O7·0.2H2O) calcd C
62.35, H 6.17; found C 62.35, H 6.12.
1
7-E: R1 = (CH3)3C-; R2 = p-C6H4CO2H. Mp = 220 °C; H NMR
(400 MHz, CD3OD) δ 1.12 (s, 9 H, C(CH3)3), 3.20 (dd, J = 18.3, 2.9
Hz, 1 H, H-3a), 3.36−3.27 (overlapped with solvent, 1 H, H-3b), 3.78
(AB q, J = 12.1 Hz, 2 H, CCH2OH), 4.31(AB q, J = 11.9 Hz, 2 H,
CCH2OC(O)C(CH3)3), 7.56 (t,
J
=
3.0 Hz, 1H, C
1
12-E: R1 = -CCH3(CH2OH)2; R2 = p-C6H4CH[CO2C(CH3)3]2H. H
CHC6H4COOH), 7.71 (d, J = 8.4 Hz, 2H, Ar), 8.13 (d, J = 8.4 Hz,
2H, Ar); FAB-MS (m/z, relative intensity) 361 (M − H, 100). Anal.
(C19H22O7·0.1H2O) calcd C 62.35, H 6.17; found C 62.26, H 6.25.
NMR (400 MHz, CDCl3) δ 0.80−0.84 (m, 12 H, CH2CH[CH2CH-
(CH3)2]2), 0.10−1.15 (m, 4 H, 2 × CH2CH[CH2CH(CH3)2]2), 1.46
(s, 18 H, CH[CO2C(CH3)3]2), 1.57 (septuplet, J ≈ 6.7 Hz, 2 H,
CH2CH[CH2CH(CH3)2]2), 1.90 (pentet, J ≈ 6.7 Hz, 1 H,
CH2CH[CH2CH(CH3)2]2), 2.22 (d, J = 6.5 Hz, 2 H, CH2CH-
[CH2CH(CH3)2]2), 3.03 (dd, J = 17.7, 2.8 Hz, 1 H, H-3a), 3.21 (dd, J
= 17.7, 2.9 Hz, 1 H, H-3b), 3.73 (AB q, J = 12.1 Hz, 2 H, CCH2OH),
4.26 (AB q, J = 11.9 Hz, 2 H, CCH2OC(O)CH2CH(CH(CH3)2)2),
4.46 (s, 1 H, CH[CO2C(CH3)3]2), 7.47 (two doublets, J = 8.8 Hz, 4
H, Ar), 7.55 (t, J = 2.8 Hz, 1 H, CCHC6H4CH[CO2C(CH3)3]2);
13C NMR (100 MHz, CDCl3) δ 173.25, 170.99, 166.90, 136.83,
135.51, 133.91, 130.09, 129.97, 124.19, 83.20, 82.40, 65.22, 64.77,
59.87, 44.00, 39.27, 32.21, 30.58, 27.85, 25.09, 22.82, 22.81, 22.54,
22.52; FAB-MS (m/z, relative intensity) 617 (MH+, 1), 57 (100).
Anal. (C35H52O9·0.6H2O) calcd C 66.98, H 8.54; found C 66.74, H
8.32.
1
8-E: R1 = [(CH3)2CHCH2]2CHCH2-; R2 = m-C6H4CO2H. H NMR
(400 MHz, CDCl3) δ 0.78−0.82 (m, 12 H, CH2CH[CH2CH-
(CH3)2]2), 0.97−1.15 (m, 4 H, CH2CH[CH2CH(CH3)2]2), 1.54
(septuplet, 2 H, J ≈ 6.4 Hz, CH2CH[CH2CH(CH3)2]2), 1.88 (pentet,
J ≈ 6.7 Hz, 1 H, CH2CH[CH2CH(CH3)2]2), 2.21 (d, J = 6.4 Hz, 2 H,
CH2CH[CH2CH(CH3)2]2), 3.07 (dd, J = 17.8, 2.4 Hz, 1 H, H-3a),
3.35 (dd, J = 17.9, 2.4 Hz, 1H, H-3b), 3.81 (AB q, J = 12.2 Hz, 2 H,
CCH2OH), 4.29 (AB q, J = 12.0 Hz, 2 H, CCH2OC(O)CH2CH-
(CH(CH3)2)2), 7.52 (t, J = 7.8 Hz, 1 H, Ar), 7.57 (br s, 1 H, C
CHC6H4COOH), 7.69 (d, J = 7.8 Hz, 1 H, Ar), 8.08 (d, J = 7.8 Hz, 1
H, Ar), 8.20 (s, 1 H, Ar); 13C NMR (100 MHz, CDCl3) δ 173.40,
171.27, 170.62, 135.86, 135.18, 134.87, 131.45, 131.28, 130.35, 129.37,
126.06, 83.95, 65.45, 64.77, 44.11, 39.42, 32.16, 30.74, 25.22, 22.94,
22.66, 22.63; FAB-MS (m/z, relative intensity) 445 (M − H, 100).
Anal. (C25H34O7·0.3H2O) calcd C 66.44, H 7.72; found C 66.31, H
7.55.
1
13-E: R1 = (HOCH2)2(CH3)C-; R2 = -CH2CH[CH2CH(CH3)2]2. H
NMR (400 MHz, CDCl3) δ 0.86 (d, J = 6.6 Hz, 12 H,
CH2CH[CH2CH(CH3)2]2), 0.99−1.18 (m, 7 H, 2 × CH2CH-
[CH2CH(CH3)2]2 and C(CH2OH)2CH3), 1.61 (septuplet, J ≈ 6.9
Hz, 2 H, CH2CH[CH2CH(CH3)2]2), 1.70 (pentet, J ≈ 6.9 Hz, 1 H,
CH2CH[CH2CH(CH3)2]2), 2.13 (dd, J = 7.2, 6.0 Hz, 2 H,
CH2CH[CH2CH(CH3)2]2), 2.63 (dd, J = 17.3, 2.4 Hz, 1 H, H-3a),
2.83 (dm, J = 17. 3 Hz, 1 H, H-3b), 3.33 and 3.41 (br s, 2 H,
C(CH2OH)2CH3), 3.60 (br s, 1 H, CCH2OH), 3.64−3.77 (m, 4 H,
C(CH2OH)2CH3), 3.85 (t, J = 11.3 Hz, 2 H, CCH2OH), 4.21 (dd, J =
12.0, 2.6 Hz, 1 H, CCHHOC(O)C(CH2OH)2CH3), 4.40 (dd, J =
11.9, 2.9 Hz, 1 H, CCHHOC(O)C(CH2OH)2CH3), 6.80 (m, 1 H,
CCH); 13C NMR (100 MHz, CDCl3) δ 175.16, 170.01, 141.70,
126.24, 83.11, 67.71, 67.67, 65.83, 64.43, 49.64, 43.82, 34.86, 32.75,
30.39, 25.18, 25.17, 22.92, 22.62, 22.60, 17.05; FAB-MS (m/z, relative
intensity) 415 (MH+, 95), 299 (100). Anal. (C22H38O7) calcd C 63.74,
H 9.24; found C 63.71, H 9.25.
1
9-E: R1 = [(CH3)2CHCH2]2CHCH2-; R2 = p-C6H4CO2H. H NMR
(400 MHz, CD3OD) δ 0.78−0.83 (m, 12 H, CH2CH[CH2CH-
(CH3)2]2), 1.01−1.10 (m, 4 H, CH2CH[CH2CH(CH3)2]2)), 1.55
(septuplet of doublets,
J ≈ 6.7, 2.7 Hz, 2H, CH2CH-
[CH2CH(CH3)2]2), 1.88 (pentet, J ≈ 6.6 Hz, 1 H, CH2CH[CH2CH-
(CH3)2]2), 2.18 (dd, J = 6.5, 0.9 Hz, 2 H, CH2CH[CH2CH(CH3)2]2),
3.24 (AB quartet of doublets, J = 18.3, 2.8 Hz, 2 H, H-3a,b), 3.75 (AB
q, J = 12.0 Hz, 2 H, CCH2OH), 4.31 (d, J = 2.8 Hz, 2 H,
CCH2OC(O)CH2CH(CH(CH3)2)2), 7.53 (t, J = 2.8 Hz, 1H, C
CHC6H4COOH), 7.69 (d, J = 8.4 Hz, 2 H, Ar), 8.10 (d, J = 8.4 Hz, 2
H, Ar); 13C NMR (100 MHz, CD3OD) δ 172.85, 171.38, 167.57,
138.64, 134.13, 129.77, 129.64, 128.04, 84.21, 65.40, 63.75, 43.80,
38.74, 31.76, 30.44, 24.81, 21.84, 21.47, 21.45; FAB-MS (m/z, relative
intensity) 445 (M − H, 100). Anal. (C25H34O7·0.2H2O) calcd C
66.71, H 7.70; found C 66.64, H 7.61
Modeling. Structures for the single arginine mutants of the PKCδ
C1b domain were generated as described previously.17 Briefly, the
selected residue in the crystal structure14 was mutated to arginine, then
subjected to a conformational search of its four χ angles to identify a
small set of low-energy conformers. Structures for the double, triple,
and quadruple mutants were built using the lowest-energy conformer
found for each single arginine side chain.
Docking of the DAG-lactones was performed using the software
program GOLD 5.0.40 The binding site was defined by atoms within a
10.0 Å sphere around the Nε atom of the C1 domain residue Gln 27
(Gln 257 in full-length PKCδ). Ligand flexibility flags included internal
10-E: R1 = [(CH3)2CHCH2]2CHCH2-; R2 = o-C6H4CH[CO2C-
(CH3)3]2H. 1H NMR (400 MHz, CDCl3) δ 0.83−0.86 (m, 12 H,
CH2CH[CH2CH(CH3)2]2), 0.10−1.16 (m, 4 H, CH2CH[CH2CH-
(CH3)2]2), 1.46 and 1.47 (s, 18 H, CH[CO2C(CH3)3]2), 1.59
(septuplet, J ≈ 6 Hz, 2 H, CH2CH[CH2CH(CH3)2]2), 1.93 (pentet, J
≈ 6.7 Hz, 1 H, CH2CH[CH2CH(CH3)2]2), 2.24 (d, J = 6.5 Hz, 2 H,
CH2CH[CH2CH(CH3)2]2), 2.89 (dd, J = 17.7, 2.8 Hz, 1 H, H-3a),
3.04 (dd, J = 17.7, 2.9 Hz, 1 H, H-3b), 3.67 (AB q, J = 12.1 Hz, 2 H,
CCH2OH), 4.22 (AB q, J = 11.9 Hz, 2 H, CCH2OC(O)CH2CH-
(CH(CH3)2)2), 4.75 (s, 1 H, CH[CO2C(CH3)3]2), 7.37 (m, 2 H, Ar),
7.55 (d, J = 7.5 Hz, 2 H, Ar), 7.74 (t, J = 2.8 Hz, 1 H, C
3842
dx.doi.org/10.1021/jm500165n | J. Med. Chem. 2014, 57, 3835−3844

Journal of Medicinal Chemistry
Article
hydrogen bond detection and ring corner flipping, and the default
torsion angle distributions were used. The GoldScore scoring function
was used with the default parameter file. The genetic algorithm
settings were automatically optimized according to ligand flexibility
with the search efficiency set to 100%.
After docking, the structures of the DAG-lactone−mutant PKCδ
complexes underwent conformational searching using systematic
torsional sampling in MacroModel41,42 to identify low-energy salt-
bridged conformers for the charged ligand and protein side chains.
The searches used 10 000 steps of sampling on the rotatable bonds in
the DAG-lactone R1 and R2 side chains and in the side chains of
residues 7−13, 20−25, and 27 in the mutated C1 domain.
Minimization of each conformer found was done using the OPLS
2005 force field with octanol implicit solvent. All atoms in the DAG-
lactone and residues 7−13, 20−25, and 27 in the binding site were free
to move during minimization, and the rest of the protein was held
fixed.
(5) Yang, C.; Kazanietz, M. G. Divergence and complexities in DAG
signaling: looking beyond PKC. Trends Pharmacol. Sci. 2003, 24, 602−
608.
(6) Brose, N.; Rosenmund, C. Move over protein kinase C, you’ve
got company: alternative cellular effectors of diacylglycerol and
phorbol esters. J. Cell Sci. 2002, 115, 4399−4411.
(7) Singer, W. D.; Brown, H. A.; Sternweis, P. C. Regulation of
eukaryotic phosphatidylinositol-specific phospholipase C and phos-
pholipase D. Annu. Rev. Biochem. 1997, 66, 475−509.
(8) Becker, K. P.; Hannun, Y. A. Protein kinase C and phospholipase
D: intimate interactions in intracellular signaling. Cell. Mol. Life Sci.
2005, 62, 1448−1461.
(9) Griner, E. M.; Kazanietz, M. G. Protein kinase C and other
diacylglycerol effectors in cancer. Nat. Rev. Cancer 2007, 7, 281−294.
(10) Reyland, M. E. Protein kinase C isoforms: multi-functional
regulators of cell life and death. Front. Biosci. 2009, 14, 2386−2399.
(11) Martin, G.; Swanson, N. Clinical findings using ingenol
mebutate gel to treat actinic keratoses. J. Am. Acad. Dermatol. 2013,
68, S39−48.
(12) Ruan, B. F.; Zhu, H. L. The chemistry and biology of the
bryostatins: potential PKC inhibitors in clinical development. Curr.
Med. Chem. 2012, 19, 2652−2664.
(13) Blumberg, P. M.; Kedei, N.; Lewin, N. E.; Yang, D.; Czifra, G.;
Pu, Y.; Peach, M. L.; Marquez, V. E. Wealth of opportunitythe C1
domain as a target for drug development. Curr. Drug Targets 2008, 9,
641−652.
(14) Zhang, G.; Kazanietz, M. G.; Blumberg, P. M.; Hurley, J. H.
Crystal structure of the cys2 activator-binding domain of protein
kinase C delta in complex with phorbol ester. Cell 1995, 81, 917−924.
(15) Canagarajah, B.; Leskow, F. C.; Ho, J. Y.; Mischak, H.; Saidi, L.
F.; Kazanietz, M. G.; Hurley, J. H. Structural mechanism for lipid
activation of the Rac-specific GAP, beta2-chimaerin. Cell 2004, 119,
407−418.
(16) Leonard, T. A.; Rozycki, B.; Saidi, L. F.; Hummer, G.; Hurley, J.
H. Crystal structure and allosteric activation of protein kinase C βII.
Cell 2011, 144, 55−66.
(17) Pu, Y.; Peach, M. L.; Garfield, S. H.; Wincovitch, S.; Marquez, V.
E.; Blumberg, P. M. Effects on ligand interaction and membrane
translocation of the positively charged arginine residues situated along
the C1 domain binding cleft in the atypical protein kinase C isoforms.
J. Biol. Chem. 2006, 281, 33773−33788.
(18) Geczy, T.; Peach, M. L.; El Kazzouli, S.; Sigano, D. M.; Kang, J.
H.; Valle, C. J.; Selezneva, J.; Woo, W.; Kedei, N.; Lewin, N. E.;
Garfield, S. H.; Lim, L.; Mannan, P.; Marquez, V. E.; Blumberg, P. M.
Molecular basis for failure of “atypical” C1 domain of Vav1 to bind
diacylglycerol/phorbol ester. J. Biol. Chem. 2012, 287, 13137−13158.
(19) Rapley, J.; Tybulewicz, V. L.; Rittinger, K. Crucial structural role
for the PH and C1 domains of the Vav1 exchange factor. EMBO Rep.
2008, 9, 655−661.
(20) Mott, H. R.; Carpenter, J. W.; Zhong, S.; Ghosh, S.; Bell, R. M.;
Campbell, S. L. The solution structure of the Raf-1 cysteine-rich
domain: a novel Ras and phospholipid binding site. Proc. Natl. Acad.
Sci. U.S.A. 1996, 93, 8312−8317.
(21) Zhou, M.; Horita, D. A.; Waugh, D. S.; Byrd, R. A.; Morrison, D.
K. Solution structure and functional analysis of the cysteine-rich C1
domain of kinase suppressor of Ras (KSR). J. Mol. Biol. 2002, 315,
435−446.
(22) Harjes, E.; Harjes, S.; Wohlgemuth, S.; Muller, K. H.; Krieger,
E.; Herrmann, C.; Bayer, P. GTP-Ras disrupts the intramolecular
complex of C1 and RA domains of Nore1. Structure 2006, 14, 881−
888.
(23) Bandyopadhyay, G.; Standaert, M. L.; Sajan, M. P.; Kanoh, W.;
Miura, A.; Braun, U.; Kruse, F.; Leitges, M.; Farese, R. V. Protein
kinase C-lambda knockout in embryonic stem cells and adipocytes
impairs insulin-stimulated glucose transport. Mol. Endocrinol. 2004, 18,
373−383.
(24) Fields, A. P.; Regala, R. P. Protein kinase Cι: human oncogene,
prognostic marker, and therapeutic target. Pharmacol. Res. 2007, 55,
487−497.
Biology. [20-3H]Phorbol 12,13-dibutyrate (PDBu) was obtained
from PerkinElmer, Waltham, MA. PDBu was from LC Laboratories
(Woburn, MA). Phosphatidyl-L-serine was from Avanti Polar Lipids
(Alabaster, AL). We have previously described the preparation of the
GST-fusion construct with the PKCδ C1b domain, together with the
construction, production, and purification of the variants incorporating
the single amino acid substitutions N7R, S10R, P11R, L20R, along
with those incorporating the multiple substitutions N7R/P11R, N7R/
L20R, N7R/S10R/P11R, and N7R, S10R, L20R.17 Binding affinities of
the various compounds to the wild-type and mutated GST-C1b
domain constructs were determined in competitive binding assays with
[20-3H]PDBu as described previously43,44 in the presence of 100 μg/
mL phosphatidylserine.
AUTHOR INFORMATION
Corresponding Author
*Phone: 301-496-3189. Fax: 301-496-8709. E-mail:
blumberp@dc37a.nci.nih.gov.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported in part through the Intramural
Research Program of the National Institutes of Health, Center
for Cancer Research, National Cancer Institute (Project Z1A
BC 005270) and in part with Federal funds from the Frederick
National Laboratory for Cancer Research, National Institutes of
Health, under Contract HHSN261200800001E. The content of
this publication does not necessarily reflect the views or policies
of the Department of Health and Human Services, nor does
mention of trade names, commercial products, or organizations
imply endorsement by the U.S. Government.
ABBREVIATIONS USED
PDBu, phorbol 12,13-dibutyrate; PKC, protein kinase C; DAG,
sn-1,2-diacylglycerol
■
REFERENCES
■
(1) Hurley, J. H.; Newton, A. C.; Parker, P. J.; Blumberg, P. M.;
Nishizuka, Y. Taxonomy and function of C1 protein kinase C
homology domains. Protein Sci. 1997, 6, 477−480.
(2) Stahelin, R. V. Lipid binding domains: more than simple lipid
effectors. J. Lipid Res. 2009, 50, S299−S304.
(3) Colon-Gonzalez, F.; Kazanietz, M. G. C1 domains exposed: from
diacylglycerol binding to protein interactions. Biochim. Biophys. Acta
2006, 1761, 827−837.
(4) Kazanietz, M. G. Targeting protein kinase C and “non-kinase”
phorbol ester receptors: emerging concepts and therapeutic
implications. Biochem. Biophys. Acta 2005, 1754, 296−304.
3843
dx.doi.org/10.1021/jm500165n | J. Med. Chem. 2014, 57, 3835−3844

Journal of Medicinal Chemistry
Article
(25) Murray, N. R.; Kalari, K. R.; Fields, A. P. Protein kinase Cι
expression and oncogenic signaling mechanisms in cancer. J. Cell.
Physiol. 2011, 226, 879−887.
lactones containing aryl groups at the sn-1 and sn-2 positions. J. Med.
Chem. 2006, 49, 3185−3203.
(40) Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor, R.
Development and validation of a genetic algorithm for flexible docking.
J. Mol. Biol. 1997, 267, 727−748.
(26) Diaz-Meco, M. T.; Moscat, J. The atypical PKCs in
inflammation: NF-κB and beyond. Immunol. Rev. 2012, 246, 154−167.
(27) Lopez-Garcia, L. A.; Schulze, J. O.; Frohner, W.; Zhang, H.;
Suss, E.; Weber, N.; Navratil, J.; Amon, S.; Hindie, V.; Zeuzem, S.;
Jorgensen, T. J. D.; Alzari, P. M.; Neimanis, S.; Engel, M.; Biondi, R.
M. Allosteric regulation of protein kinase C PKCζ by the N-terminal
C1 domain and small compounds to the PIF-Pocket. Chem. Biol. 2011,
18, 1463−1473.
(41) Goodman, J. M.; Still, W. C. An unbounded systematic search of
conformational space. J. Comput. Chem. 1991, 12, 1110−1117.
(42) MacroModel, version 9.9; Schrodinger, LLC: New York, NY,
̈
2011.
(43) Lewin, N. E.; Blumberg, P. M. [3H]Phorbol 12,13-dibutyrate
binding assay for protein kinase C and related proteins. Methods Mol.
Biol. 2003, 233, 129−156.
(28) Diaz-Meco, M. T.; Municio, M. M.; Sanchez, P.; Lozano, J.;
Moscat, J. λ-Interacting protein, a novel protein that specifically
interacts with the zinc finger domain of the atypical protein kinase C
isotype λ/ι and stimulates its kinase activity in vitro and in vivo. Mol.
Cell. Biol. 1996, 16, 105−114.
(44) Wang, Q. J.; Fang, T. W.; Nacro, K.; Marquez, V. E.; Wang, S.;
Blumberg, P. M. Role of the hydrophobic residues in the C1b domain
of protein kinase C delta on ligand and phospholipid interactions. J.
Biol. Chem. 2001, 276, 19580−19587.
(29) Moscat, J.; Diaz-Meco, M. T.; Albert, A.; Campuzano, S. Cell
signaling and function organized by PB1 domain interactions. Mol. Cell
2006, 23, 631−640.
(30) Diaz-Meco, M. T.; Municio, M. M.; Frutos, S.; Sanchez, P.;
Lozano, J.; Sanz, L.; Moscat, J. The product of par-4, a gene induced
during apoptosis, interacts selectively with the atypical isoforms of
protein kinase C. Cell 1996, 86, 777−786.
(31) Marquez, V. E.; Blumberg, P. M. Diacylglycerol (DAG) and
DAG-lactones as selective activators of protein kinase C (PK-C). Acc.
Chem. Res. 2003, 36, 434−443.
(32) Duan, D.; Sigano, D. M.; Kelley, J. A.; Lai, C. C.; Lewin, N. E.;
Kedei, N.; Peach, M. L.; Lee, J.; Abeyweera, T. P.; Rotenberg, S. A.;
Kim, H.; Kim, Y. H.; El Kazzouli, S.; Chung, J. U.; Young, H. A.;
Young, M. R.; Baker, A.; Colburn, N. H.; Haimovitz-Friedman, A.;
Truman, J. P.; Parrish, D. A.; Deschamps, J. R.; Perry, N. A.; Surawski,
R. J.; Blumberg, P. M.; Marquez, V. E. Conformationally constrained
analogues of diacylglycerol. 29. Cells sort diacylglycerol-lactone
chemical zip codes to produce diverse and selective biological
activities. J. Med. Chem. 2008, 51, 5198−5220.
(33) Nacro, K.; Bienfait, B.; Lee, J.; Han, K. C.; Kang, J. H.; Benzaria,
S.; Lewin, N. E.; Bhattacharyya, D. K.; Blumberg, P. M.; Marquez, V.
E. Conformationally constrained analogues of diacylglycerol (DAG).
16. How much structural complexity is necessary for recognition and
high binding affinity to protein kinase C? J. Med. Chem. 2000, 43,
921−944.
(34) Lee, J. Design and synthesis of bioisosteres of ultrapotent
protein kinase C (PKC) ligand, 5-acetoxymethyl-5-hydroxymethyl-3-
alkylidene tetrahydro-2-furanone. Arch. Pharmacal Res. 1998, 21, 452−
457.
(35) Choi, Y.; Kang, J. H.; Lewin, N. E.; Blumberg, P. M.; Lee, J.;
Marquez, V. E. Conformationally constrained analogues of diacylgly-
cerol. 19. Synthesis and protein kinase C binding affinity of
diacylglycerol lactones bearing an n-hydroxylamide side chain. J.
Med. Chem. 2003, 46, 2790−2793.
(36) Kang, J. H.; Siddiqui, M. A.; Lewin, N. E.; Sigano, D. M.;
Blumberg, P. M.; Lee, J.; Marquez, V. E. Conformationally constrained
analogues of diacylglycerol. 24. Asymmetric synthesis of a chiral R-
DAG-lactone template as a versatile precursor for highly functionalized
DAG-lactones. Org. Lett. 2004, 6, 2413−2416.
(37) Nacro, K.; Sigano, D. M.; Yan, S.; Nicklaus, M. C.; Pearce, L. L.;
Lewin, N. E.; Garfield, S. H.; Blumberg, P. M.; Marquez, V. E. An
optimized protein kinase C activating diacylglycerol combining high
binding affinity (Ki) with reduced lipophilicity (log P). J. Med. Chem.
2001, 44, 1892−1904.
(38) Sigano, D. M.; Peach, M. L.; Nacro, K.; Choi, Y.; Lewin, N. E.;
Nicklaus, M. C.; Blumberg, P. M.; Marquez, V. M. Differential binding
modes of diacylglycerol (DAG) and DAG lactones to protein kinase C
(PK-C). J. Med. Chem. 2003, 46, 1571−1579.
(39) Kang, J. H.; Benzaria, S.; Sigano, D. M.; Lewin, N. E.; Pu, Y. M.;
Peach, M. L.; Blumberg, P. M.; Marquez, V. E. Conformationally
constrained analogues of diacylglycerol. 26. Exploring the chemical
space surrounding the C1 domain of protein kinase C with DAG-
3844
dx.doi.org/10.1021/jm500165n | J. Med. Chem. 2014, 57, 3835−3844