Synthesis and Biological Evaluation of Fluorescent Bryostatin Analogues

Article abstract of DOI:10.1002/cbic.201700655

To investigate the cellular distribution of tumor-promoting vs. non-tumor-promoting bryostatin analogues, we synthesized fluorescently labeled variants of two bryostatin derivatives that have previously shown either phorbol ester-like or bryostatin-like b

Full text of DOI:10.1002/cbic.201700655

Accepted Article
Title: Synthesis and Biological Evaluation of Fluorescent Bryostatin
Analogues
Authors: Thomas J. Cummins, Noemi Kedei, Agnes Czikora, Nancy E.
Lewin, Sharon Kirk, Mark E. Petersen, Kevin M. McGowan,
Jin-Qiu Chen, Xiaoling Luo, Randall C. Johnson, Sarangan
Ravichandran, Peter M. Blumberg, and Gary E. Keck
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to ensure accuracy of information. The authors are responsible for the
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To be cited as: ChemBioChem 10.1002/cbic.201700655
Link to VoR: http://dx.doi.org/10.1002/cbic.201700655
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10.1002/cbic.201700655
ChemBioChem
Synthesis and Biological Evaluation of Fluorescent Bryostatin
Analogues
Thomas J. Cummins Ph.D,[b] Noemi Kedei MD,[a] Agnes Czikora Ph.D,[a] Nancy E. Lewin,[a]
Sharon Kirk Ph.D,[b] Mark E. Petersen Ph.D,[b] Kevin M. McGowan Ph.D,[b] Jin-Qiu Chen
Ph.D,[c] Xiaoling Luo, [c] Randall C. Johnson, [d] Ph.D, Sarangan Ravichandran, Ph.D, [d] Peter
M. Blumberg Ph.D, [a]* and Gary E. Keck Ph.D.[b]
[a]Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer
Institute, 37 Convent drive 4048, Bethesda, MD, 20892, Bethesda, MD 20892, [b]University of
Utah, Department of Chemistry, 315 South 1400 East, RM 2020, Salt Lake City, Utah 84112,
USA, and [c]Collaborative Protein Technology Resource, Center for Cancer Research, National
Cancer Institute, 37 Convent drive 1044, Bethesda, MD 20892, [d]Advanced Biomedical and
Computational Sciences Biomedical Informatics and Data Science (BIDS) Directorate Frederick
National Laboratory for Cancer Research (FNLCR) Leidos Biomedical Research, Inc., Bldg 430,
Miller Drive, Fort Detrick, Frederick, MD 21702
Corresponding author:
*Email: blumberp@dc37a.nci.nih.gov.
Phone: (+1) 240-760-6632
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ABSTRACT
To investigate the cellular distribution of tumor-promoting vs nontumor-promoting bryostatin
analogues, we synthesized fluorescently labeled variants of two bryostatin derivatives that have
previously shown either phorbol ester-like or bryostatin-like biological activity on U937 cells.
These new fluorescent analogues both displayed high affinity for PKC binding and retained the
basic properties of the parent, unlabeled compounds in U937 assays. The fluorescent compounds
showed similar patterns of intracellular distribution in cells, however, arguing against an existing
hypothesis that varying patterns of intracellular distribution contributed to the difference in
biological activity. In further characterization, the fluorescent compounds revealed a slow rate of
cellular uptake; correspondingly, they showed reduced activity for cellular responses that are only
transient upon treatment with phorbol ester or bryostatin 1.
INTRODUCTION
The 1,2-diacylglycerols (DAG) are important secondary messengers that regulate numerous
cellular functions such as apoptosis, differentiation, cell growth, and tumor-promotion. Among the
best characterized mediators of DAG signaling are the family of protein kinase C (PKC) isoforms,
which play important roles in cell proliferation, differentiation, and apoptosis[1] and afford an
interesting target for the treatment of a variety of diseases including cancer,[2] human
immunodeficiency virus HIV,[3] diabetes,[4] and Alzheimer’s disease.[5] Ligand binding drives a
conformational change of PKC, leading both to enzymatic activation and to membrane
association.[6] The membrane association, in turn, influences enzyme specificity by controlling
proximity to the different target proteins present in different subcellular locations.[7] In addition to
DAG, numerous potent exogenous ligands have been identified (Scheme 1), such as the phorbol
and ingenol esters (tetracyclic diterpenes), aplysiatoxins (polyacetates), teleocidins (indolactams),
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and the bryostatins (macrocyclic lactones).[8] These ligands all function by inserting into a
hydrophilic cleft on the C1 domain of PKC, thus completing a hydrophobic surface as well as
contributing new hydrophobic or hydrophilic elements depending on the ligand.[9] Binding of a
ligand to this C1 domain promotes membrane insertion and activation of the enzyme.
While various ligands share this mechanism of PKC activation, the complicated downstream
signaling pathways of the family of PKC isoforms, and of the related classes of signaling proteins
with homologous C1 domains (e.g. RasGRPs, chimaerins, PKDs), result in different ligands
having dramatically different and even contrasting biological outcomes. For example, although
PMA (phorbol 12-myristate 13-acetate) (Scheme 1) is the archetypical skin tumor promoter,[10]
prostratin (12-deoxyphorbol 13-acetate) is not tumor-promoting.[11] Bryostatin 1 has attracted
particular attention because it is unique among C1 domain ligands. Not only does bryostatin 1 fail
to induce most of the typical cellular responses that are induced by PMA, but it also blocks those
biological responses that it itself does not induce.[8] PMA inhibits cell growth and induces
attachment of U937 leukemia cells and inhibits the growth of the human prostate cancer cell line
LNCaP; bryostatin 1 has little effect on any of these endpoints but when co-administered with
PMA will antagonize the response to PMA. Our group has utilized the U937 and LNCaP systems
in an effort to link structural features of bryostatin 1 and closely related analogues to this unique
pattern of ‘bryostatin-like’ biology.[12] Because the molecule is non-tumor promoting[13] and acts
in many instances as an antagonist of PKC activity, bryostatin 1 has attracted substantial interest
as a therapeutic candidate (> 80 phase I and phase II trials).[14]
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Scheme 1. Comparison of structurally distinct PKC ligands.
The binding affinities, taken from the literature, were measured by competitive binding assays
using a variety of PKC preparations and non-parallel conditions, so they are not directly
comparable, but are included to show that these various structures all possess potent activity on
PKC.[15]
Bryostatin 1 shows multiple mechanistic differences compared to PMA. For some responses, it
shows transient activity compared to PMA followed by suppression of response.[16] In multiple
cell types, it displays a biphasic dose response curve for down regulation of PKCδ, with partial
protection from down regulation at higher bryostatin 1 concentrations,[17] while PMA, in contrast,
induces monophasic down regulation. Another difference that has been studied in detail is the
effect on translocation. PMA causes PKCδ to initially translocate to the plasma membrane,
followed by partial translocation to the internal membranes and the nuclear membrane.
Contrastingly, bryostatin 1 causes PKCδ to translocate to the internal membranes and the nuclear
membrane but not to the plasma membrane.[18] Finally, whereas PMA induces phosphorylation of
PKCδ at Y311, among other sites, bryostatin 1 has little effect on phosphorylation at this site.[12j]
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Each of these mechanistic differences may contribute to a different pattern of biological
response, depending on the specific cell type and the specific biological endpoint. A critical tool
for dissecting the relative roles of these different mechanistic features has been the recent advances
in bryostatin chemistry, now making available analogues which can permit correlation between
retention or loss of a particular mechanistic feature and downstream biological responses.[19] Our
current view is that transient response is the predominant mechanism responsible for the
differential effect of bryostatin 1 and PMA in the U937 and LNCaP cell systems.[12i] Although the
pattern of translocation of PKCδ correlates well with the reported tumor promoting ability of
compounds in whole animal studies, its contribution to the unique behavior of bryostatin 1 as an
antagonist of typical PMA responses remains unresolved.
We have previously shown that ligand lipophilicity is an important determinant for the pattern
of PKCδ translocation and that the less lipophilic ligands, like bryostatin 1, tend to cause the
translocation of PKCδ to internal membranes and the nuclear membrane and also tend to be non-
tumor promoting.[20] Moreover, using a series of fluorescent phorbol esters, we showed that
compounds with long lipophilic hydrocarbon chains first accumulated in the plasma membrane
before slowly equilibrating with internal membranes.[21] Recruitment of PKC to a plasma
membrane associated ligand affords a plausible mechanism for localized PKC activation at the
plasma membrane leading to phosphorylation of specific substrates. Likewise, localized activation
of PKC at internal membranes may lead to phosphorylation of different substrates. We have also
shown that, for a series of phorbol esters and related analogues of varying lipophilicities, some of
the less lipophilic compounds had a diminished ability to inhibit LNCaP growth or to induce tumor
necrosis factor α in the LNCaP cells compared to PMA; these effects are like those of bryostatin
1 albeit of considerably lesser magnitude.[22] Bryostatin 1 however is still unique in its ability to
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antagonize the effects of other C1 ligands, and, unlike bryostatin 1, all of the phorbol esters tested
inhibited U937 cell growth comparably although a few caused less cellular attachment.[22] While
[23]
[24]
the cellular distribution of phorbol esters
and indole alkaloids
has been studied, little is
known about the distribution of the much less lipophilic bryostatins and analogues.
Our goal in the present study was to assess whether differences in cellular distribution could
explain the different biological profiles of a pair of bryostatin derivatives, one PMA-like and the
other bryostatin-like in U937 cells. Since the addition of the fluorescent tag of course modified the
structure of the parent compounds, a related objective was to characterize the patterns of biological
response of the compounds.
Scheme 2. Side by side comparison of synthetic bryostatin analogues.
The binding affinities reported here were measured by competitive binding assays using purified
PKCα.[12b-e]
RESULTS AND DISCUSSION
In order to identify a suitable attachment site for a fluorescent dye we utilized existing bryostatin
structure function relationships. Contributions by several research groups have led to the synthesis
of over 100 bryostatin analogues, providing a detailed understanding of the functional role for
many of the bryostatin substituents.[12b-f, 25] Our group, specifically, has prepared over 20
analogues, many of which rival the potency of bryostatin 1 itself (Scheme 2).[12d] For example,
Merle 28, which lacks the B-ring methyl ester, was shown to have a binding affinity of 0.52 ± 0.06
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nM for PKCα while the affinity of bryostatin 1 in comparable assays was 0.73 ± 0.05 nM. Early
analogue work established that binding of bryostatin, or a bryopyran analogue, to PKC is primarily
mediated through functionality on the C-ring (southern) portion of the molecule,[26] although the
A- and B-ring portions also contribute.[12g] The A-, and B-ring (northern) portions make their
primary contribution by influencing the pattern of biological responses, provided that the
compound possesses sufficient PKC binding affinity. As described above, tumor promoting
ligands such as PMA inhibit proliferation and induce attachment in the U937 leukemia cells
whereas bryostatin 1 has little effect on either response, but when co-administered, bryostatin 1
will block the effects of PMA in a dose dependent manner. We have described analogues as either
PMA-like or bryostatin-like based on their behavior in these assays. Merle 23[12d] and Merle 27[12c]
were shown to be PMA-like while Merle 28[12b] and Merle 30[12e] were bryostatin-like. The most
notable difference between these analogues is the number of polar functional groups displayed on
the A-, and B-rings. Both Merle 28 and Merle 30 contain 2 oxygen substituents while Merle 23
and 27 possess 0 and 1 respectively. Since our current binding model has the A- and B-rings
forming a cap on the top of the C1 domain[12e], we hypothesize that differences in polar substituents
on these rings influence membrane interaction and subcellular distribution of the ligands, with
consequent effects on the downstream responses. To investigate this hypothesis, the PMA-like
Merle 23 and the bryostatin-like Merle 28 were selected for further study using fluorescent
versions of these bryostatin analogues to help us assess their membrane interactions.
Scheme 3. Retrosynthetic analysis of Merle 44 and Merle 45.
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Synthesis of Merle 45: Retrosynthetically we anticipated that Merle 45 could be formed through
a highly convergent coupling of fully functionalized A ring 2 and C ring 3 using a pyran annulation
and Yamaguchi esterification (Scheme 3). Based on previous experience we anticipated that
having the C9 ketal in place would be problematic due to formation of a spiroketal byproduct under
conditions typically used for an intermolecular pyran annulation (TMS-OTf, Et2O), so we decided
to first connect the two pieces through a Yamaguchi esterification followed by more mild acid
mediated pyran annulation conditions.[27] Based on an earlier reported docking structure of
bryostatin in the C1 domain of PKC and SAR data on the C20 ester position, we reasoned that the
C20 position would be well suited for addition of a bulky fluorophore.[12d-e, 28] The C20 ester, which
is mostly solvent exposed and does not make crucial interactions with the protein, has been shown
previously to tolerate substantial variations in structure, including bulky substituents.[23h] The
fluorescent BODIPY FL tag was envisioned to be installed at a late stage using click chemistry
between a BODIPY FL containing azide[29] and alkyne 1.
The synthesis started from the hydrolysis of previously described thioester 4[30] using NBS and
water to give carboxylic acid 5 (Scheme 4).[31] The β-hydroxyallylsilane was installed using the
Williams reaction between (S,S)-allylboronate 6 and aldehyde 5 to give a homoallylic alcohol,
which was protected as the TES ether.[32] This protected allylsilane 7 was obtained in 77% yield
as a 6:1 mixture of diastereomers over the two steps.
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Scheme 4. Synthesis of the A-ring portion of Merle 45.
The southern portion of the bryostatin analogue started from previously described C-ring 3.[27a]
The acetate was removed using K2CO3/MeOH, the resulting alcohol was esterified with 5-
hexynoic acid, and the TBS ether was removed to give allylic alcohol 8. The unmasked alcohol
was oxidized and the C25 PMB was deprotected to produce the free C25 alcohol. At this point,
the intramolecular pyran annulation was investigated.
Scheme 5. Synthesis of the C-ring portion of Merle 45
At first it was found that the intramolecular pyran annulation did not work even though a similar
substrate was used during the synthesis of bryostatin 9 (Scheme 6).[27b] When compound 10 was
subjected to PPTS in MeOH only acetal 11 was formed and no macrocycle was observed.
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Scheme 6. Attempt to cyclize using the intramolecular pyran annulation reaction.
On the other hand, after removal of the C19 methyl ketal and Yamaguchi esterification to form
ester 12, (Scheme 7) subjection to the same reaction conditions resulted in macrocyclization to
give tricyclic macrolactone 13 in a 70% yield with no spirocyclic side product observed. Thus the
macrocyclization fails with a methoxy group at C19, but proceeds in good yield with OH at this
position. Dependence on the presence of the C19 hydroxyl is not fully understood; however, in
the crystal structure of bryostatin 1[33] the C19 alcohol donates a hydrogen bond to the C3 alcohol
which is within H-bonding distance to both the A-, and B-ring pyran oxygens. This internal H-
bonding network is required for biological activity, and partial formation of this network may serve
to orient the aldehyde and β-hydroxyallylsilane for the pyran annulation. It thus seems highly likely
that the intramolecular pyran annulation reaction is in fact a hydroxyl directed event.
The synthesis was continued by removing the protecting groups, with exposure of BPS ether 13
to HFpy followed by a global deprotection using LiBF4 to give alcohol 14 in a 66% yield over 2
steps. Finally, azide 15 (see supporting information) was coupled with 14 in the presence of
copper(I) iodide and (iPr)2NEt to afford Merle 45 in an 80% yield.
Scheme 7. Synthesis of Merle 45.
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Me
Me
MeO
Me
MeO
Me
OAc
TMS
OAc
O
1) HF/H2O,
MeCN
2) 2,4,6-Cl3PhCOCl,
7, Et3N, THF;
OTES
O
O
O
O
O
H
O
O
PPTS,
1) HF•Py, THF
BPSO
H
OH
MeOH, 70%
OBPS
H
2) LiBF4, CH3CN/ H2O,
80°C, 66% 2 steps
9
OH
Me
Me
O
then alcohol,
DMAP, toluene,
70% (2 steps)
Me
Me
O
O
Me
CO2Me
OBOM
O
Me
OBOM
O
O
CO2Me
13
12
Me
Me
HO
Me
HO
Me
Me
OAc
OAc
Me
N
Me
O
O
O
O
O
O
O
O
B
F
N
15
F
O
O
N
N
N3
B
OH
OH
OH
OH
H
H
Me
F
F
Me
Me
O
Me
O
Me
CuI, i-Pr2NEt,
N
N
Me
CO2Me
OH
Me
CO2Me
OH
THF, 80%
N
O
O
Merle 45
14
Synthesis of Merle 44: Using our pyran annulation methodology[12d,34] to couple A-ring 16[12d, 30a]
with fully functionalized C-ring 17[27a] we were able to produce 18 in a 92% yield (Scheme 8).
Unlike the PPTS mediated pyran annulation, TMSOTf mediated cyclization does not require a
C19 hydroxyl group. Since the top portion of Merle 44 has less functionality, the stronger TMSOTf
can be used to construct the B-ring with the C19 methoxy group still present. Closing the
macrocycle began by first deprotecting the C1 BPS ether, followed by consecutive oxidations
using Parikh-Doering and Pinnick oxidations to give the C1 carboxylic acid 19. The C25 TBS
ether was then removed, followed by a Yamaguchi macrolactonization to afford the desired
tricyclic macrolactone 20. The C20 acetate was removed and the resultant alcohol was esterified
with 5-hexynoic acid to give the desired ester 21. The PMB ether was removed with DDQ, and
global deprotection with LiBF4 gave the bryopyran intermediate, which was coupled with azide
15 to afford Merle 44 in an 80% yield.
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Scheme 8. Synthesis of Merle 44.
TMS
H
OH
O
1) TBAF, AcOH,
DMF, 75%
2) SO3•Py, iPr2NEt
DMSO, CH2Cl2
O
O
16
OBPS
O
1
OBPS
OPMB
+
TMSOTf,
OMe
O
OPMB
H
OMe
H
OTBS
3) 2-methyl-2-butene
tBuOH, KH2PO4,
NaClO2
Et2O, 92%
Me
Me
Me
O
OTBS
Me
AcO
Me
CO2Me
OBOM
AcO
Me
CO2Me
OBOM
90% (2 steps)
18
17
1) K2CO3,MeOH, 1h
O
O
O
O
O
1
1) HF•Py,
THF
O
2)
CO2H
OH
OMe
OMe
OPMB
OPMB
H
H
OTBS
O
O
O
2) 2,4,6-Cl3PhCOCl
Et3N, THF; then
DMAP, toluene
70%
2,4,6-Cl3PhCOCl,
DMAP, Et3N,
toluene 70% (2 steps)
Me
Me
Me
Me
25
OBOM
AcO
AcO
Me
CO2Me
Me
CO2Me
OBOM
20
19
Me
1) DDQ, H2O
CH2Cl2
Me
N
2) LiBF4, CH3CN/ H2O
80 °C, 70% (2 steps)
Me
O
O
O
O
O
O
F
B
N
F
3)
O
O
OH
OMe
15
OH
OPMB
H
H
Me
Me
O
Me
O
O
N
N
N3
B
Me
Me
F
F
N
O
Me
CO2Me
OH
Me
CO2Me
OBOM
N
CuI, iPr2NEt,
N
O
O
THF, 80%
21
Merle 44
Biological evaluation of Merle 44 and Merle 45: Consistent with the tolerance of the C20
position for bulky substitutions, the bulky fluorophore did not substantially affect the in vitro
binding of Merle 44 and Merle 45 to PKCα. In a competition binding assay with [3H]PDBu and
purified PKCα, Merle 44 bound with a Ki of 0.64 ± 0.08 nM (n = 3), which was very similar to
that for Merle 23 (Ki = 0.70 ± 0.01 nM). Merle 45 bound with a Ki of 0.32 ± 0.03 nM (n = 3),
which was also very similar to the binding affinity of Merle 28 (Ki = 0.52 ± 0.06 nM). These
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values are likewise similar to that for bryostatin 1 (Ki = 0.73 ± 0.05 nM), showing that inclusion
of the fluorescent side chain at the C20 position has very little effect on the ligand binding to PKCα
in vitro.
The motivation for the preparation of Merle 44 and Merle 45 was to prepare fluorescent analogues
of Merle 23 and Merle 28, respectively. Merle 23 is largely PMA-like in its biological activity[12d]
in U937 cells whereas Merle 28 is bryostatin-like.[12b] A crucial issue was whether the fluorescent
compounds retained the patterns of biological activity of their non-fluorescent congeners.
Merle 45, like Merle 28 and bryostatin 1, did not inhibit proliferation (Figure 1) or induce
attachment (Figure 2) of U937 cells. Moreover, like Merle 28 and bryostatin 1, Merle 45
antagonized the effects of PMA when co-administered (p=0.1767 for any difference between the
effect of any of the three compounds). Interestingly, Merle 45 induced 7.1% less attachment than
was seen with Merle 28 (p = 0.0002) and was a more potent antagonist of attachment induced by
PMA, resulting in a pattern that appears even more bryostatin-like than that of Merle 28 (p =
0.8671 for comparison of M45 with Bryo 1; p = 0.0200 for comparison of M28 with Bryo 1).
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Figure 1. Growth of U937 Cells.
U937 cells were treated with the indicated concentrations of ligands alone, or in the presence of
10 nM PMA. Cell number was counted at 60 hr post treatment and expressed relative to a DMSO
control. Values represent the mean and 95% confidence intervals (shaded region) from a linear
regression model including at least four (n = 4-6) independent experiments per drug/concentration
combination, with the exception of M45 (3000 nM), which only has 2 observations.
Just as the fluorescent Merle 45 preserved the bryostatin-like biology activity of Merle 28,
fluorescent Merle 44 largely preserved the PMA-like biological behavior of Merle 23. Like Merle
23 and PMA, Merle 44 inhibited growth (Figure 1) and induced attachment (Figure 2) of the
U937 cells, although it should be noted that the level of inhibition was 9.6% less than that achieved
by the optimal dose of Merle 23 (p = 0.0060). Additionally, like Merle 23, Merle 44 did not
antagonize the effects of PMA in U937 cells when co-administered (p = 0.0034 for comparison of
M44 with Bryo 1; p = 0.0016 for comparison of M23 with Bryo 1).
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Figure 2. Attachment of U937 Cells.
U937 cells were treated with the indicated concentrations of ligands or with the indicated ligands
in the presence of 10 nM PMA. Attachment was measured at 60 hr of treatment. Values represent
the mean and 95% confidence intervals (shaded region) from a log-linear regression model
including at least four (n = 4-6) independent experiments per drug/concentration combination, with
the exception of M45 (3000 nM), which only has 2 observations.
Secretion of tumor necrosis factor α (TNFα) provides another differential response of the U937
cells to PMA and bryostatin 1, with bryostatin 1 inducing limited release compared to PMA.[12j, 35]
Once again, Merle 45 behaved very similarly to bryostatin 1 and Merle 28 for inducing tumor
necrosis factor α release (Figure 3). Merle 44 more closely resembled PMA and Merle 23 in
inducing a higher level of release, albeit lower than that induced by Merle 23. Interestingly, the
maximal response was shifted to higher concentrations relative to Merle 23 by an order of
magnitude. A similar shift was evident in the maximal doses of Merle 44 versus Merle 23 for
inhibition of proliferation and for induction of attachment of the U937 cells. In all three biological
assays, Merle 44 was 2 orders of magnitude less potent than was Merle 45. This is in marked
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contrast to the in vitro binding assays, in which Merle 44 and Merle 45 were very similar and
illustrates that other factors, in addition to PKC binding affinity, contribute to biological efficacy.
Figure 3. Secretion of TNFα.
U937 cells were treated for 60 hr with the indicated concentrations of the ligands. In each
experiment, the level of secreted TNFα under each treatment condition was normalized to the level
of secreted TNFα for the cells treated with 100 nM PMA. Values represent the mean of at least
four (n = 4-6) independent experiments. Bars, SE.
While the U937 cells provide a robust measure of the difference in biological response to PMA
and bryostatin 1 (or its analogues) as do several other cell lines such as K562 and MV4-11,[12f] the
Toledo cells, derived from a non-Hodgkin’s leukemia, show growth inhibition to both PMA and
bryostatin 1. They thus provide a convenient and straightforward measure of intrinsic activity,
and they also show somewhat greater sensitivity to PMA and bryostatin 1 than do the U937 cells.
In this system, Merle 44 once again stood out as being an order of magnitude less potent than
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Merle 23 (Figure 4). All the other compounds were relatively close to one another. IC50 values
were as follows: PMA (IC50 = 0.57 ± 0.27 nM), bryostatin 1 (IC50 = 0.091 ± 0.026 nM), Merle 28
(IC50 = 0.111 ± 0.009 nM), Merle 45 (IC50= 0.334 ± 0.068 nM), Merle 23 (IC50= 0.33 ± 0.11 nM),
and Merle 44 (IC50 = 3.17 ± 0.67 nM).
Figure 4. Growth of Toledo Cells.
Cell number of Toledo cells was determined after treatment with the indicated concentrations of
the ligands for 72 hrs and expressed as % of control. Values represent the mean of at least 3
independent experiments (n = 3-5). Bars, SE.
Localization of Merle 44 and Merle 45 in LNCaP cells: We have previously described that
different PKC ligands can drive translocation of multiple PKC isoforms to different subcellular
locations. In particular, the tumor promoting phorbol ester PMA induced initial localization of
PKCδ to the plasma membrane, with subsequent relocalization to internal membranes, whereas
non-promoting phorbol esters and bryostatin 1 caused the initial translocation of PKCδ to internal
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membranes.[18] The fluorescently labeled Merle 44 and Merle 45 provided an opportunity to
evaluate if ligand localization could determine whether a ligand would have bryostatin-like
biology. The localization and rate of uptake of Merle 44 and Merle 45 were visualized in real
time by confocal microscopy in LNCaP cells. These cells were used because they are attached
cells, unlike U937, but like U937 cells show differential biological response to PMA and to
bryostatin 1.[12j] Both fluorescent compounds showed similar internal membrane distribution with
no selective presence in the plasma membrane (Figure 5A). This contrasts with the initial plasma
membrane that has been described for PMA.[18] We conclude that differential membrane
localization of the ligand, with PMA-like behavior (as with Merle 44) reflecting an early
predominantly plasma membrane localization and bryostatin-like behavior (as with Merle 45)
reflecting an early internal localization, cannot account for the differential biological response.
Their pattern of ligand localization was consistent with our finding that initial plasma membrane
localization of a ligand is characteristic of more lipophilic compounds (higher CLogP) and initial
internal localization is characteristic of less lipophilic compounds.[21] The calculated CLogP value
of 3.71 for Merle 44 and of 4.68 for Merle 45 is higher than that of the parents compounds (Merle
23 (CLogP 2.67), Merle 28 (CLogP 1.70)) but lower than that of PMA (CLogP 6.65).
A further striking feature of the compounds was their slow rate of uptake. To better quantitate
the rate of uptake, uptake of 100 nM Merle 44 and Merle 45 was determined to avoid saturation
of the optical signal and Cos 7 cells were used rather than LNCaP cells, since the Cos 7 cells do
not change their shape or show mobility on the culture dish after ligand addition (Figure 5B).
Merle 45 showed modestly faster uptake than did Merle 44. Times for half-maximal uptake of
100 nM Merle 45 and Merle 44 into the Cos 7 cells were 16.3 ± 2.8 and 22.8 ± 1.6 min (980 ±
170 and 1368 ± 93 sec), respectively. Such slow uptake will necessarily impose uncertainty in
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efforts to measure dose response relations for early biological endpoints because the internal
concentration of the ligand is changing. In addition, to the degree that different PKC isoforms
have different EC50 values for stimulation, the gradient of rising ligand levels with time will predict
that there will be sequential activation of those PKC isoforms, at least at lower levels of ligand.
We have previously described that a series of DAG-indololactones, which also function as ligands
for PKC, showed rapid uptake, with half-times in the range of 1 min.[36] In contrast, the slow rate
of uptake of Merle 44 and Merle 45 is consistent with the slow rates of uptake that we previously
have reported for several fluorescent phorbol ester derivatives.[21] Finally, we can conclude that
the limited difference in the patterns of substitution in the A- and B-rings of Merle 44 and Merle
45 (and correspondingly of Merle 23 and Merle 28) had only a modest effect on the rates of uptake.
B
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Figure 5. Localization of Merle 44 and Merle 45 in LNCaP cells and their uptake into Cos7 cells.
A. LNCaP cells were transfected with mCherry PKCδ and imaged after treatment for the indicated
times with 1000 nM of Merle 44 or Merle 45. Results are from one representative experiment (5
independent experiments were performed under these conditions). B. Uptake of Merle 44 and
Merle 45 into Cos7 cells as a function of time. Cos7 cells were transfected with mCherry PKCδ
or mCherry PKCε and imaged after treatment for the indicated times with 100 nM Merle 44 or
Merle 45. Values represent the fluorescent intensity of the whole field of view normalized to the
maximal signal of each individual experiment and represent the mean of 7-8 experiments. Bars,
SE. The arrow indicates the addition of the drug.
Synthesis and biological evaluation of fluorescent phorbol esters: To provide fluorescent
phorbol esters for comparison with the fluorescent Merle 44 and Merle 45, we prepared phorbol
esters containing the same BODIPY tag, taking advantage of previous phorbol analogue
chemistry.[37] Starting with commercially available phorbol 12-myristate 13-acetate, the primary
alcohol was protected as a TBS ether and the C12 and C13 esters were hydrolyzed to give tetrol
23. The C13 alcohol could then be selectively acylated because of its placement on the convex
face of this highly substituted ring system to give ester 24.[38] 5-Hexynoic acid was then used in a
Yamaguchi esterification to install the alkyne moiety at the C12 position to construct 25.
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Deprotection of the TBS ether to expose the terminal alcohol on C20 was achieved using HF-
pyridine, and finally, a copper catalyzed click cycloaddition was performed to furnish FPMA1
and FPMA2.[39]
Scheme 9. Synthesis of FPMA1 and FPMA2.
Unlike the bryostatin analogues, the Ki of FPMA1 was greatly affected (Ki = 201 ± 24 nM vs
Ki = 2.6 nM for PMA) by the replacement of the 12-tetradecanoate ester of PMA with the
fluorescent group and linker. Correspondingly, the potency of phorbol FPMA1 in the U937 and
Toledo cell assays was dramatically (about 10,000 fold) reduced, with growth in the Toledo cells
only beginning to show inhibition at 3,000 nM and effects on the U937 cells only beginning to be
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induced at 10,000 nM, with possible toxicity at 30,000 nM (Supplementary Figure 1). Based on
these observations FPMA1 appears to be PMA-like, but the weak activity permitted limited
characterization. The localization of FPMA1 in the LNCaP cells was examined. Its localization
is similar to that of Merle 44 and Merle 45, translocating mostly to internal membranes (data not
shown).
Along with FPMA1 (ClogP=3.42), we prepared the fluorescent derivative FPMA2
(CLogP=5.54), in which we enhanced the lipophilicity by exchanging the C13 acetate for a longer
aliphatic ester (Scheme 9). The Ki value of FPMA2 for PKCα was much improved (Ki = 2.21 ±
0.36 nM, n = 4).
FPMA2 inhibited U937 cell growth and induced attachment of U937 cells to a similar level but
with different potency as did PMA (Figure 6). The combination of FPMA2 (1000 nM) and PMA
gave growth inhibition similar to that for PMA alone and induced attachment to a similar level as
seen for PMA alone. At 10,000 nM FPMA2, a little less attachment was seen for the combination
than for PMA alone, suggestive of limited toxicity at this concentration and incubation time (60
hr). Like PMA and bryostatin 1, FPMA2 inhibited Toledo cell growth. In these cellular assays
FPMA2 remained appreciably less potent than PMA, with EC50 values between 100 and 1000 nM.
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Figure 6. Biological analysis of FPMA2.
Growth (A) and attachment (B) of U937 cells after treatment for 60 hr with the indicated
concentrations of PMA, bryostatin 1, and FPMA2. C) Inhibition of growth of Toledo cells after
treatment for 72 hr. All values represent the mean and S.E. of at least 5 independent experiments
(n = 5-7). D) Comparison of structures of PMA and FPMA2.
PKC translocation in LNCaP cells in response to treatment with Merle 44, Merle 45, and
FPMA2. Merle 44 (Figure 7A), along with FPMA2 (Figure 7B), failed to induce clear
translocation of PKCδ. As a positive control, 1 µM PMA was added after 10 min incubation with
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1 µM FPMA2 and yielded the characteristic translocation of PKCδ to the plasma and nuclear
membranes, as well as to internal membranes (Figure 7B). Merle 45 induced weak translocation
of PKCδ at later times (Figure 7A) visible mostly in cells that showed nuclear PKCδ staining. For
PKCε, 1 µM Merle 45 induced partial translocation to the plasma membrane. 3 µM FPMA2
induced some translocation, although not as much as 1 µM Merle 45. As with PKCδ, 1 µM Merle
44 failed to induce translocation of PKCε in this time range (Figure 7C).
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Figure 7: Imaging of mCherry tagged PKCδ (A/B) and PKCε (C) in LNCaP cells after treatment
with Merle 44 and Merle 45 (A/C) or FPMA2 (B/C).
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The red signal represents mCherry; the green signal the BODIPY FL labeled ligands. Results are
representative of 3 – 6 independent experiments. C) Imaging of mCherry tagged PKCε in LNCaP
cells after treatment with Merle 44, Merle 45 and FPMA2. Two representative set of images from
3-6 independent experiments are shown. The red signal represents mCherry.
PKC isozyme selectivity of Merle 44 and Merle 45: Although the specific goal of the
development of Merle 44 and Merle 45 was to assess whether the ligands localized differently in
the cells, while retaining PMA-like or bryostatin-like biological activity, we also did further
characterization of these novel compounds. In U937 cells, the prominent isoforms of PKC are
PKCβII, PKCδ, PKCε, while in LNCaP cells they are PKCα, PKCδ and PKCε.[40] We had shown
previously that in U937 cells PKCβII and PKCδ are modulated by bryostatin 1 and PMA, while
there was no downregulation of PKCε when treated with these ligands.[12i] Bryostatin 1 and similar
analogues are unique in inducing a biphasic downregulation of PKCδ in these and other cells with
maximum downregulation at 1 and 10 nM bryostatin 1 and partial protection from down regulation
at higher bryostatin 1 concentrations. Here, we found that both PMA and the bryostatin-like
compounds downregulated PKCβII at 24 hr but with very different potencies (Figure 8A). For
PKCδ downregulation (Figure 8B), Merle 45 did not protect PKCδ from down regulation at high
concentrations as did bryostatin 1 or Merle 28. Merle 23 and Merle 44 were less potent than Merle
45 for downregulation but again showed no protection of PKCδ down regulation at high doses.
The difference between Merle 45 and bryostatin 1 as well as other Merle compounds emphasizes,
as we have already described for Merle 23, that different derivatives can dissect different endpoints
of behavior.[12j]
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Figure 8. Downregulation of PKCβII and PKCδ in U937.
Downregulation of PKCβII (A) and PKCδ (B) in U937 cells after treatment for 24 hr with the
indicated concentrations of compounds. Values represent the mean of at least 4 independent
experiments (n = 4-7). Bars, SE. Values represent signal intensity (AU) normalized to the DMSO
control (100%).
Signal transduction induced by the different compounds in LNCaP cells. We have shown
that the fluorescent derivatives based on the bryostatin-like Merle 28 and the PMA-like Merle 23
retain the bryostatin-like or PMA-like biology of their parent compounds in U937 cells although
their intracellular localization is very similar in LNCaP cells, another cell line in which PMA and
bryostatin 1 have markedly different effects on growth and other biological responses .[22] We
have also shown evidence for unusually slow uptake of the fluorescent compounds into the cells.
This slow uptake might be predicted to influence the signal transduction events, especially the
early phosphorylation events, induced by the compounds. We therefore evaluated a series of signal
transduction responses including phosphorylation of PKCδ, PKD1, MEK1/2 and ERK1/2 and
changes in the levels of the early response proteins cFos and EGR1 in LNCaP cells treated for 30
min, 60 min and 150 min. While we have previously characterized differential effects of PMA and
bryostatin 1 on both the U937 cells and the LNCaP cells,[12i] here we focused on the LNCaP cells
because the effects of the compounds on gene expression were somewhat more marked.
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While we observed a complicated pattern of signaling responses for the different compounds,
two clear conclusions emerge (Figure 9). The first clear conclusion was that PMA, bryostatin 1,
Merle 45, and Merle 44 each had a different pattern of response, as shown by comparison of the
patterns of phosphorylation of PKCδ S299, PKCδ Y311, and PKD1 S744/S748. The second
conclusion was that, for responses that were characterized by transiency, the responses to Merle
45 and Merle 44 never achieved the maximal levels achieved by PMA (Figure 9). The pattern of
behavior fits with the model that the transient responses are turning off before a fully stimulatory
dose of the slowly penetrating ligand is achieved.
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Figure 9. Signal transduction in LNCaP cells treated for the indicated times with different drugs.
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