Design, synthesis, and evaluation of potent bryostatin analogs that modulate PKC translocation selectivity

Article abstract of DOI:10.1073/pnas.1015270108

Modern methods for the identification of therapeutic leads include chemical or virtual screening of compound libraries. Nature's library represents a vast and diverse source of leads, often exhibiting exquisite biological activities. However, the advancem

Full text of DOI:10.1073/pnas.1015270108

Design, synthesis, and evaluation of potent bryostatin
analogs that modulate PKC translocation selectivity
Paul A. Wender1, Jeremy L. Baryza, Stacey E. Brenner, Brian A. DeChristopher, Brian A. Loy,
Adam J. Schrier, and Vishal A. Verma
Departments of Chemistry and of Chemical and Systems Biology, Stanford University, Stanford, CA 94305
Edited by Stuart L. Schreiber, Broad Institute, Cambridge, MA, and approved February 9, 2011 (received for review December 14, 2010)
Modern methods for the identification of therapeutic leads in-
clude chemical or virtual screening of compound libraries. Nature’s
library represents a vast and diverse source of leads, often exhibit-
ing exquisite biological activities. However, the advancement of
natural product leads into the clinic is often impeded by their
scarcity, complexity, and nonoptimal properties or efficacy as well
as the challenges associated with their synthesis or modification.
Function-oriented synthesis represents a strategy to address these
issues through the design of simpler and therefore synthetically
more accessible analogs that incorporate the activity-determining
features of the natural product leads. This study illustrates the
application of this strategy to the design and synthesis of func-
tional analogs of the bryostatin marine natural products. It is spe-
cifically directed at exploring the activity-determining role of
bryostatin A-ring functionality on PKC affinity and selectivity. The
resultant functional analogs, which were prepared by a flexible,
modular synthetic strategy, exhibit excellent affinity to PKC and
differential isoform selectivity. These and related studies provide
the basic information needed for the design of simplified and thus
synthetically more accessible functional analogs that target PKC
isoforms, major targets of therapeutic interest.
desired structural, synthetic, pharmacokinetic, and activity goals.
In essence, FOS focuses on function, the approach to which can
be achieved through design, diversity-oriented synthesis, biology-
oriented synthesis, diverted total synthesis, and others.
Our group has employed this approach in the design and
development of numerous simplified agents that harness the
functional activities of structurally complex natural leads. This
strategy is also finding increasing success in other laboratories
(12–16) and resonates in part with the goals and related concept
of diverted total synthesis (17, 18). The starting point of FOS
is function and the view that function could be emulated with
a wide range of designed structures, as many structural types
could mimic a natural product’s shape and electron density fea-
tures. This view draws validation from many sources including,
for example, early studies on synthetic β-lactams. An early exam-
ple of this FOS strategy in our own studies was the demonstration
that the activity of the phorbol esters (29-step synthesis) could be
emulated in part by highly simplified functional analogs available
in only 7 steps (Fig. 1) (19). Bryostatin is another noteworthy
example of this approach and forms the focus of this study.
The bryostatins are complex, scarce natural products produced
by a bacterial symbiont (20, 21) of the marine bryozoan Bugula
neritina. Extracts of this marine organism were reported to pos-
sess anticancer activities by Pettit et al. in 1970 (22), but it was
not until 1982 that the structure of bryostatin 1 (Fig. 2), a primary
active constituent of these extracts, was elucidated (23). Nineteen
additional bryostatins have since been described.
The most thoroughly investigated member of this family is
bryostatin 1, which possesses a remarkable if not unique portfolio
of biological activities relevant to cancer therapy including mod-
ulation of apoptotic function (24), reversal of multidrug resis-
tance (25, 26), and stimulation of the immune system (27).
Bryostatin has been investigated for anticancer activity in over
35 phase I and II clinical trials (see http://clinicaltrials.gov). Its
efficacy as a single agent has varied as a function of cancer type.
More recently, studies have shown that it can enhance the clinical
efficacy of known oncolytics in several cases (28–30). Addition-
ally, bryostatin or its tunable analogs provide a starting point for
promising immunotherapeutic approaches to cancer treatment
(31). Notably, the required clinical dose of bryostatin is extraor-
dinarily low (ca. 50 μg∕m2); only approximately 1 mg is needed
for an eight-week clinical treatment cycle.
herapeutic leads are derived from or inspired by natural,
T
nonnatural, and virtual sources. Nature’s collection, a library
representing 3.8 billion years of chemical evolution, is vast, struc-
turally diverse, and remarkably rich with therapeutically promis-
ing molecules (1). This collection can also be readily expanded
by harnessing the powerful capabilities of biosynthetic machinery
using, for example, phage display (2), engineered biosynthesis
(3–5), and synthetic biology. Complementing nature’s library,
fully synthetic libraries (6, 7) offer the notable advantage of being
derivable from a more varied collection of bond construction
methods, atoms, and building blocks than those used in nature.
Virtual libraries (8, 9) provide a third and comprehensive source
of lead structures and are limited only by bonding theory and
self-imposed structure generation and selection criteria.
Although natural products have proven to be a very effective
source of library leads, traditional medicines, and modern drugs
(10), they are neither evolved nor optimized for human use.
Furthermore, they are often scarce, too complex to synthesize
in a practical fashion, or too difficult to derivatize as needed
to optimize for therapeutic function. Function-oriented synthesis
(FOS) offers a strategy to address these issues (11). FOS draws
on the view that a specific function of a natural or nonnatural
compound, whether it be a therapeutic, material, probe, nanode-
vice, imaging agent, diagnostic, catalyst, conductor, or a molecule
of theoretical interest, can be achieved with many even simplified
structures through synthesis-informed design. As it applies to
therapeutic leads, FOS is based on the understanding that the
activity (function) of a natural product is often determined by
only a subset of its functionality and that such activity could thus
be reproduced or improved by incorporating these function-
determining features into simplified and therefore synthetically
more accessible structures. The goal of FOS is to design for
both optimal function and ease of synthesis, and an attractive
aspect of this approach is that one can creatively select for
Of additional clinical significance, bryostatin enhances
learning and extends memory in animal models (32, 33), which
is attributable in part to its ability to induce synaptogenesis
(34). These activities suggest that bryostatin or a more readily
Author contributions: P.A.W., J.L.B., S.E.B., B.A.D., B.A.L., A.J.S., and V.A.V. designed
research; P.A.W., J.L.B., S.E.B., B.A.D., B.A.L., A.J.S., and V.A.V. analyzed data; J.L.B.,
S.E.B., B.A.D., B.A.L., A.J.S., and V.A.V. performed research; and P.A.W., B.A.L., A.J.S.,
and V.A.V. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. E-mail: wenderp@stanford.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/
doi:10.1073/pnas.1015270108/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1015270108
PNAS ∣ April 26, 2011 ∣ vol. 108 ∣ no. 17 ∣ 6721–6726

and novel (see SI Appendix) (50): Conventional PKCs (α, βI,
βII, and γ) require both DAG and Ca2þ, whereas novel PKCs
(δ, ϵ, θ, and η) are activated by DAG alone.
An initial goal of this program was to identify the functional
groups in bryostatin that contribute to its activity. In collaboration
with the groups of Pettit and Blumberg, we advanced the hypoth-
esis based on structure–function analyses and computer structure
comparisons that, of all possible pharmacophores in bryostatin,
the C1-carbonyl, C19-OH, and C26-OH groups were compelling
candidates for controlling its affinity to PKC (51). This hypothesis
was supported by chemical derivatization studies, comparisons of
natural product activities, and systematic structural comparison
of bryostatin with structurally unrelated yet competitive PKC
ligands (e.g., DAG and phorbol ester derivatives). This analysis
suggested that bryostatin’s southern or C-ring region directly
contacts PKC and thus serves as its “recognition domain.” It
was additionally proposed (52) that the northern A- and B-ring
regions serve as an important scaffolding element, or “spacer
domain,” that influences the presentation of bryostatin’s pharma-
cophoric elements and possibly influences intracellular traffick-
ing and membrane association.
Fig. 1. The function-oriented synthetic approach to simplified analogs of
the phorbol esters that possess affinity to PKC.
accessible designed analog (35) could serve as a first-in-class
clinical lead for the treatment of Alzheimer’s disease (36) and
other neurodegenerative disorders (37). Bryostatin also exhibits
neuroprotective capabilities in animal models of cerebral ische-
mia, indicating its potential use for minimizing the destructive
neurological effects of stroke (38). Of special recent significance,
bryostatin, not unlike prostratin (39), has been found to activate
latent HIV reservoirs, providing a potential strategy for the
eradication of HIV (40).
This hypothesis led to the design of macrocycles containing
bryostatin-like recognition domains but structurally simplified
spacer domains (Fig. 2). In 1998, we reported an initial series
of potent, fully synthetic bryologs that were prepared according
to these design principles (53). We have since reported numerous
potent, tunable bryologs exemplified by analog 1 (54). This com-
pound possesses excellent affinity for PKC (Ki ¼ 3 nM), is orders
of magnitude more potent than bryostatin against several human
cancer cell lines, and can be synthesized in a step-economical
fashion (29 total steps) scalable to meet clinical demand. Recent
work by Keck and coworkers has provided further examples of
the effectiveness of this design strategy (55–58).
Notwithstanding its great therapeutic potential, research on
and clinical development of bryostatin has been impeded by its
low natural abundance. The GMP production of bryostatin for
clinical use provided only 18 g of pure bryostatin 1 from 14 tons
of B. neritina (0.00014%) (41). Although this supply was sufficient
to initiate preclinical and clinical research on bryostatin, econom-
ic and environmental factors have limited further development
of this source. The yields of bryostatin’s related congeners are
similarly low (10−3 to 10−8%) (42), and seasonal variability has
further complicated isolation. Although chemical synthesis offers
a supply possibility, published syntheses of the clinical lead or
similarly potent congeners (Ki < 10 nM) have thus far been
too long (>55 steps) to impact cost-effective supply (43–47).
Similarly, engineered biosynthesis, although promising, is still in
the early stages of development (48). A further consideration is
that these strategies collectively provide only bryostatin or closely
related derivatives, which are neither evolved nor optimized for
human therapeutic use.
Step economy is enabled through design, both by simplification
and careful scaffold engineering; substitution of the natural pro-
duct’s B-ring tetrahydropyran motif with a dioxane substructure
allows for the mild, modular union of appropriately functiona-
lized recognition and spacer domain elements by esterification
followed by acid-promoted macrotransacetalization (Fig. 2). This
dioxane for pyran substitution has also found value in other target
simplifications (59). The modularity inherent to this convergent
fragment coupling strategy renders it highly amenable to library
synthesis. We have recently extended our convergent fragment
coupling approach to the natural B-ring tetrahydropyran scaffold
using a mechanistically related Prins macrocyclization reaction
(60). To date, we have developed a library of over 100 bryologs,
In 1986, our group set out to address both the supply and
therapeutic optimization goals associated with bryostatin by
using an FOS strategy. Bryostatin’s activities are mediated by its
interaction with several intracellular targets (49), including the
diacylglycerol (DAG)-dependent isoforms of PKC, a family of
kinases that transduce endogenous DAG and Ca2þ signals. DAG-
dependent PKC is subdivided into two classes, conventional
Fig. 2. (Left) Structures of bryostatin 1 and 2 and general analog design principle. Pharmacophoric atoms are in blue. Conformational depictions are derived
from crystal structure (bryo 1) or Monte Carlo MM3 conformational search (analog 1). For clarity, C20 side chains are excluded (bryo) or were abbreviated for
modeling efficiency (analog 1). (Right) General synthetic strategy for B-ring dioxane analogs. Analogs are modularly assembled via a late-stage esterification/
macrotransacetalization coupling protocol.
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activation and subcellular localization: Inactive PKC is located
primarily in the cytosol and undergoes translocation to mem-
branes when activated by bryostatin or other C1 domain modu-
lators (50). The rate and extent of PKC activation induced by
synthetic agents was measured in real time by changes in subcel-
lular fluorescence intensity, and the localization of the activated
kinase was directly observed. Additionally, preliminary isoform
selectivity was investigated by comparing the relative activities
of our agents for different PKC-GFP isoforms. For this study,
we utilized PKCβ1-GFP and PKCδ-GFP as representative con-
ventional and novel isoforms, respectively.
In accord with literature reports, bryostatin 1 was found to
induce rapid translocation of PKCβ1 and PKCδ at a concentra-
tion of 200 nM (Fig. 3). The kinetics and extent of translocation
are indistinguishable between isoforms, and activation is com-
plete after several minutes following treatment. The dominant
localization of both isoforms is the cellular membrane. Synthetic
analog 1 at 200 nM possesses a similar ability to activate PKCδ
as bryostatin 1; however, its ability to translocate PKCβ1 was
attenuated relative to the natural product (Fig. 3, G–I). The
activity of 1 at 2,000 nM was nearly indistinguishable from results
obtained at 200 nM (see SI Appendix).
The differential translocation activities of bryostatin 1 and
analog 1 for conventional PKC were also observed in NIH 3T3
mouse fibroblasts (71). In this system, the cellular localization
of the conventional-PKCα isoform and novel isoforms PKCδ
and ϵ was assessed via Western blot in response to incubation with
100 nM bryostatin 1 or analog 1. Results are expressed as the
ratio of cytosolic to membrane-bound PKC. Bryostatin 1 and ana-
log 1 exhibit identical abilities to activate novel PKC isoforms in
this system; complete membrane association of PKCδ and PKCϵ
was observed after a 5-min incubation with either agent (Fig. 4 A
and B). Mirroring the PKC-GFP observations, analog 1 exhibited
a diminished ability to activate the conventional-PKCα isotype.
Whereas bryostatin 1 induced complete membrane associa-
tion of this isoform, analog 1 induced only 39 ꢀ 4% transloca-
tion following a 30-min incubation. These selectivities were also
observed for longer incubations; bryostatin 1 and analog 1 possess
significantly (ρ ¼ 0.0057) different abilities to down-regulate
endogenous PKCα (Fig. 4 C and D), down-regulation being a
phenomenon that typically follows prolonged PKC activation
(50). After a 24-h incubation with 200 nM bryostatin 1, the
majority of endogenous PKCα was degraded; however, 73 ꢀ 11%
of PKCα remained following similar exposure to analog 1.
Fig. 3. (A–C) Translocation of PKCβ1-GFP by 200 nM bryostatin 1 in transi-
ently transfected CHO-k1 cells, (A) Predose, (B) 5 min postdose, and (C) 38 min
postdose. (D–F) Translocation of PKCδ-GFP by 200 nM bryo 1, same time
points. (G–I) PKCβ1-GFP, 200 nM analog 1, same time points. (J–L) PKCβ1-
GFP, 200 nM bryostatin 2, same time points. See Fig. 6 A and B for quantifica-
tion and time-course analysis.
more than 30 of which exhibit single-digit nanomolar or subna-
nomolar activities in PKC affinity or in vitro assays (61, 62). The
most promising of these agents (e.g., 1) are currently undergoing
preclinical investigation for their use in cancer therapy (63),
Alzheimer’s disease (35), and latent HIV activation.
Because PKC plays a role in numerous biological processes
ranging from apoptosis to cognition, an emerging goal of great
importance is to understand whether analog affinity can be main-
tained while controlling functional isoform selectivity. Although
PKC isoforms are in some cases functionally redundant, unique
roles can be ascribed to each (64); thus, isotype selectivity is likely
to be important in optimizing agents for desired functions while
designing against unwanted activities or toxicities. Although some
isoform- or class-selective PKC inhibitors have been developed
(65, 66), they target the highly conserved ATP binding site of
the human kinome. A promising complementary approach to
isoform-selective PKC modulation is interference with, or simu-
lation of, protein∶protein interactions involved in the activation
and localization of specific PKC isoforms (67). However, the
general lack of selective modulators of the PKC C1 regulatory
domain, for which gain and loss of function are possible, has
slowed investigation of this signaling pathway and its clinical
ramifications (68).
As described herein, the design, synthesis, and biological
evaluation of systematically functionalized A-ring analogs 4–6
were investigated to better understand the relationship between
A-ring functionality and PKC isoform selectivity. We find that
the C8-geminal dimethyl unit incorporated into these analogs
enhances their potency relative to des-methyl analogs 2 and 3.
Additionally, we find that the A-ring motif of these agents
modulates their functional selectivity for representative PKC
isoforms, providing a strong lead for development of more selec-
tive agents based on translocation kinetics.
Results and Discussion
Spacer Domain Structure Influences Conventional-PKC Activity. As a
reference point for this study, we measured, using a previously
described protocol (69, 70), the ability of natural bryostatins
and synthetic analog 1 to translocate GFP fusion constructs of
representative conventional and novel PKC isoforms in CHO-k1
cells. This assay relies on the mechanistic link between PKC
Fig. 4. (A and B) Band density analysis of endogenous PKC translocation
in NIH 3T3 fibroblasts following incubation with 100-nM test compound.
Results are an average of two to four experiments; error bars indicate
SEM. (C) Endogenous PKC remaining relative to control in NIH 3T3 fibroblasts
following a 24-h incubation with 200-nM test compound. (D) Representative
experiment from 24-h degradation study.
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As bryostatin 1 and analog 1 have similar PKC affinities,
these combined results suggested that functional selectivity for
novel PKC isoforms could be realized without sacrificing overall
potency. Similar results have been observed for other synthetic
analogs (72). Structurally, as the recognition domain elements of
bryostatin 1 and analog 1 are very similar, we considered the
possibility that the basis for these differential activities is con-
tained in spacer domain functionality.
Pertinent to this hypothesis, we found that bryostatin 2, which
is similarly potent (PKC Ki ¼ 5.9 nM) to bryostatin 1 and differs
structurally only by lack of a C7-acetate group, is unable to acti-
vate PKCβ1-GFP at 200 nM in CHO-k1 cells (Fig. 3). This finding
implicated the A-ring region, particularly the C7 position, as a
structural contributor to conventional-PKC activity and thus
overall PKC selectivity.
exquisite anti selectivity to provide diol 12 in 85% yield after
recrystallization. Acid-promoted lactonization differentiated the
resulting diol motif to afford crystalline hydroxylactone 8. This
common intermediate was silylated for the synthesis of C7-oxy
analogs 4 and 5. Alternatively, deoxygenation gave lactone 14
in two steps and 91% yield en route to C7-deoxy analog 6.
Lactones 13 and 14 both cleanly alkylated the dienolate of
ethyl acetoacetate. The C9-lactol products were deoxygenated
with TFA and Et3SiH to give pyrans 15 and 16 in 50% and 51%
yield over two steps, respectively. Noyori hydrogenation with
½ðRÞ-BINAPꢁRuCl2 afforded the corresponding hydroxyesters
with excellent diastereoselectivity (>95∶5). These products were
further reduced with LiBH4 and protected as acetonides 17 and
18 in excellent overall yield. Debenzylation and oxidation pro-
vided the completed carboxylic acid spacer domain fragments
19 and 20.
Analog Design and Synthesis. For an initial investigation of the
influence of C7 functionality on PKC selectivity, we recently
prepared simplified C7-OAc and C7-OH bryologs 2 and 3 (Fig. 2)
(73). Although C7-OAc analog 2 (Ki ¼ 13 nM) has good affinity
for PKC, C7-OH analog 3 (Ki ¼ 1;000 nM) was found to be
orders of magnitude less potent than bryostatin 2. As bryostatin
2 represents a significant lead in the development of novel iso-
form-selective PKC activators, we were prompted to determine
what structural features might account for this potency differ-
ence. We reasoned that the C8-gem dimethyl moiety present in
the natural product might mitigate the deleterious impact of
its C7-OH moiety on potency. We therefore designed C8 gem-
dimethyl bryologs 4–6 to test this hypothesis as well as to further
probe the role of A-ring functionality on PKC affinity and selec-
tivity. The C7-OAc and C7-OH analogs 4 and 5 were designed to
probe the influence of C7 functionality on PKC selectivity, and
the C7-deoxy analog 6 was prepared to parse out any individual
contribution of the C8-gem dimethyl moiety.
We approached the syntheses of analogs 4–6 by way of com-
mon A-ring lactone 8 (Fig. 5), which was derived from gem-
dimethyl ketone 9 and known aldehyde 10 (see SI Appendix).
A diastereoselective aldol reaction between these partners pre-
ferentially gave hydroxyketone 11. Although the 1,3-anti diaster-
eoselectivity of this reaction was poor (1.5∶1) using the lithium
enolate of 9, the cyclohexyl boron enolate gave an improved
diastereomeric ratio (3∶1). The anti∶syn ratio was further im-
proved to 9∶1 by use of isopinocampheyl boron ligands. On a
5-g scale this process delivered hydroxyketone 11 in 64% isolated
yield. Reduction with Me4NBHðOAcÞ3 (74) proceeded with
The highly convergent coupling of spacer domains 19 and 20 to
previously described recognition domain 7 (54) was accomplished
using Yamaguchi’s esterification (Fig. 5B). Treatment of the
resulting products with HF·pyridine effected, in one subsequent
step, removal of all silyl protecting groups and macrotransaceta-
lization to afford bryostatin 2-like C7-OH analog 4 and C7-deoxy
analog 6 in 65% and 83% yield over two steps, respectively.
Analog 4 was readily converted into bryostatin 1-like C7-OAc
analog 5 in two steps and 88% yield by in situ silylation of
C26 followed by acylation of C7 and subsequent desilylation.
Analog PKC Affinity and Selectivity. C8-gem dimethyl bryologs 4, 5,
and 6 are potent ligands for a rat-brain PKC isozyme mixture
(75), with Ki’s of 19 nM, 2.0 nM, and 1.4 nM, respectively. When
compared to des-methyl analogs 1–3, the C8-dimethyl moiety
enhances the PKC affinity of analogs bearing polar C7 function-
ality. C7-OAc analog 5 is 6.5-fold more potent than des-methyl
analog 2 (Ki ¼ 13 nM). More dramatically, C7-OH analog 4
is 50-fold more potent than the des-methyl analog 3 (Ki ¼
1;000 nM). This affinity augmentation is less in the C7-deoxy ser-
ies; the potencies of dimethyl analog 6 and analog 1 are quite
similar. The origin of the large potency difference between 3
and 4 remains to be fully elucidated, but one possible explanation
is that the sterically demanding C8-dimethyl moiety partially des-
olvates the neighboring C7-OH, diminishing penalties associated
with protein binding and/or membrane association.
PKC-GFP translocation assays demonstrate that C7 modifi-
cations affect functional selectivity for PKC isoforms. As de-
scribed above, the abilities of analogs to activate PKCδ-GFP
Fig. 5. (A) Synthesis of spacer domains for analog synthesis. Reagents and conditions: (a) Ketone 9, ðþÞ-Ipc2BCl, Et3N, Et2O, 0 °C, then 10, −98 °C, then H2O2,
MeOH, pH 7 Buffer, 90∶10 d.r., 64% isolated 11; (b) Me4NBHðOAcÞ3, 1∶1 HOAc∶MeCN, −15 °C, 85% recrystallized yield; (c) ( )-CSA, PhH, reflux, 90% recrys-
tallized yield; (d) when X ¼ OTBS: TBS-OTf, 2,6-Lutidine, CH2Cl2; when X ¼ H: (i) Im2CS, CH2Cl2, (ii) Bu3SnH, AIBN, PhCH3, 110 °C; (e) ethyl acetoacetate, LDA,
THF, −78 °C; (f) TFA, Et3SiH, CH2Cl2; (g) Ru½ðRÞ-BINAPꢁCl2 (2 mol%), EtOH, H2 (78 bar), 35–40 °C; (h) LiBH4, THF, (i) 2,2-dimethoxypropane, PPTS, DMF; (j) lithium
naphthalenide, THF; (k) TEMPO (cat.), NaClO (cat.), NaClO2, MeCN, pH 7 buffer. (B) Completion of bryologs 4–6. Reagents and conditions: (l) 2,4,6-trichlor-
obenzoyl chloride, Et3N, PhCH3, then 7, DMAP; (m) HF·pyridine, THF; (n) TESCl, DMAP, CH2Cl2, then Ac2O; (o) HF·pyridine, THF.
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The observed selectivities of these agents for novel PKCδ thus
mimic that observed for analog 1. As in the case of 1, a 10-fold
higher dose of 5 or 6 did not significantly enhance their PKCβ1
activity (see SI Appendix).
These combined observations indicate that the A ring of
bryostatin and analogs modulates the activity of these agents
for conventional PKC. We find that incorporation of a C8-gem
dimethyl group is important for enhancing the PKC affinity of
C7-OH analogs (e.g., analog 4); this is significant as such com-
pounds appear to have high functional selectivity for novel PKC
isoforms. Intriguingly, synthetic analogs 1, 5, and 6 are capable of
activating conventional-PKC although to a lesser extent than
bryostatin 1. This difference contributes to bryostatin’s ability
to more effectively down-regulate the conventional-PKCα iso-
zyme (Fig. 4). These and previous studies provide a collection
of designed analogs covering a range of selectivities: Some mem-
bers emulate bryostatin, whereas others exhibit complementary
selectivities. Thus, the natural product and its synthetic analogs
comprise a valuable set of tools to probe relationships between
desired biological functions and selective modulation, activation,
and down-regulation of the PKC family members.
Conclusions
The path of research progresses from information acquisition to
understanding to the creative use of the resulting knowledge to
produce solutions and products. Screening structures for sought-
after function provides a point of entry along this path that is of-
ten required when structural leads are limited or absent. Natural
products often provide a more advanced point of entry as activity
(function) is often known. However, many are too scarce or com-
plex to be synthesized and tuned for research or clinical advance-
ment. As exemplified above, these problems can be addressed in
part through an FOS strategy. In many cases, only a portion of a
natural structure is needed for sought-after function. Knowledge
of such structure–function dependencies can thus be used to
design simpler compounds with comparable or superior activities
that can be synthesized in a practical, step-economical fashion.
The above studies show that simplified bryostatin analogs can
be prepared that have potencies comparable or superior to the
natural product. Additionally, these agents exhibit PKC selectiv-
ities comparable or complementary to the natural product. The
syntheses of these analogs are up to 25 steps shorter than pub-
lished syntheses of the highly potent natural product congeners.
Although this gap will no doubt decrease as more step-economic-
al syntheses of bryostatins are reported, access to the simplified
analogs will also improve. Given the comparable potency of
bryostatin and the bryologs, the synthetic route to lead analog
1 involving 29 steps could now be used to supply clinical studies.
Importantly, these studies allow rapid access to designed, tunable
analogs and the opportunity to investigate their rich chemical
biology, selectivity in target activation, and therapeutic potential
as related to cancer, Alzheimer’s disease, and HIV eradication.
Fig. 6. (A and B) Normalized cytosolic fluorescence readings from transloca-
tion studies. Cells were dosed at 2.5 min. Decrease in cytosolic fluorescence
indicates PKC activation. (C–O) Representative confocal images from cellular
PKC-GFP translocation assays. (C–E, F–H, and L–N) Translocation of PKCβ1 by
200 nM 5, 6, or 4 at 0, 5, and 38 min postdose, respectively. (I–K) Translocation
of PKCδ by 200 nM 4 at the same time points. (O) Translocation of PKCβ1 by
200 nM bryo 1 following initial treatment with 200 nM 4.
and PKCβ1-GFP were measured in CHO-k1 cells. Analogs 4, 5,
and 6 induced significant activation of novel PKCδ (Fig. 6A) at
200 nM. Translocation of fluorescence from the cytosol was
immediately induced and was sustained over the 38-min obser-
vation period. The dominant localization of this isoform was the
cellular membrane, although some intracellular compartmenta-
lization was also observed (Fig. 6, I–K).
On the other hand, synthetic bryologs 4–6 differentially acti-
vated the conventional-PKCβ1 isoform. Most dramatically, in
contrast to its ability to activate PKCδ, C7-OH analog 4 at a
200-nM concentration induced only minimal translocation of
PKCβ1 (Fig. 6, B and L–N). Thus, at a therapeutically relevant
concentration, analog 4 shows functional selectivity for the novel
PKCδ isoform over the conventional-PKCβ1 isoform. Following
incubation with 4, translocation of PKCβ1 could be induced by
administration of 200 nM of bryostatin 1 (Fig. 6O), indicating
the presence of responsive kinase in these experiments.
Materials and Methods
Synthetic procedures and compound characterization data for all previously
undescribed compounds, along with experimental protocols for PKC affi-
nity, activation, translocation, and degradation assays, can be found in
SI Appendix.
ACKNOWLEDGMENTS. We thank Professor Tobias Meyer (Stanford University,
Stanford, CA) for the generous gift of PKC-GFP DNA plasmids and Professors
Daria Mochly-Rosen and Lynette Cegelski for support with biological
materials and equipment. Support of this work through a grant (CA31845)
provided by the National Institutes of Health is gratefully acknowledged.
In contrast, C7-OAc analog 5 and C7-deoxy analog 6 were able
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