Highly potent, synthetically accessible prostratin analogs induce latent HIV expression in vitro and ex vivo

Article abstract of DOI:10.1073/pnas.1302634110

Highly active antiretroviral therapy (HAART) decreases plasma viremia belowthe limits of detection in the majority of HIV-infected individuals, thus serving to slow disease progression. However, HAART targets only actively replicating virus and is unable

Full text of DOI:10.1073/pnas.1302634110

Highly potent, synthetically accessible prostratin
analogs induce latent HIV expression in vitro
and ex vivo
Elizabeth J. Beansa,b, Dennis Fournogerakisa,b, Carolyn Gauntletta,b, Lars V. Heumanna,b, Rainer Kramera,b
Matthew D. Marsdenc, Danielle Murrayd, Tae-Wook Chund, Jerome A. Zackc,e,1, and Paul A. Wendera,b,1
,
Departments of aChemistry and bChemical and Systems Biology, Stanford University, Stanford, CA 94305; cDepartment of Medicine, Division of Hematology
and Oncology, and eDepartment of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095; and dNational
Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
Edited by Malcolm A. Martin, National Institute of Allergy and Infectious Diseases, Bethesda, MD, and approved June 3, 2013 (received for review
February 8, 2013)
Highly active antiretroviral therapy (HAART) decreases plasma
viremia below the limits of detection in the majority of HIV-infected
individuals, thus serving to slow disease progression. However,
HAART targets only actively replicating virus and is unable to
eliminate latently infected, resting CD4+ T cells. Such infected cells
are potentially capable of reinitiating virus replication upon cessa-
tion of HAART, thus leading to viral rebound. Agents that would
eliminate these reservoirs, when used in combination with HAART,
could thus provide a strategy for the eradication of HIV. Prostratin
is a preclinical candidate that induces HIV expression from latently
infected CD4+ T cells, potentially leading to their elimination
through a virus-induced cytopathic effect or host anti-HIV immunity.
Here, we report the synthesis of a series of designed prostratin
analogs and report in vitro and ex vivo studies of their activity
relevant to induction of HIV expression. Members of this series
are up to 100-fold more potent than the preclinical lead (prostratin)
in binding to cell-free PKC, and in inducing HIV expression in a la-
tently infected cell line and prostratin-like modulation of cell surface
receptor expression in primary cells from HIV-negative donors. Sig-
nificantly, selected members were also tested for HIV induction in
resting CD4+ T cells isolated from infected individuals receiving
HAART and were found to exhibit potent induction activity. These
more potent agents and by extension related tunable analogs now
accessible through the studies described herein should facilitate
research and preclinical advancement of this strategy for HIV/
AIDS eradication.
harbors about 1 million such cells, which, with an estimated half-
life of 44 mo for elimination, would take over 70 y to be naturally
depleted (7).
Increasing research attention has been directed at the devel-
opment of strategies that would eliminate the latent viral reservoir,
which with concomitant HAART would provide for HIV eradi-
cation or a functional cure. For this approach, it has been pro-
posed that certain agents might be used in combination with
HAART to induce HIV activation and therefore depletion of
reservoir cells through a cytopathic effect associated with virus
production. It is also possible that a combination of inducers and
HIV-specific agents would provide a complementary or synergistic
strategy for clearance of infected cells (8, 9). Immunotoxins, for
example, consisting of an antibody to target the HIV envelope
protein on the surface of productively infected cells (10) and toxins
to kill the cell, represent one strategy to eliminate reservoirs more
effectively in conjunction with latency activating agents. Several
agents have been evaluated for their capacity to activate HIV from
latency, including general immune activators such as IL-2 and anti-
CD3 antibody, and histone deacetylase inhibitors (valproic acid).
Although promising, these inducing agents suffer from toxicity,
lack of potency, or both (11). Despite the advances over the last
decade in our understanding of factors contributing to HIV la-
tency, there has been a paucity of novel small-molecule agents that
potently induce reservoir clearance. Very recently, however, it was
demonstrated that administration of suberoylanilide hydroxamic
acid to HIV-infected subjects with undetectable viremia led to
induction of HIV expression in vivo (12). In addition, prostratin
and more recently bryostatin and its analogs have also emerged
as lead candidates in such studies directed at reservoir clearance
(13–15).
HIV latency NF-κB PKC-δ bryostatin
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IV/AIDS is a catastrophic pandemic (1). Over 34 million
Hpeople worldwide are living with HIV (2). Over 2.7 million
new infections were estimated for 2010. In the same year, 1.8
million infected individuals died of the disease. Current therapy
for the treatment of HIV involves administration of a combination
of antiretroviral agents, collectively referred to as highly active
antiretroviral therapy (HAART), which serves to reduce HIV
plasma viremia of many infected individuals to undetectable lev-
els, thereby slowing disease progression (3). HAART, however, is
not curative. Reservoirs of HIV-infected cells persist in infected
individuals receiving HAART despite years to decades of therapy.
As a result, interruption of HAART could potentially lead to
plasma viral rebound that putatively is supplied by the latent
provirus in infected CD4+ T cells and other persistent HIV res-
ervoirs (4, 5). As such, HAART must be used chronically, leading
to concerns regarding side effects, compliance, cost, and the
generation of viral resistance to the drugs.
The natural product prostratin 3 has figured prominently in
studies directed at purging latently infected, resting CD4+ T cells
and is currently in preclinical development (15, 16). Although
prostratin was originally isolated from Pimelea prostrata by Hecker
and coworkers in the 1970s (17), interest in its therapeutic po-
tential was intensified in the early 1990s when it was found by Cox
and a team of National Institutes of Health scientists to be the
active component of the Samoan medicinal plant Homalanthus
nutans (18). Prostratin is thought to elicit its biological effects
Author contributions: E.J.B, D.F., C.G., L.V.H., R.K., M.D.M., J.A.Z., and P.A.W. designed
research; .E.J.B., D.F., C.G., L.V.H., R.K., M.D.M., and D.M. performed research; E.J.B., D.F.,
C.G., L.V.H., R.K., M.D.M., D.M., T.-W.C., J.A.Z., and P.A.W. analyzed data; and E.J.B., D.F.,
M.D.M., D.M., T.-W.C., J.A.Z., and P.A.W. wrote the paper.
The authors declare no conflict of interest.
The most extensively studied persistent HIV reservoirs identi-
fied to date are latently infected, resting memory CD4+ T cells (6).
These long-lived cells are activated when presented with a specific
antigen or by cytokine stimulation, leading to concomitant bursts
of viral production. It is estimated that an HIV-positive individual
This article is a PNAS Direct Submission.
1To whom correspondence may be addressed. E-mail: wenderp@stanford.edu or jzack@
ucla.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1302634110/-/DCSupplemental.
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consistent with the observation that 12-deoxyphorbol-13-phenyl-
acetate (DPP) (4) is 10-fold more potent than prostratin in PKC
binding assays (19), 20–40 times more potent at inducing viral
production in latently infected cell lines (30), and at least five
times more potent at inducing viral expression from human pe-
ripheral blood mononuclear cells (huPBMCs) from HAART-
suppressed patients (31). With this understanding, we sought to
investigate whether modifications at C13 could be used to achieve
more potent analogs with better pharmacological properties. To
achieve this goal, we designed a diversification strategy in which
a readily available precursor can be chemoselectively derivatized
at C13 and the resultant product deprotected in one step to
rapidly produce analogs. Overall, this now successful strategy
provides unique access to a wide range of analogs for research and
clinical advancement from a readily available and renewable
source, i.e., phorbol derived from croton oil.
Our synthetic route to this diversifiable precursor is given in Fig.
2. Previous work in our group addressed the problem of prostratin
supply by converting phorbol (5), a relatively abundant natural
product, to prostratin in five synthetic steps (28). Drawing on this
strategy, the synthesis described herein is designed to furnish a
stable late-stage intermediate containing the desired free C13
hydroxyl (10), which can be converted to analogs in only two steps
(Fig. 2). In this sequence, phorbol is subjected to acid hydrolysis,
which effectively removes the undesired C12 hydroxyl group, and
cleaves the cyclopropane ring to afford crotophorbolone (6). The
isopropenyl moiety resulting from the opening of the D-ring
provides the needed functional handle to reclose this ring. At this
point, the C20 hydroxyl group of crotophorbolone is converted to
a trityl ether, thereby serving to remove the more reactive C20
alcohol from competing with the C13 hydroxyl group for de-
rivatization. Tritylation also renders compounds in the synthetic
route soluble in a broader variety of solvents than the parent
alcohols. Treatment of the ketone (7) with hydrazine hydrate
forms a hydrazone, which upon heating with diisopropylethyl
amine in toluene cyclizes to an air-sensitive pyrazoline. After
cyclization is complete, the reaction mixture is cooled to −78 °C
and treated with a solution of lead tetraacetate in dichloro-
methane to afford the oxidized cyclic diazene (8) in an isolated
yield of up to 59% over two steps from trityl-crotophorbolone. The
use of toluene in combination with diisopropylethyl amine instead
of pyridine allows the cyclization and oxidation reactions to be
conducted in one flask, thus shortening the synthetic route. Dia-
zene (8) is then photolyzed to extrude nitrogen, providing C20-
O-trityl-prostratin (9). Saponification of the C13 acetate moiety
using barium hydroxide in methanol reveals the desired C13 al-
cohol (10), the diversification point for analog synthesis. Steglich
Fig. 1. Pharmacophore model highlighting triad of oxygenation proposed
for binding to the PKC-C1 regulatory domain in diacylglycerol (1), phorbol-
12-myristate-13-acetate (2), prostratin (3; R = CH3), and DPP (4; R = CH2Ph).
wholly or in part by binding to the diacylglycerol binding domain of
protein kinase C (PKC), leading to its activation, translocation to
cellular membranes, and downstream signaling (19). Unlike the
structurally related phorbol esters, prostratin is not a tumor pro-
moter or an irritant, and furthermore, it protects against the
tumor-promoting effects of these agents (20, 21). In addition to
having selective inhibitory activity against specific cancer cell
lines (22), prostratin has been found to have unique activity
against HIV.
Prostratin had been shown to elicit three distinct effects relevant
to HIV treatment. It causes down-regulation of CD4, C-X-C che-
mokine receptor type 4 (CXCR4), and in some cases C-C che-
mokine receptor type 5 (CCR5), thereby protecting CD4+ T cells
from HIV-1 entry (23, 24). Other cell surface receptors, such as the
early activation marker CD69, are up-regulated, whereas the late
activation marker CD25 is little affected by prostratin treatment
(25). In acutely infected cells, prostratin enhances cell survival
possibly due to cytostatic effects (18). Most importantly, in latently
infected cells, prostratin stimulates viral replication putatively
through PKC-mediated phosphorylation of IκB kinase, which
enables release and penetration of the transcription factor NF-κB
into the cell nucleus (26), where its binding to the HIV LTR leads
to viral gene expression and subsequent replication of the virus.
Until recently, research on prostratin has relied exclusively on
plant sources that produce prostratin but generally in only vari-
able and low isolation yields (27). In 2008, we reported a step-
economical (five steps) synthesis of prostratin from phorbol 5,
a readily available constituent of croton oil (28). This synthesis not
only provides a reliable and scalable supply of prostratin but it also
allows access to derivatives including nonnatural analogs needed
to establish the structural basis for its activity and thereby to
identify superior candidates. Herein, we describe an adaptation of
the reported synthesis designed to rapidly access prostratin ana-
logs and disclose the biological activities of these agents pertinent
to latency induction, including PKC binding, modulation of cell
surface receptor levels in cells from healthy donors, and induction
of latent virus in a model cell line and in cells obtained from
patients on suppressive therapy. These agents outperform pros-
tratin, with some being 100-fold more potent than the current lead
clinical candidate.
Results and Discussion
Designed Analog Synthesis. Previously, we proposed that the PKC
affinities exhibited by tigliane natural products (e.g., phorbol
esters) could be attributed to a subset of hydrogen bond donors
and acceptors, with priority given to the oxygens at C20, C3/C4,
and C9, which colocate spatially with similar donors and accept-
ors in the endogenous ligand, diacylglycerol (1) (Fig. 1) (29). In
this model, the lipids of the CD-ring system are thought to in-
fluence membrane insertion (both depth and orientation) of the
bound PKC–ligand complex and thus indirectly the conformation,
affinity, and catalytic function of the complex. This analysis is
Fig. 2. Synthesis of C20-O-trityl-prostratin-ol (10). Reagents and con-
ditions: (a) H2SO4, H2O, 90 °C, 40% yield; (b) trityl chloride, pyridine, 50 °C,
94–96% yield; (c) N2H4•H2O, AcOH, MeOH; (d) iPr2NEt, toluene, 140 °C,
then Pb(OAc)4, CH2Cl2, −78 °C → room temperature, 50–59% yield over
two steps; (e) hν (350 nM), EtOAc, 64% yield; and (f) Ba(OH)2•8H2O, MeOH,
89% yield.
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C13 ester introduction. Additionally, this diversification sequence
requires only two simple steps (selective C13 esterification and
C20 hydrolysis) from the diversification point 10, whereas the
original sequence involved a more restrictive introduction of C13
through oxidative formation of a C13–O bond.
PKC Binding Affinity. Given that PKC is proposed to mediate latent
virus activation, we evaluated the PKC affinities of the analogs
relative to the lead compounds, prostratin and DPP, by testing for
binding to PKC-δ (Table 1) using a standard radiolabeled ligand
displacement assay. In this assay, prostratin and DPP exhibit PKC
binding affinities of 21.8 and 3.1 nM, respectively, consistent with
previous reports (19). Significantly, each analog bound PKC-δ with
a higher affinity than either prostratin or DPP, all exhibiting Ki
values of less than 2 nM. Two analogs (11b and 11c) showed
a binding affinity in the picomolar potency range, corresponding to
a 33-fold increase in potency compared with the current clinical
lead, prostratin. A correlation between PKC-δ binding affinity and
cLogP is apparent, with maximal binding affinity corresponding to
a cLogP range of 3.47 and 3.75. As prostratin operates via a PKC-
dependent pathway for the induction of the latent virus, these
studies show that variation in the C13 position can be used to
improve potency while providing options for controlling metabo-
lism and biodistribution.
Latent HIV Induction. Having scalable and tunable access now to
analogs that possess higher affinities for PKC than prostratin and
DPP, we next evaluated whether they could induce latent HIV in
vitro. For this purpose, the latently infected U1 cell line, a de-
rivative of the promonocytic U937 cell line was selected. These
cells harbor two copies of HIV provirus with a TAT defect that
prevents viral production as the cells replicate. Upon treatment
with prostratin or other inducing agents, these cells produce new
virions, which can be quantified by an ELISA for the viral p24 gag
protein. Similar to exposure to prostratin, when U1 cells are
treated with the analogs, significant levels of p24 production are
observed after a 48-h incubation (SI Appendix, Fig. S1). All of our
compounds produced p24 with an EC50 in the nanomolar to
single-digit nanomolar range (Table 1). In agreement with the
PKC binding assay, all of our compounds outperformed pros-
tratin and DPP in the U1 cell assay, with our most potent com-
pounds being over 20-fold more potent than DPP and greater
than 130-fold more potent than the clinical lead, prostratin.
These findings demonstrate that increased PKC binding affinity
correlates with increased viral induction. These studies further
show that unnatural prostratin analogs can activate latent HIV
Fig. 3. Summary of analogs synthesized.
esterification with various carboxylic acids and subsequent C20
deprotection affords a range of prostratin analogs in two steps
from the diversification point 10. The structures produced in this
study are summarized in Fig. 3. It is worth noting that the current
sequence introduces the photolytic extrusion of nitrogen before
esterification of C13, thereby avoiding problems that could arise
with photolabile C13 esters if photoextrusion were done after
Table 1. Summary of biological activities for the natural products and analogs
Analog
Side chain
PKC-δ Ki, nM*
U1 EC50, nM†
CD69 EC50, nM‡
cLogP§
Prostratin
DPP
11a
11b
11c
11d
11e
11f
11g
Acetate
Phenyl acetate
Cyclohexyl acetate
Pentafluorophenyl acetate
1-Naphthyl acetate
2-Naphthyl acetate
(5,6,7,8)Tetrahydro-1-naphthyl acetate
Biphenyl acetate
Adamantyl acetate
p-Benzyl phenyl acetate
21.8 (13.6–35.1)
3.1 (1.1–8.7)
1.2 (0.77–1.9)
0.61 0.03{
0.65 (0.39–1.1)
1.2 (0.63–2.2)
1.3 (0.7–2.7)
1.75 (0.87–3.5)
1.5 (0.82–3.1)
1.8 (0.88–3.7)
>1,000
163.2 (122.5–217.6)
127.4 (94.1–172.5)
19.5 (11.4–33.6)
10.2 (4.8–21.9)
7.3 (3-17.9)
25.6 (10-60)
9.0 (3.5–23)
13.1 (4.8–35.4)
5.8 (3.3–10)
398.7
33.8
33.2
32.5
3.2
3.9
ND
26.8
24.3
ND
1.20
2.66
3.40
3.47
3.75
3.75
4.00
4.23
4.29
4.44
11h
*PKC-δ affinities from competitive binding assay using recombinant human protein; results are from a single triplicate dilution
experiment, and values in parentheses indicate 95% confidence intervals unless otherwise noted.
†U1 EC50 from triplicate stimulation studies incubating cells with compound for 48 h, p24 levels in culture supernatants read by ELISA;
values in parentheses indicate 95% confidence intervals.
‡CD69 induction tested in primary resting CD4+ T cells from HIV-negative donors and analyzed by flow cytometry.
§cLogP as calculated using VCCLAB: Virtual Computational Chemistry Laboratory (32, 33).
{Average of two runs; error is reported as SEM.
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with potencies that significantly exceed the natural system and
clinical lead.
the binding data, our analogs induced the expression of CD69 with
one to two orders of magnitude greater potency than did pros-
tratin (Fig. 4). Importantly, our compounds induce CD69 activa-
tion in greater than 90% of the cells tested after only a single dose,
suggesting that prolonged exposure or repeated dosing could ef-
fect activation of all cells harboring latent virus as preferred for an
eradication strategy.
Cell Surface Receptors Expression. Given the encouraging PKC af-
finities and U1 cell performance of our analogs, attention was next
turned to the performance of these compounds in primary CD4+ T
cells from healthy individuals. Prostratin is proposed to work via
an NF-κB–related pathway; therefore, it was of interest to know
whether our compounds could activate NF-κB in CD4+ T cells.
We selected the cell surface receptor CD69 for study as a surro-
gate marker for NF-κB activation, given that CD69 has three
known NF-κB binding sites in its promoter region (34), and is up-
regulated when CD4+ T cells are stimulated with prostratin (25).
Primary resting CD4+ cells were isolated from HIV-negative
donors, exposed to our compounds for 48 h, and evaluated for cell
Following demonstration of CD69 up-regulation on CD4+
T
cells, we then examined the effect of our compounds on other
activation markers to determine whether the activation is selec-
tive. Importantly, although our compounds induce expression of
CD69, an early activation marker, they do not induce the high
expression levels of the late activation marker CD25 that is a
characteristic of costimulated T cells, suggesting that our com-
pounds generate lower overall levels of cellular activation than
surface expression of CD69 by flow cytometery. As expected from costimulation does (Fig. 4).
Fig. 4. Treatment of resting CD4+ T cells with costimulation (via bead-based antibody ligation of CD3 and CD28), natural products, or analogs. Results are
from a single experiment and are representative of two to four similar experiments.
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Treatment of uninfected CD4+ T cells with prostratin can
reduce HIV infection by transcriptional and/or translational
down-regulation of CD4 and other cell surface receptors used by
HIV to enter the cell (23, 24). To further assess the prostratin-
like activity of our compounds on the expression of cell surface
markers, we examined the level of CD4 expression on resting
CD4+ T cells. Supporting the other trends that we have ob-
served, our compounds caused the down-regulation of CD4 and
did so at concentrations of 10–100 times lower than prostratin
(data shown in SI Appendix, Fig. S2). Based on the three cell
surface markers observed in resting CD4+ T cells from healthy
donors, our compounds display prostratin-like activity, but with
superior potency.
11f induced high levels of viral expression at 50 nM, whereas
50 nM prostratin was indistinguishable from the control. Pros-
tratin, although active at 500 nM, required a 5 μM dose to induce
viral expression to the same extent as 11c at 50 nM. Encouraged
by these results, we examined 11c in resting CD4+ T cells isolated
from multiple patients, and found that it demonstrated consis-
tently high levels of HIV production across the board. Variability
from patient to patient could either be due to differential sensitivity
to our compounds or in varying number of latently infected cells
that could give rise to HIV particles. This finding is highly sig-
nificant because it shows that the activity of these compounds ob-
served in immortal cell line models of HIV latency translates to
potent activation from latently infected primary cells from HIV-
positive donors.
HIV Induction in Resting CD4+ T Cells from Infected Individuals Receiving
HAART. Given the positive results obtained in cell lines mimicking
HIV latency and CD4+ T cells obtained from healthy donors, our
next focus was the immunologic and virologic effects of our
compounds on CD4+ T cells from infected individuals receiving
HAART. Resting CD4+ T cells were isolated from the blood of
HIV-infected individuals who were on suppressive HAART, and
treated with either analog 11c or 11f. The cells were evaluated for
induction of virion-associated HIV RNA at 2 and 4 d following
treatment. Both compounds were exceptionally potent in this as-
say, with 11c exhibiting high levels of virion production at both
time points at 50 nM (Fig. 5). Although compound 11f induced
lower overall levels of viral RNA expression, at day 2, viral in-
duction was clearly evident even at 5 nM concentration. In a side-
by-side comparison with the clinical lead, prostratin, both 11c and
Conclusion
Prostratin represents a clinical lead for HIV eradication, an as-yet-
unachieved goal. Here, we report a highly efficient, scalable, and
diversifiable synthetic route to prostratin analogs. These analogs
exhibit superior potency over prostratin, the parent compound and
current clinical lead for an eradication strategy. Based on a re-
newable resource, croton oil, from which phorbol is readily ob-
tained, the synthetic route developed allows for great variation in
accessing C13 analogs, requiring only two simple and selective
steps (esterification and hydrolysis) from an advanced interme-
diate. The analogs prepared in this way exhibited higher affinities
to a representative PKC isoform and mode of action target than
does prostratin. They induced expression of HIV in a latently
Fig. 5. Summary of viral induction from huPBMC from fully suppressed HIV-positive donors. (A) Amount of viral induction after 2 d of treatment with
compounds 11c and 11f at four different concentrations. (B) Amount of viral induction after 4 d of treatment with compounds 11c and 11f at four different
concentrations. (C) Amount of viral induction after 3 d of exposure to compound 11c (50 nM) in samples from six different patients. (D and E) Amount of viral
induction after 3 d of exposure to compound 11c (50 nM), 11f (50 nM), and prostratin (multiple doses).
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infected model cell line, again with potencies better than pros- Materials and Methods
tratin. When treated with our compounds, resting CD4+ T cells
Additional supplemental figures, synthetic procedures and characterization
from healthy donors consistently showed the same patterns of cell
surface marker modulation as is elicited by prostratin, but with up
to 100-fold greater potency. Significantly, a lead analog was also
found to potently induce the expression of latent virus from
PBMCs isolated from HIV-infected individuals receiving HAART
at concentrations in which prostratin elicits no effect. This study
provides a versatile synthetic route to analogs of the clinical lead
prostratin and several analogs with activities superior to the cur-
rent clinical lead. These analogs, their promising activities, and the
potential of this synthesis to access related systems as needed to
evaluate preclinical performance open a broad range of oppor-
tunities for research and for the advancement of this approach to
HIV eradication.
data for compounds along with protocols for PKC-δ binding affinity, U1 cell
stimulation, surface receptor expression analysis in primary CD4+ T cells, and
HIV induction in CD4+ T cells from infected individuals receiving HAART can
be found in SI Appendix.
ACKNOWLEDGMENTS. We thank Prof. Lynette Cegelski for support with
biological materials and equipment. Support of this work through National
Institutes of Health Grants AI70010 (to J.A.Z.) and R01 CA031841 and R01
CA031845 (to P.A.W.), and University of California, Los Angeles, Center for
AIDS Research Grant AI28697 is gratefully acknowledged. This research was
also supported in part by the Intramural Research Program of the National
Institute Allergy and Infectious Diseases. Further support through fellow-
ships by Novartis (to E.J.B.), Fulbright Commission (to C.G.), American Cancer
Society (to L.V.H.), and Alexander von Humboldt Foundation (to R.K.) is
gratefully acknowledged.
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