Designed, synthetically accessible bryostatin analogues potently induce activation of latent HIV reservoirs in vitro

Article abstract of DOI:10.1038/nchem.1395

Bryostatin is a unique lead in the development of potentially transformative therapies for cancer, Alzheimer's disease and the eradication of HIV/AIDS. However, the clinical use of bryostatin has been hampered by its limited supply, difficulties in access

Full text of DOI:10.1038/nchem.1395

ARTICLES
PUBLISHED ONLINE: 15 JULY 2012 | DOI: 10.1038/NCHEM.1395
Designed, synthetically accessible bryostatin
analogues potently induce activation of latent
HIV reservoirs in vitro
1
1
2
1
2
†
Brian A. DeChristopher †, Brian A. Loy †, Matthew D. Marsden , Adam J. Schrier †, Jerome A. Zack
*
and Paul A. Wender1
*
Bryostatin is a unique lead in the development of potentially transformative therapies for cancer, Alzheimer’s disease and
the eradication of HIV/AIDS. However, the clinical use of bryostatin has been hampered by its limited supply, difficulties
in accessing clinically relevant derivatives, and side effects. Here, we address these problems through the step-economical
syntheses of seven members of a new family of designed bryostatin analogues using a highly convergent Prins-
macrocyclization strategy. We also demonstrate for the first time that such analogues effectively induce latent HIV
activation in vitro with potencies similar to or better than bryostatin. Significantly, these analogues are up to 1,000-fold
more potent in inducing latent HIV expression than prostratin, the current clinical candidate for latent virus induction. This
study provides the first demonstration that designed, synthetically accessible bryostatin analogues could serve as superior
candidates for the eradication of HIV/AIDS through induction of latent viral reservoirs in conjunction with current
antiretroviral therapy.
IV/AIDS is a global pandemic1. The Joint United Nations depletion and provide a strategy to eradicate HIV when used in
Programme on HIV/AIDS has estimated that the combination with HAART. In this approach, which is now being
H
number of people living with HIV, the virus that causes actively pursued11, the deliberate induction of viral replication
AIDS, totalled 33.3 million in 2009 (see http://unaids. from its latent state is proposed to eliminate HIV-harbouring cells
org/globalreport/Global_report.htm). In the same year, there either by direct viral cytopathic effects or by rendering those cells
were 1.8 million AIDS-related deaths.
susceptible to immune system regulation. Concomitant HAART
The leading treatment for HIV is highly active antiretroviral would prevent infection of healthy cells from the released virions.
therapy (HAART), a combination of drugs that halts viral prolifer- This combined approach would thus eliminate both the latent
ation by inhibiting several stages of the viral life cycle. For many and active viral pools.
patients, this treatment strategy has transformed HIV into a man-
One experimental strategy for latent viral reactivation is direct
ageable, chronic disease by reducing their plasma viral loads, immunological modulation of resting memory T cells. However,
often to undetectable levels. However, HAART is not curative, as administration of cytokines and/or antibodies has thus far lacked
it does not address genomically integrated latent viral reservoirs clinical efficacy12,13. Another approach makes use of the pharmaco-
that slowly resupply replication-competent active virus. As such, logical modulation of the signalling pathways associated with viral
discontinuation of HAART results in viral rebound and disease pro- reactivation. To this end, protein kinase C (PKC) has emerged as
gression2. In addition, HAART is costly, has associated side effects3, an important target. Prostratin (Fig. 1), a non-tumour-promoting
and requires strict adherence to treatment regimens4 to avoid the 12-deoxy phorbol ester that binds to and activates PKC, has demon-
emergence of viral resistance5.
strated promising in vitro and ex vivo activities in reactivating latent
The most formidable obstacle to the eradication of HIV is the HIV14,15 and is currently being advanced as a clinical candidate for
persistence of various latent proviral reservoirs6–8. These are believed latent viral reservoir clearance16. A source of synthetic prostratin has
to be primarily established following integration of the HIV genome recently been reported that also provides access to even more effec-
into that of activated CD4þ T cells and other cell types. In rare cases, tive analogues17.
these infected cells transition to quiescent memory cells in a process
Like prostratin, bryostatin 1 also targets PKC, but it is signifi-
that reversibly inhibits expression of the integrated HIV provirus, cantly more potent (Ki ¼ 1.35 nM versus 50 nM) and thus rep-
rendering it unsusceptible to HAART9. As a consequence, eradica- resents a unique lead for HIV reservoir clearance. Bryostatin 1,
tion of HIV in HAART-suppressed patients would require elimin- the lead member of the bryostatin family, was reported in 1982 by
ation or inactivation of these proviral reservoirs.
Pettit and Clardy18, and has subsequently been shown to demon-
As HAART targets only actively replicating virus, it has little strate promising in vitro, in vivo and human clinical19 activities
influence on latent viral reservoirs. It is estimated that decades of (see http://clinicaltrials.gov) for a diverse array of indications20
HAART treatment would be required for depletion of the reservoir including cancer and Alzheimer’s disease21. Early reports also
source10. Therefore, agents that can controllably facilitate purging of suggested that bryostatin might induce HIV reservoir clearance22,23
,
the latent virus from these reservoirs could reduce the time for and this has been further supported in more recent studies24. An
1Departments of Chemistry and of Chemical and Systems Biology, Stanford University, Stanford, California, 94305, USA, 2Department of Medicine, Division
of Hematology and Oncology, University of California Los Angeles, Los Angeles, California, 90095, USA; †These authors contributed equally to this work.
e-mail: wenderp@stanford.edu; jzack@ucla.edu
*
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.1395
ARTICLES
steps, are currently the most synthetically accessible agents with
bryostatin-like activity, PKC translocation selectivity43 and
potency. Moreover, a related bryostatin analogue has been shown
to be well tolerated and efficacious in an in vivo mouse cancer
model44. To date, there has been no reported study of simplified
bryostatin analogues for the activation of latent HIV reservoirs as
required for disease eradication. Here, we report the convergent
syntheses of seven members (1–7) of a new family of bryostatin
HO
OAc
O
MeO2C
O
O
O
O
OH HO
O
H
O
H
OH
O
OH
analogues incorporating
a tetrahydropyranyl B-ring formed
OH
OH
O
through a versatile Prins macrocyclization. We also disclose for
the first time that such designed, synthetically accessible analogues
can induce activation of latent HIV expression in a cellular
latency model with potencies similar to or better than bryostatin
1. We also demonstrate that these simplified bryostatin analogues
share the functional activity of the clinical candidate prostratin in
this assay, but are up to four orders of magnitude more potent
than prostratin. These findings establish that bryologs, in addition
to their therapeutic potential for cancer and Alzheimer’s disease45,
could serve as synthetically accessible and potentially superior
clinical candidates as adjuvants with HAART for the eradication
of HIV infection.
Pr
O
CO2Me
Bryostatin 1
Prostratin
Y
Y
X
X
MeO2C
O
O
O
O
O
O
O
O
OH HO
O
OH HO
O
O
O
OH
OH
Results
C7H15
O
CO2Me
C7H15
O
CO2Me
The assembly of the new bryostatin analogues 1–7 was proposed to
entail a highly convergent strategy in which a spacer domain (Fig. 2:
8, 9 or 10) would be conjoined with a recognition domain (11)
through a two-step esterification/Prins-driven macrocyclization46
sequence. This process would form a readily diversifiable B-ring
pyran and set C15 stereochemistry to produce the key intermediates
20–22. Support for this strategy comes from our earlier work on the
1 X = Y = H
2 X = OH, Y = H
3 X = OAc, Y = H
5 X = Y = H
6 X = OAc, Y = H
7 X = OAc, Y = OH
4 X = OAc, Y = OH
Figure 1 | Bryostatin, prostratin and synthetic analogues.
attractive aspect of bryostatin is that it has already been tested in syntheses of simplified bryostatin analogues41 as well as bryostatin 9
human trials, albeit for other cancer-related indications. However, (ref. 33). As the name implies, the recognition domain is proposed
further underscoring the need for a reliable supply of a more effec- to directly contact the PKC, thereby influencing analogue–PKC affi-
tive agent, patient accrual in a recent bryostatin clinical trial was ter- nity, while the spacer domain is proposed to control bryolog confor-
minated by the National Cancer Institute and the clinical mation and to influence translocation of the bryolog/PKC complex
investigators ‘given the more potent bryostatin analogs in develop- and its insertion into the cellular membrane. The preparation of
ment’, a focus of this current study25.
analogues 1–7 was proposed to start from a common A-ring inter-
Unlike prostratin and its analogues, for which a practical five- mediate 12, which was prepared and elaborated as described pre-
step synthesis has been reported17, bryostatin has not been reliably viously42 into hydroxyesters 13 and 14. Hydroxyester 13 would be
available in the quantities needed for research and sustained clinical used to synthesize the C7-deoxy analogues 1 and 5, while hydroxy-
studies. Isolated yields from natural sources are low (1023 to 1028%) ester 14 would be used to prepare the C7-oxy analogues 2, 3 and 6.
and variable; the good manufacturing practices (GMP) production Analogues 4 and 7, which have bryostatin 1-like A-ring functiona-
required 14 tons of the marine bryozoan Bugula neritina to lization, would be prepared from spacer domain 10 (ref. 33).
provide just 18 g of bryostatin 1 (ref. 26). Although this supply
Our studies started with previously prepared hydroxyesters 13
was sufficient to initiate preclinical and clinical research on bryo- and 14 (ref. 42), which were converted into allylsilanes 15 and 16,
.
statin, economic and environmental factors have severely limited respectively, in three steps involving C11 silylation, CeCl3 2LiCl-
further development of this source and related aquaculture pro- mediated47 double nucleophilic addition of TMSCH2MgCl,
duction. Engineered biosynthesis, while promising, is still in devel- and Peterson olefination of the carbinol product. Knochel’s
opment and has yet to have an impact on clinical supply27. CeCl3 2LiCl salt provided optimal yields for the nucleophilic
.
Impressive progress has also recently been made in the realm of addition step33. Intermediates 15 and 16 were then separately deben-
bryostatin total synthesis28–34, but approaches to the potent bryosta- zylated with lithium naphthalenide (84–91% yield), and the resul-
tins require, at best, a total of ꢀ40 steps. Importantly, clinical studies tant C1 alcohols oxidized with TPAP/NMO followed by NaClO2
have also revealed off-target toxicities associated with bryostatin. In to provide spacer domains 8 and 9 (91–97% yield over two steps).
principle, issues of both supply and undesirable effects could be
Spacer domains 8, 9 and 10 were then each esterified with recog-
overcome by the design of simplified, clinically superior, functional nition domain 11 using Yamaguchi’s esterification to provide the
analogues that can be produced in a more step-economical fashion. Prins macrocyclization precursors 17, 18 and 19, respectively.
To address these supply and performance issues, our group pro- Significantly, we found that C11 deprotection and Prins-driven
posed in 1988 that the activities of bryostatin could arise from only a macrocyclization of 17, 18 and 19 can be promoted in one operation
subset of its functionality35. As such, bryostatin-like activity could be and in excellent yields (up to 90%) using mild conditions (catalytic
achieved with simplified and thus more synthetically accessible PPTS in alcohol solvent), providing macrocycles 20, 21 and 22,
designed analogues dubbed ‘bryologs’, which incorporate the key respectively. Importantly, only a single diastereomer is obtained in
activity-determining functionalities36,37
. Using this function- each case. The Prins-driven macrocyclizations of 17 and 18 pro-
oriented synthesis38 approach, we reported in 1998 the first simpli- ceeded rapidly with PPTS/EtOH without C26 desilylation.
fied bryostatin analogues39 and, more recently, similar bryologs that Macrocyclization of 19 with PPTS/MeOH occurred less rapidly
are even more potent than the natural product40–42. Significantly, giving macrocycle 22 and some C26-desilylated product; this
these analogues, several of which were prepared with less than 30 crude mixture of macrocycles was either further deprotected to
2
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.1395
ARTICLES
Spacer domain synthesis
X
EtO 13
O
X
O
OH
Me3Si
4–5 steps
Ref. 42
a, b, c
11
OTES O
11
9
O
7
7
A
OH
O
1
OBn
OBn
12 (7 steps)
13 X = H
15 X = H
TBDPSO
OBn
14 X = OTBS
16 X = OTBS
OTBDPS
OTBDPS
Spacer domains
Y
X
Me3Si
7
d, e, f
OTES O
1
O
TBDPSO
OH
8
9
X = Y = H
X = OTBS, Y = H
10 X = OAc, Y = OMe (ref. 33)
Fragment coupling and Prins cyclization
Y
X
Y
11
O
X
Me3Si
11
g
OTES O
8–10
O
15
OTBDPS
O
h
O
H
O
Recognition domain
OH
O
TBDPSO
OH
O
O
O
H
O
O
26
O
OTBS
OH
O
O
OTBS
C7H15
CO2Me
O
OH
C7H15
O
CO2Me
20 X = Y = H
(86%)
O
OTBS
21 X = OTBS, Y = H
22 X = OAc, Y = OMe
(90%)
17 X = Y = H
(88%)
(81%)
(83%)
(73% over 2)
C7H15
CO2Me
18 X = OTBS, Y = H
19 X = OAc, Y = OMe
11
Y
X
9
7
O
O
O
1
2
4
X = Y = H
(81%)
i
O
X = OH, Y = H
X = OAc, Y = OH
(80%)
(51% from 19)
OH HO
O
j, k
O
(53%)
3
X = OAc, Y = H
OH
C7H15
O
CO2Me
Figure 2 | Synthesis of analogues 1–4 via Prins-driven macrocyclization. Reagents and conditions. a–f, Spacer domain synthesis. When X ¼ H, TESCl,
.
imidazole, CH2Cl2, 95% (a); CeCl3 2LiCl, TMSCH2MgCl, THF (b); silica gel, CH2Cl2, 85% over 2 steps (c); lithium naphthalenide, THF, 84% (d); TPAP
(10 mol%), NMO, 4 Å MS, CH2Cl2 (e); NaClO2, NaH2PO4, 2-methyl-2-butene, 2:1 t-BuOH:H2O, 91% over 2 steps (f). When X ¼ OTBS, conditions as before:
.99% (a); 82% over 2 steps (b,c); 91% (d); 97% over 2 steps (e,f). g, Fragment coupling: 2,4,6-trichlorobenzoyl chloride, Et3N, then DMAP, recognition
domain 11, PhCH3, rt. h, Prins macrocyclization: from 17 and 18, PPTS, EtOH, rt; from 19, i. PPTS, MeOH, rt, ii. TBSCl, imidazole, CH2Cl2. i–k, Analogue
.
.
synthesis: from 20 and 21, HFpyridine, THF, rt; from 19, i. PPTS, MeOH, rt, ii. HFpyridine, THF, rt, iii. PPTS, 4:1 THF:H2O (i); i. TESCl, imidazole, CH2Cl2,
.
ii. Ac2O, DMAP, pyridine, CH2Cl2 (j); HFpyridine, THF (k).
directly provide analogue 4 (51% over three steps) or reprotected to
Stoichiometric ozonolysis of the C13-methylidene subunits of
provide intermediate 22 (73% overall yield) en route to analogue 7. bryopyrans 20, 21 and 22 followed by Horner–Wadsworth–
Analogues 1 and 2 were prepared by deprotection of macrocycles 20 Emmons olefination afforded intermediates 26, 27 and 28, respect-
and 21, respectively. The C7-OH analogue 2 was converted into the ively (Fig. 3). Although we have shown that reagent-controlled
C7-OAc analogue 3 by C26 silylation followed by C7 acylation olefination can afford greater selectivity for the Z-enoate33, prep-
and desilylation.
aration of both E- and Z-enoate isomers was required for ongoing
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NATURE CHEMISTRY DOI: 10.1038/NCHEM.1395
ARTICLES
Y
(Z)
X
MeO2C
Y
Y
O
X
X
O
O
O
MeO2C
O
O
O
O
O
O
O
OTBDPS
O
OH HO
O
O
O
O
a
b
c
20, 21
OH
O
OH HO
O
or 22
O
O
OH
OTBS
OTBS
C7H15
O
CO2Me
C7H15
CO2Me
C7H15
26 X = Y = H
O
CO2Me
5
X = Y = H
(79% comb.; 38% 5, 19% E-isomer)
29 X = OH, Y = H
23 X = Y = H
(81%)
(86%, 40:60 E:Z)
24 X = OTBS, Y = H (58%)
25 X = OAc, Y = OMe (77%)
27 X = OTBS, Y = H (66%, 37:63 E:Z)
28 X = OAc, Y = OMe (91%, 50:50 E:Z)
(84% comb.; 32% 29, 15% E-isomer)
X = OAc, Y = OH
(29% 7, 27% E-isomer)
X = OAc, Y = H (68%)
d, e
7
6
Figure 3 | Synthesis of analogues 5–7 via ozonolysis followed by Horner–Wadsworth–Emmons olefination. Reagents and conditions. a, O3, CH2Cl2, –78 8C,
.
.
then thiourea, MeOH:CH2Cl2. b, Trimethyl phosphonoacetate, NaHMDS, THF, 0 ꢁ 4 8C. c, From 26 or 27: HFpyridine, THF, rt; from 28: i. HFpyridine, THF,
.
rt, then ii. PPTS, 4:1 THF:H2O, rt. d, i. TESCl, imidazole, CH2Cl2, ii. Ac2O, DMAP, pyridine, CH2Cl2. e, HFpyridine, THF.
comparative activity studies. In this study, the activities of only the of particular agents to activate HIV from post-integration
Z-enoates are reported (vide infra). Deprotection of intermediates latency48. These models typically employ stable cell lines harbouring
26–28 followed by reverse-phase HPLC purification afforded an integrated, yet transcriptionally silent, partial HIV genome.
enoate analogues 5 and 7 and intermediate 29 en route to analogue On treatment with an appropriate agent, viral reactivation is
6. The C7-OH intermediate 29 could be converted to the C7-OAc assessed by observation of either the production of natural viral
analogue 6 using the protocol described above involving C26 silyla- proteins or other reporter proteins whose expression is coupled to
tion followed by C7 acylation and desilylation.
that of the virus. J-Lat cell lines harbour near full-length latent
Analogues 1–7 were found to exhibit excellent affinities for a rat HIV proviruses that express green fluorescent protein (GFP) upon
brain mixture of PKC isoforms with single-digit nanomolar or sub- stimulation of virus expression49. Treatment of J-Lat cells with
nanomolar Ki values (Table 1). This PKC mixture was selected certain PKC activators such as prostratin50 or bryostatin 1
because it allows for calibration of the potency of the new bryostatin (ref. 24) results in the induction of HIV expression51. Alongside
analogues 1–7 with data obtained previously for other bryostatin these benchmark agents, we examined the ability of our novel
analogues39–42. Importantly, this represents the first quantitative bryostatin analogues to induce HIV expression in J-Lat cells
comparison of the relative affinities of bryostatin 1, prostratin and (clone 10.6) at varying concentrations. In this assay, bryostatin
synthetic bryostatin analogues using a common PKC mixture. induced expression of HIV at concentrations as low as 1 nM
Significantly, all of the new bryostatin analogues (1–7) were more (Table 1). Significantly, several of the bryostatin analogues
potent in this assay than the clinical candidate prostratin. Similar displayed comparable or better potency, when compared to
to previous observations on simplified substrates41, Z-enoate ana- bryostatin, in both half maximum effective concentration (EC50)
logues 5, 6 and 7 were found to be slightly more potent than their and the percentage of cells induced to express latent HIV. For
des-enoate counterparts 1, 3 and 4. As we had observed for the example, analogues 4 and 7 both had EC50 values below 1 nM
B-ring dioxane scaffold42, and similar to the potency difference and induced HIV expression in a higher percentage of cells than
between bryostatin 1 (C7-OAc, lit. Ki ¼ 1.4 nM, ref. 35) and bryostatin bryostatin at all concentrations tested between 0.1 nM and 1 mM
2 (C7-OH, lit. Ki ¼ 5.9 nM, ref. 35), the C7-OH analogue 2, while (Supplementary Fig. S1). Similar to the results obtained in the
highly potent (Ki ¼ 3.4 nM), was less so than the C7-OAc analogue PKC binding affinity assay, Z-enoate analogues 5, 6 and 7 were
3 (Ki ¼ 0.42 nM) or the C7-deoxy analogue 1 (Ki ¼ 0.58 nM).
found to be more potent than their des-enoate counterparts 1, 3
After establishing that the novel bryostatin analogues bind and 4 in their ability to activate latent HIV expression in the J-Lat
effectively to PKC, we tested their HIV latency induction activity. cell line. Moreover, all of the bryostatin analogues evaluated in
Several cellular models have been developed to assess the ability this assay were at least 25-fold more potent than prostratin in
Table1 | Analogue PKC affinity and activity in the J-Lat cell line model of HIV latency.
Analogue
X (C7)
Y (C9)
B-ring
PKC Ki (nM)*
J-Lat EC50 (nM)†
Bryostatin 1
Prostratin
OAc
–
OH
–
Z-Enoate
–
0.28 (0.18–0.44)
6.6 (4.1–10.6)
1.61 (0.92–2.84)
.1,000
1
H
H
H
H
OH
H
Pyran
Pyran
Pyran
Pyran
Z-Enoate
Z-Enoate
Z-Enoate
0.58 (0.41–0.81)
3.4 (1.7–6.6)
37.4 (21.6–64.9)
15.2 (7.5–30.8)
32.0 (16.3–62.8)
0.46 (0.31–0.69)
1.9 (0.91–3.95)
1.15 (0.42–3.18)
0.38 (0.21–0.69)
2
3
4
5
6
7
OH
OAc
OAc
H
OAc
OAc
0.42 (0.22–0.77)
0.95 (0.67–1.4)
0.46 (0.28–1.1)
0.32 (0.17–0.60)
0.79 (0.58–1.1)
H
OH
*Determined in a rat brain isoform mixture. Results from single experiments are presented. Error ranges presented in parentheses indicate 95% confidence intervals from nonlinear regression analysis.
†EC50 for induction of GFP transcription in the J-Lat cell line. GFP expression indicates transcription of the HIV-Long Terminal Repeat and correlates with viral reactivation from latency. Error ranges presented in
parentheses indicate 95% confidence intervals.
4
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ARTICLES
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Discussion
Bryostatin 1 shows great promise as a lead candidate in the search
for transformative therapies aimed at cancer and neurodegenerative
disorders such as Alzheimer’s disease. More recently, Bryostatin 1
was shown to induce the expression of HIV from latent viral reser-
voirs, an activity of potential importance for the eradication of HIV
infection. However, a sustainable, cost-effective supply of bryostatin
1 has not been established. More importantly, an agent that does not
exhibit the side effects of bryostatin is desirable. To address these
issues of both supply and therapeutic performance, we have
designed a series of bryostatin analogues (1–7) that can be syn-
thesized on scale and can be tuned for optimal clinical performance.
Analogues 1–7 were found to exhibit excellent affinities for PKC
with single-digit nanomolar or subnanomolar Ki values, and all
were more potent in this assay than the preclinical candidate pros-
tratin. Significantly, these simplified bryologs were shown to induce
the expression of latent HIV in vitro, with potencies similar to or
better than the natural product, bryostatin 1, and at doses up to
1,000-fold lower than that of prostratin.
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The study presented here demonstrates that bryostatin analogues
potently activate latent HIV reservoirs in vitro. Notably, none of the
compounds evaluated in this assay showed overt toxicity at concen-
trations up to 1 mM based on flow cytometry forward and side
scatter profiles. While in vivo studies of these bryostatin analogues
1–7 in an animal viral induction model are in progress, a related
analogue has been shown to be well tolerated and efficacious in a
mouse cancer model, with no toxicity observed at doses in excess
of that reported here (up to 1 mg kg21)44. Moreover, the convergent
nature of this strategy allows for additional tuning if needed to
improve therapeutic performance. Further evaluation of these com-
pounds in additional in vitro, ex vivo and in vivo latency induction
assays is in progress.
This study is the first demonstration that designed, simplified
analogues of bryostatin can serve as therapeutic leads for the eradi-
cation of HIV/AIDS. Given the problems associated with the supply
of bryostatin 1 and with its side effects, this study provides a reliable
synthetic source of agents that are comparable or superior to bryo-
statin in activity and can be tuned to accommodate clinical needs.
Viral reactivation with these agents, performed in combination
with HAART, would purge latent viral reservoirs while simul-
taneously depleting active virus. Coupling this approach with a tar-
geted therapeutic such as an immunotoxin to more rapidly kill any
resulting virus-expressing cell represents an additional therapeutic
opportunity52. This type of approach, now enabled by the avail-
ability of highly potent and tunable analogues, could be used to
clear all replication-competent HIV from infected individuals,
thereby providing a strategy for disease eradication.
Methods
Experimental details for the synthesis of all new compounds, including experimental
procedures, characterization and spectral data are provided in the Supplementary
Information. Assay protocols for the PKC competitive binding assay and HIV
latency activation assay are also provided in the Supplementary Information.
Received 27 February 2012; accepted 28 May 2012;
published online 15 July 2012
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Acknowledgements
This research was supported by the National Institutes of Health (CA31845 to P.A.W.,
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funding was provided by the ACS Division of Organic Chemistry Graduate Fellowship
sponsored by Bristol-Myers Squibb, the Eli Lilly Organic Chemistry Graduate Fellowship,
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Author contributions
B.A.D., B.A.L., M.D.M., A.J.S., J.A.Z. and P.A.W. conceived and designed the experiments.
B.A.D., B.A.L., M.D.M. and A.J.S. performed the experiments and analysed the data. B.A.L.,
M.D.M., A.J.S. and P.A.W. co-wrote the paper. All authors commented on the manuscript.
45. Khan, T. K., Nelson, T. J., Verma, V. A., Wender, P. A. & Alkon, D. L. A cellular
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Additional information
The authors declare no competing financial interests. Supplementary information and
chemical compound information accompany this paper at www.nature.com/
naturechemistry. Reprints and permission information is available online at http://www.
nature.com/reprints. Correspondence and requests for materials should be addressed to
J.A.Z. and P.A.W.
6
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