Marinopyrrole A target elucidation by acyl dye transfer

Article abstract of DOI:10.1021/ja903149u

(Figure Presented) The targeting of marinopyrrole A to actin was identified using a fluorescent dye transfer strategy. The process began by appending a carboxylic acid terminal tag to a phenol in the natural product. The resulting probe was then studied i

Full text of DOI:10.1021/ja903149u

Published on Web 08/12/2009
Marinopyrrole A Target Elucidation by Acyl Dye Transfer
Chambers C. Hughes,† Yu-Liang Yang,‡ Wei-Ting Liu,‡ Pieter C. Dorrestein,*,‡ James J. La Clair,*,§
and William Fenical*,†
Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, UniVersity of California,
San Diego, La Jolla, California 92093-0204, Departments of Chemistry and Biochemistry, the Skaggs School of
Pharmacy and Pharmaceutical Science, UniVersity of California, San Diego, La Jolla, California 92093-0204, and
Xenobe Research Institute, 3371 Adams AVenue, San Diego, California 92164-4073
Received April 24, 2009; E-mail: wfenical@ucsd.edu; pdorrest@ucsd.edu; i@xenobe.org
While a variety of methods exist,1 target identification continues
to pose a fundamental challenge in elucidating a natural product’s
mode of action.2 The development of solutions to this problem is
essential to advancing drugs into the discovery pipeline. The
complex structural diversity that characterizes natural products
complicates the adoption of routine protocols for these studies.3 A
systematic approach to target elucidation would both facilitate the
introduction of drugs into clinical trials and challenge leads early
on should undesired targets be identified.4
use of bio-orthogonal approaches6 (Huisgen cycloaddition reactions
or Click chemistry,7 Diels-Alder cycloaddition,8 or olefin metath-
esis9), wherein the reporter is added bio-orthogonally after the
natural product binds to its target. Though sufficient for cellular
studies, the lack of a covalent link between the labeled natural
product and its target commonly thwarts enrichment strategies (e.g.,
immunoprecipitation).
Cross-linking or photo-cross-linking,10 a common solution to this
problem, is frequently plagued by additional complications. First,
the preparation of a dual tag results in an enlarged probe,11 which
often exhibits only a fraction of the activity of its parent natural
product.2b Second, cross-linking methods are generally low yielding
and unselective, increasing the complexity of downstream mass
spectrometric (MS) analyses.12 Finally, and perhaps most critically,
the cross-linked target still bears a covalently linked natural product
that is subject to xenobiotic metabolism (e.g., cytochrome P450
oxidation).13 Such metabolic processing leads to the formation of
multiple products, each of which is detected by MS, obscuring
subsequent protein identification and binding-site mapping studies.
In the present study, the interaction of a natural product with its
target(s) is addressed via an elimination or displacement reaction,
wherein the natural product is removed from the reaction milieu,
acting as a leaving group.14
Scheme 1. Structures of Marinopyrrole A (6) and Marinopyrrole B
(7), IAF Labels 9-11, Control 12 and the Synthesis of Bis-acetate
8, Mono-Labeled Probe 13, and Bis-Labeled Probe 14
Figure 1. Application of acyl dye transfer to natural product mode of action
studies. (a) A dye (L) labeled natural product (NP) probe 1 binds to target
protein 2 forming complex 3. Ligation-directed acyl transfer in complex 3
results in the displacement of phenol 4 and formation of labeled protein 5.
(b) Typical workflow associated with antitumor mode of action studies on
a natural product. The process begins by verifying the activity of the probe
from (a) in live cells. The process continues with steps that apply the probe
for protein affinity purification or immunoprecipitation (IP) studies, gel
analysis, and mass spectral based protein identification.
Given the use of acyl phenols in protein reactive dyes,15 we
examined the feasibility of this motif as a transfer agent for the
immunoaffinity fluorescent (IAF) tag (Figure 1a). Though the
attachment of a phenol-containing linker would enable this method
to be applied to natural products in a general sense, the marinopy-
rroles, a family of phenolic 1,3′-bipyrroles including 6 and 7, were
selected for this study.16 We chose these materials based on (1)
their availability from culture (strain CNQ-418) at mg/L scale; (2)
their unknown mode of action; (3) their activity in tumor cell lines
(GI50 values for 6 and 7 were 10 µM in HCT-116 cells and 620
and 500 nM for 6 and 7, respectively, in A549 breast cancer cells);
and (4) their bis-phenol structure, previously shown to undergo clean
The process of determining the therapeutic mode of action of a
natural product typically begins by developing probes bearing an
affinity and/or fluorescent tag.5 The aim in this exercise is to
construct a probe with activity comparable to that of its parent
natural product. Often a loss in activity can be circumvented by
† Scripps Institution of Oceanography, University of California, San Diego.
‡ The Skaggs School of Pharmacy and Pharmaceutical Science, University of
California, San Diego.
§ Xenobe Research Institute.
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C O M M U N I C A T I O N S
acylation to 8 with acetic anhydride (Scheme 1) without a loss in
activity (GI50 value of 0.8 µM for 8 in HCT-116 cells, an ∼12-
fold increase over 6 or 7).16
Based on this evidence, mono-labeled 13 (45% yield) and bis-
labeled 14 (traces) were prepared and analyzed using a combination
of MS and NMR analysis. HMBC analysis confirmed the position
of the tag in mono-labeled 13 (see Scheme 1). The low yield of
the bis-labeled 14 was due to its instability, rapidly converting to
13 in aqueous media with a half-life of t1/2 ) 40 min in PBS pH
7.2 at 32 °C. Mono-labeled 13 was much less prone to hydrolysis
with a half-life of t1/2 ) ∼2 d in PBS pH 7.2 at 32 °C.
Figure 3. Marinopyrrole probes target actin. (a) A fluorescent gel showing
cell lysate obtained from HCT-116 cells (108 cells) treated with 10 µM 13
in L1 and 10 µM 14 in L2 for 12 h. (b) A Silver Blue stained SDS-PAGE
gel depicting proteins immunoprecipitated from the lysate in (a) using
Affigel 10 resin containing 3.5 mg/mL of an anti-IAF antibody XRI-TF35.
(c) SDS-PAGE gel depicting the HCT-116 cell lysate used in (b). (d) LC/
MS/MS analysis of the IP product in (b) containing actin and actin binding
proteins. (e) A fluorescent SDS-PAGE gel of 5 µM rabbit muscle actin
(protein ) a, lanes L1-L10) treated with IAF tags 10-11 and probes 13-14
at 32 °C. Reactions were conducted under conditions wherein probe 13 did
not label a lane marker (NativeMark, Invitrogen; protein ) lm, lane LM).
Fluorescent gels were imaged by excitation at 360 nm.
HCT-116 cells (∼108 cells) were incubated with 10 µM 13 for 12 h.
The cells were collected, lysed, and screened for proteins that were
labeled with the IAF tag. Two fluorescent bands at 40-45 kDa
were apparent in the crude lysate (Figure 3a). After IP, three bands
were obtained (Figure 3b). Mass spectral analysis (Figure 3d)
indicated that the two lower bands were actin, while the upper band
arose from actinin. The fact that actinin was observed during IP
experiments in the lysate, while not fluorescent, is expected given
its tight complexation with actin.
The selective binding of the probe to actin was validated with
purified proteins. Samples of rabbit muscle actin (Worthington
Biochemicals) were treated with 13 or 14 to yield comparable
fluorescently labeled bands (lanes L4-L7, Figure 3e). Even with
a 50-fold increase in concentration (lanes L1, L2, L3, L9, and L10
in Figure 3e), the nonspecific labels 10 and 11 failed to stain actin.
The reactivity observed in Figure 3e was indeed due to a specific
binding event as pretreatment of actin with 6 effectively blocked
the uptake of dye from 14 (lane L8, Figure 3e).
Finally, mass spectral methods were applied to determine the
site of dye transfer to actin. Protein from the reaction in lane
L6 (Figure 3e) was trypsin digested, purified by HPLC based
on the absorbance of the IAF dye at 350 nm, and subjected to
LTQ-MS/S with a NanoMate direct infusion system. Evaluation
of the resulting spectra using the proteomics search tool,
InSpecT, to find peptides that were modified by the IAF tag
(M+286 Da), resulted in the identification of the modified
peptide V98APEEHPTLLTEAPLNPKANR118. Because InSpecT
Figure 2. Uptake and subcellular localization of probes 13 and 14 in HCT-
116 cells. Confocal fluorescent images depicting the red fluorescence from
the uptake of 6 in cells treated with (a) 10 µM 6 for 1 h or (b) 10 µM 6 for
6 h. A set of confocal fluorescent images showing blue (447 nm), red (692
nm), and two color mixed (mix) fluorescence in HCT-116 cells treated with
(c) 10 µM 13 for 6 h or (d) 10 µM 14 for 6 h. Probe 14 likely hydrolyzed
to 13 under these conditions. (e) Image of cells treated with 10 µM 13 for
6 h and then washed, fixed, and stained for nucleic acids with Syto-60 (red)
and actin with FITC-phalloidin (green). (f) Identical cells as in (e) stained
for nucleic acids (red) and transfer of the IAF tag to actin (blue). Note the
overlap of green in (e) with blue in (f). All cells were treated under
conditions wherein control 12 was completely washed from cells (inset
images). Wavelengths denote the center of emission filter. Bars denote 10
µm.
Probes 13 and 14 mimicked the activity of marinopyrrole A (6).
Probe 14, with a GI50 value of 1 µM, was ∼10-fold more active
than the parent natural products 6 and 7. Using two-color confocal
microscopy, we compared the native red fluorescence from
marinopyrrole A (6) at 692 nm (Figure 2a,b) to the blue fluorescence
from the IAF tag in 13 (Figure 2d) and 14 (Figure 2c). The presence
of a similar pattern of red and blue staining confirmed that probes
13 and 14 shared the same subcelluar localization in HCT-116 cells
and, therefore, provided a sufficient mimic of 6. The colocalization
of natural product 6 and dye-labeled probes 13 and 14 also showed
that cells were not stained from the hydrolytic release of 9.
Importantly, control 12 was readily washed from cells under each
study (insets, Figure 2a-d).
annotated the modification to be anywhere from K115 to R118
,
we manually annotated the data to determine the site of
localization. The manual annotation of the low-resolution data
suggested that the modification could be found at K115. To further
The putative protein target of the marinopyrrole probe was
initially established using an immunoprecipitation (IP) protocol.2c
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improve the localization of the probe, the same sample was
reanalyzed by LTQ-FTICR-MS, giving rise to a mass of
861.7839 Da within 6.6 ppm of the calculated mass of the
modified peptide. Furthermore, all the MS2 b and y fragment
ions observed by LTQ-FTICR-MS matched to within 0.02 Da
enabling us to localize the site of modification to K115. The
unmodified peptide V98APEEHPTLLTEAPLNPK115 was also
identified, which indicates that this residue was only partially
modified.
were blue fluorescent (Figure 2f) from the binding of 13, as evident
by costaining with FITC-phalloidin (Figure 2e).
This investigation provides definitive support for the use of a
dye transfer protocol via an acyl phenol motif for mode of action
studies. By transferring the dye from the natural product to its
protein target, we were able to streamline cellular, target elucidation,
and binding site determination studies into a single workflow (see
Figure 1). Because bioactive natural products bind to proteins in
selective binding pockets, the process of natural product mediated
ligation may also be used to direct protein modifications site
specifically. Studies are now underway to evaluate the scope of
this reaction centered on the electronic nature of the phenol and its
ability to transfer its payload.
Acknowledgment. We thank Qishan Lin (CFG at University
of Albany) for preliminary protein ID analyses. Support was
provided by the NCI under Grant R37 CA44848 (to W.F.), the
V-foundation (to P.C.D.), and NIH R01 GM086283 (to P.C.D.).
Supporting Information Available: Synthetic methods, copies of
NMR spectra, protocols for cell imaging studies, IP studies and actin
assays, detailed methods for the mass spectral studies, copies of original
images in Figures 2-3 as well as additional references are available
free of charge via the Internet at http://pubs.acs.org.
References
(1) (a) Leslie, B. J.; Hergenrother, P. J. Chem. Soc. ReV. 2008, 37, 1347. (b)
Wang, X.; Imber, B. S.; Schreiber, S. L. Bioconjugate Chem. 2008, 19,
585.
(2) Recent examples: (a) Bowers, A.; West, N.; Taunton, J.; Schreiber, S. L.;
Bradner, J. E.; Williams, R. M. J. Am. Chem. Soc. 2008, 130, 11219. (b)
Kotake, Y.; Sagane, K.; Owa, T.; Mimori-Kiyosue, Y.; Shimizu, H.; Uesugi,
M.; Ishihama, Y.; Iwata, M.; Mizui, Y. Nat. Chem. Biol. 2007, 3, 570. (c)
Hughes, C. C.; MacMillan, J. B.; Gaudeˆncio, S. P.; Fenical, W.; La Clair,
J. J. Angew. Chem., Int. Ed. 2009, 48, 728.
(3) (a) Gough, J. D.; Crews, C. M. Ernst Schering Res. Found. Workshop.
2006, 61. (b) Drahl, C.; Cravatt, B. F.; Sorensen, E. J. Angew. Chem., Int.
Ed. 2005, 44, 5788.
(4) Altmann, K. H.; et al. ChemBioChem. 2009, 10, 16.
(5) (a) Peddibhotla, S.; Dang, Y.; Liu, J. O.; Romo, D. J. Am. Chem. Soc.
2007, 129, 12222. (b) Alexander, M. D.; et al. ChemBioChem. 2006, 7,
409.
(6) (a) Carrico, I. S.; Carlson, B. L.; Bertozzi, C. R. Nat. Chem. Biol. 2007, 3,
321. (b) Sadaghiani, A. M.; Verhelst, S. H.; Bogyo, M. Curr. Opin. Chem.
Biol. 2007, 11, 20. (c) Heal, W. P.; Wickramasinghe, S. R.; Leatherbarrow,
R. J.; Tate, E. W. Org. Biomol. Chem. 2008, 7, 2308. (d) Channon, K.;
Bromley, E. H.; Woolfson, D. N. Curr. Opin. Struct. Biol. 2008, 18, 491.
(7) (a) Colombo, M.; Peretto, I. Drug DiscoV. Today 2008, 13, 677. (b) Hein,
C. D.; Liu, X. M.; Wang, D. Pharm. Res. 2008, 25, 2216. (c) Moorhouse,
A. D.; Moses, J. E. ChemMedChem. 2008, 3, 715.
(8) (a) Weisbrod, S. H.; Marx, A. Chem. Commun. 2008, 30, 5675. (b) de
Arau´jo, A. D.; Palomo, J. M.; Cramer, J.; Seitz, O.; Alexandrov, K.;
Waldmann, H. Chemistry 2006, 12, 6095.
(9) (a) Binder, J. B.; Raines, R. T. Curr. Opin. Chem. Biol. 2008, 12, 767. (b)
Kirshenbaum, K.; Arora, P. S. Nat. Chem. Biol. 2008, 4, 527.
(10) Tanaka, Y.; Bond, M. R.; Kohler, J. J. Mol. Biosyst. 2008, 4, 473.
(11) Guizzunti, G.; Brady, T. P.; Malhotra, V.; Theodorakis, E. A. Bioorg. Med.
Chem. Lett. 2007, 15, 320.
Figure 4. Marinopyrrole probe 13 transfers an IAF tag selectively to residue
K115 of actin. (a) Structure of actin (green) noting the position of the targeted
K115 residue (blue) and other lysine residues (red). (b) Side and (c) frontal
views of the putative marinopyrrole binding pocket (aqua shaded). Images
were developed using a complex between rabbit skeletal muscle actin and
latrunculin A at 2.85 Å (pdb id: 1IJJ). The side chain amine of K115 (star)
and the latrunculin A binding pocket (sphere) are noted.
Lysine 115 occupies a distinct site on actin (star, Figure 4a) that
neither overlaps the ATP, gelsolin, and profilin binding sites (see
Supporting Information) nor overlaps with the pocket targeted by
other known natural products such as latrunculin A (dot, Figure
4a), a marine natural product that binds to the nucleotide binding
cleft of actin. Structural modeling identified a channel that exists
adjacent to the amine terminus of K115, suggesting a putative
marinopyrrole-binding site (Figure 4b,c).
Transfer of the IAF dye from the natural product to actin was
further supported by secondary analyses. First, microcalorimetry
analyses verified the binding between marinopyrrole A (6) and
rabbit muscle actin with a Kd of 0.12 ( 0.2 µM. The strength of
this complex compares favorably to a reported value of 0.2 µM
for latrunculin A (albeit at a different binding site, see Figure 4a).17
Second, using a fluorescence assay (Cytoskeleton Inc.),18 6 was
shown to inhibit the polymerization of a pyrene-labeled G-actin
with an IC50 value of 39.5 ( 6.2 µM (see Supporting Information).
Lastly, actin fibers in live HCT-116 cells treated with probe 13
(12) Hurst, G. B.; Lankford, T. K.; Kennel, S. J. J. Am. Soc. Mass Spectrom.
2004, 15, 832.
(13) (a) Dekant, W. EXS 2009, 99, 57. (b) Johnson, W. W. Drug Metab. ReV.
2008, 40, 101.
(14) During the preparation of this manuscript a team led by Hamachi disclosed
a similar concept using ligand-directed tosylation: Tsukiji, S.; Miyagawa,
M.; Takaoka, Y.; Tamura, T.; Hamachi, I. Nat. Chem. Biol. 2009, 5, 341.
(b) Tsukiji, S.; Wang, H.; Miyagawa, M.; Tamura, T.; Takaoka, Y.;
Hamachi, I. J. Am. Chem. Soc. 2009, 131, 9046.
(15) Gee, K. R.; Archer, E. A.; Kang, H. C. Tetrahedron Lett. 1999, 40, 1471–
1474.
(16) Hughes, C. C.; Prieto-Davo´, A.; Jensen, P. R.; Fenical, W. Org. Lett. 2008,
10, 629.
(17) Coue´, M.; Brenner, S. L.; Spector, I.; Korn, E. D. FEBS Lett. 1987, 213,
316.
(18) (a) Zhai, L.; Zhao, P.; Panebra, A.; Guerrerio, A. L.; Khurana, S. J. Biol.
Chem. 2001, 276, 36163. (b) Pollard, T. D. Anal. Biochem. 1983, 134,
406.
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