Systematic chemical mutagenesis identifies a potent novel apratoxin A/E hybrid with improved in vivo antitumor activity

Article abstract of DOI:10.1021/ml200176m

Apratoxins are cytotoxic marine natural products that prevent cotranslational translocation early in the secretory pathway. We showed that apratoxins downregulate receptors and growth factor ligands, giving a one"two punch to cancer cells, particularly those that rely on autocrine loops. Through total synthesis, we tested the effects of amino acid substitutions, including alanine scanning, on the downregulation of receptor tyrosine kinases and vascular endothelial growth factor A (VEGF-A) and probed the stereospecificity of target engagement by epimerization of selected chiral centers. Differential effects on two types of secretory molecules suggest that the apratoxins' substrate selectivity with respect to inhibition of secretion may be tuned through structural modifications to provide tailored therapy. Our structure"activity relationship studies and medicinal chemistry efforts led to a potent inhibitor with in vivo efficacy in a colorectal tumor xenograft model without irreversible toxicity exerted by apratoxin A, demonstrating that this novel mechanism of action has therapeutic potential.

Full text of DOI:10.1021/ml200176m

LETTER
pubs.acs.org/acsmedchemlett
Systematic Chemical Mutagenesis Identifies a Potent Novel Apratoxin
A/E Hybrid with Improved in Vivo Antitumor Activity
Qi-Yin Chen,† Yanxia Liu,† and Hendrik Luesch*
Department of Medicinal Chemistry, University of Florida, Gainesville, Florida 32610, United States
S Supporting Information
b
ABSTRACT: Apratoxins are cytotoxic marine natural products
that prevent cotranslational translocation early in the secretory
pathway. We showed that apratoxins downregulate receptors
and growth factor ligands, giving a oneꢀtwo punch to cancer
cells, particularly those that rely on autocrine loops. Through
total synthesis, we tested the effects of amino acid substitutions,
including alanine scanning, on the downregulation of receptor
tyrosine kinases and vascular endothelial growth factor A (VEGF-A) and probed the stereospecificity of target engagement by
epimerization of selected chiral centers. Differential effects on two types of secretory molecules suggest that the apratoxins' substrate
selectivity with respect to inhibition of secretion may be tuned through structural modifications to provide tailored therapy. Our
structureꢀactivity relationship studies and medicinal chemistry efforts led to a potent inhibitor with in vivo efficacy in a colorectal
tumor xenograft model without irreversible toxicity exerted by apratoxin A, demonstrating that this novel mechanism of action has
therapeutic potential.
KEYWORDS: Antitumor agents, natural products, receptor tyrosine kinases, growth factors, secretory pathway, total synthesis
ancer cells are characterized by aberrant pro-growth signal-
ing, usually induced upon ligand binding to their corre-
hypothesis through a combination of medicinal chemistry based
on the apratoxin scaffold, molecular in vitro and in vivo pharma-
cology, and colorectal tumor xenograft efficacy studies.
C
sponding receptor tyrosine kinases (RTKs), which are over-
expressed in many cancers.1 Consequently, RTKs have become
prime therapeutic targets resulting in the development of specific
small molecules (e.g., erlotinib) and antibodies (e.g., cetuximab
and panitumumab) targeting epidermal growth factor receptors.2
However, drug resistance is a clinical problem, and combination
therapy or the use of broader-spectrum RTK inhibitors (e.g.,
sorafenib and sunitinib), targeting vascular endothelial growth
factor (VEGF) receptor (VEGFR) and platelet-derived growth
factor receptor (PDGFR), may be more advantageous.3ꢀ5 Alter-
natively, the ligands themselves may be targeted, which has led to
the development and FDA approval of an anti-VEGF-A antibody
(bevacizumab) for colorectal cancer treatment.6,7 While studying
the mechanism of action of the marine natural product apratoxin A
(Figure 1),8 belonging to a class of potent cytotoxins produced by
marine cyanobacteria,9ꢀ12 we have raised the possibility that
inhibition of the secretory pathway at the level of cotranslational
translocation by apratoxins and, in general, may be exploited for
anticancer therapy.13 Inhibition of this process prevents export of
receptors and secretory molecules from the cytoplasm, leading to
receptor (including RTK) depletion and additionally should
prevent secretion of the corresponding ligands, growth factors,
and cytokines.14 This oneꢀtwo punch is expected to have
unusually potent antiproliferative activity and may be an effective
alternative to combination therapy with multiple (or broad-
spectrum) RTK and growth factor inhibitors and may be particu-
larly useful to treat cancers where autocrine loops, that is,
uncontrolled proliferation stimulated by secreted growth factors,
play a major role (e.g., colorectal cancer).15 Here, we test this
Since we found that apratoxin A activity was reversible,13 it
became evident that apratoxins have to be administered on a
chronic schedule at a low dose rather than given as a bolus
injection; the latter was initially reported to cause toxicity without
significant antitumor activity.8 Our preliminary data for chronic
administration of apratoxin A (Figure S3 in the Supporting
Information), recently confirmed by others,12 indicate that this
compound shows some antitumor efficacy in vivo but is not well
tolerated. The therapeutic window is small; we found a 50% death
rate (day 16) at the “therapeutic” concentration of 0.25 mg/kg
(daily ip) with irreversible toxicity occurring after 2 weeks of
treatment (Figure S3 in the Supporting Information). The main
question remained whether this toxicity is mechanism-related or
an off-target effect. Here, we describe the synthesis and biological
evaluation of synthetic analogues of apratoxin A, one of which
shows greater potency and efficacy than the parent compound and
is much better tolerated in vivo, providing the groundwork for
second-generation apratoxins with more promising anticancer
potential and validating this novel mechanism of action for cancer
therapy.
Our goal was 3-fold: We aimed (1) to define critical and
tunable elements for inhibition of cotranslational translocation,
(2) to extend our previous receptor studies and prove that
transport of receptors and their corresponding ligands are
Received: July 20, 2011
Accepted: August 21, 2011
Published: August 31, 2011
r
2011 American Chemical Society
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LETTER
Scheme 1. Preparation of the Analogues of Apratoxin Aa
Figure 1. Selected known apratoxins and synthetic targets 1aꢀe
produced through alanine or valine incorporations within apratoxin A
at various positions (apratoxins F and S1ꢀS3) or through “hybridiza-
tion” of apratoxins A and E (apratoxin S4). Me, methyl; Bu, butyl; Pr,
propyl; and PMB, p-methoxybenzyl. Two epimers of 1e (2-epi-1e,
apratoxin S5; and 34-epi-1e, apratoxin S6), minor reaction products
during 1e synthesis, were included in our SAR studies.
inhibited by apratoxins, and (3) to design a synthetic analogue
with improved potency and less toxic side effects in vivo. After
the report of the first total synthesis,16 structureꢀactivity
relationship (SAR) studies by other groups have focused on
the polyketide (C33ꢀC43) chain.17,18 Apratoxin cytotoxicity is
highly sensitive to certain structural modifications to this unit.
Reversal of the configuration at C37 from 37S to 37R or
removal of the methyl group at C37 abolished the antiproli-
ferative activity of apratoxins, shown for the oxazoline
analogue;17 however, the C-34 epimer of apratoxin A showed
equipotent activity.18 Depending on the cell line, the C34ꢀC35
dehydration product of apratoxin A possessed 12ꢀ29-fold
reduced cytotoxicity.11 However, the potency of apratoxin E
(Figure 1), while also unsaturated at C34ꢀC35, is only 6ꢀ15-
fold less cytotoxic than apratoxin A,11 suggesting that the
modification in the moCys unit (C27ꢀC31), viz. removal of
the methyl group and/or α,β-unsaturation, may increase activ-
ity if the original, hydrated configuration of C34ꢀC35 as in
apratoxin A can be restored. We also hypothesized that the α,β-
unsaturated system in the moCys unit may be responsible for
nonspecific (off-target) toxicity due to conjugate addition of
cellular nucleophiles. Thus, we aimed to synthesize such an
apratoxin A/E hybrid (1e) with hopefully improved anticancer
activity and also tumor selectivity. Furthermore, we interro-
gated modifications in the peptide portion, where it is known
that lack of N-methylation of isoleucine reduces activity
(apratoxin B)9 and replacement of proline by N-methyl alanine
largely retains activity (1a, apratoxin F)12 (Figure 1). We
carried out an alanine scan (without changing the N-alkylation
pattern) and synthesized a valine analogue to more broadly
probe the hydrophobicity requirements of the R3 position
(Ile f Val f Ala; Figure 1), which led to the first synthesis of
the natural product apratoxin F (1a) and positional congeners
(1bꢀd), providing new insight into the SAR of apratoxins.
a Reagents and conditions: (a) MS 4 Å, toluene, ꢀ78 °C. (b) TrocCl,
DMAP, pyridine, CH2Cl2. (c) DDQ, CH2Cl2ꢀH2O. (d) Cl3C6H2COCl,
DIEA, THF, DMAP, toluene. (e) OsO4, oxone, NaIO4. (f) HATU, DIEA,
CH2Cl2. (g) Ph3P(O), Tf2O, CH2Cl2, 0 °C. (h) Zn, NH4OAc, THF. (i)
Pd(PPh3)4, N-methylaniline, THF. (j) HATU, DIEA, CH2Cl2. (k) Pd-
(PPh3)4, N-methylaniline, THF. (l) Et2NH, MeCN. (m) HATU, DIEA,
CH2Cl2. Troc, 2,2,2-trichloroethoxycarbonyl; DMAP, 4-(dimethylamino)-
pyridine; DDQ, 2,3-dichloro-4,5-dicyanobenzoquinone; Fmoc, 9-fluorenyl-
methoxycarbonyl; Trt, triphenylmethyl; HATU, O-(7-azabenzotriazol-1-
yl)-N,N,N0,N0-tetramethyluronium hexafluorophosphate; DIEA, diisopro-
pylethylamine; and Tf, trifluoromethylsulfonyl.
Scheme 1 illustrates the main syntheticprocedures to obtain the
desired apratoxin analogues, closely paralleling reported synthesis
strategies and retrosynthetic analysis for apratoxin A with minor
modifications.19,20 Aldehyde 219ꢀ21 was treated with Roush's (E)-
crotylboronate 322 to give 4. Protection of the hydroxyl group with
Troc gave 5, and removal of PMB group with DDQ, followed by
esterification with Fmoc-Pro-OH/Fmoc-N-Me-Ala-OH using the
Yamaguchi method,23 provided ester 6. Acid 7 was obtained when
6 was oxidized with OsO4/oxone/NaIO4 oxidation system.24
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Table 1. Activities of Apratoxins on HCT116 Cell Viability
and VEGF-A Secretion
apratoxin
IC50 (nM)a cell viability IC50 (nM)b VEGF-A secretion
A
E
5.97
184
4.92
373
4.13
1700
1.14
258
1.58
1.49
9.10
0.461
F (1a)
S1 (1b)
20.7
0.322
340
0.308
112
0.391
S2 (1c)
S3 (1d)
S4 (1e)
S5 (2-epi-1e)
S6 (34-epi-1e)
a Determined after 48 h (n = 4). b Determined after 12 h (n = 3).
Coupling of 6 with modified cysteine amine 8 gave the key
intermediates 9. Using the Kelly method (PPh3(O)/Tf2O in
CH2Cl2),17,19,20,25 the thiazoline ring was formed, and Troc was
removed immediately thereafter. Subsequent removal of allyl from
10 with Pd(PPh3)4/N-methylaniline provided acid 11, which was
coupled with tripeptide 12(see the SupportingInformation) using
HATU/DIEA to give protected linear compounds 13. Cleavage of
O-allyl ester by Pd(PPh3)4/N-methylaniline and removal of Fmoc
by Et2NH/MeCN afforded cyclization precursors, whose final
macrolactamization with HATU/DIEA under high dilution con-
centration provided apratoxins 1aꢀe in 18ꢀ52% yield. We used
the same strategy to synthesize apratoxin A for direct comparison
in our biological assays.
We found that apratoxin F (1a) was the most potent apratoxin
A analogue that arose from an alanine scan; both compounds
showed comparable effects on HCT116 colorectal carcinoma cell
viability (Table 1) and met proto-oncogene (MET) receptor
levels (Figure 2a). In contrast, the other two alanine scan
“mutants” 1b and 1d exhibited strongly reduced activity in both
assays. Replacement of Ile with Val (1c) did not affect activity.
However, apratoxin S4 (1e)—the apratoxin A/E hybrid
(Figure 1)—was superior as compared with all other apratoxins
(Figure 2a,b and Table 1), consistent with our hypothesis. As a
result of synthesis scale-up, we also identified two minor reaction
products that we characterized as 2-epi-1e and 34-epi-1e
(Figure 1 and see the Supporting Information), which we
included in our SAR studies. While 2-epi-1e lost activity by ca.
200-fold, the two 34-epimers of 1e were almost equally active. In
all cases, cell viability (Table 1) correlated well with the reduction
of receptor levels based on immunoblot analysis (Figure 2a),
suggesting that the configuration at C-2 is crucial for potent
cytotoxicity, while the C-34 configuration is irrelevant as recently
found for apratoxin A.18
Next, we extended our SAR studies to our hypothesized
effects of apratoxins on growth factor secretion and focused on
the angiogenic drug target VEGF-A.26ꢀ28 Indeed, all apratox-
ins inhibited the secretion of VEGF-A (Table 1 and Figure 2c),
confirming our hypothesis that apratoxins prevent the export
of receptors and the secretion of ligands, which is also
consistent with our data for the antiangiogenic properties of
apratoxins.29 Furthermore, depending on the apratoxin struc-
ture, the IC50 values were 2ꢀ20-fold lower than the IC50
values for cell viability, suggesting that VEGF-A is more
susceptible to inhibition of secretion as compared with
receptor proteins tested. This indicates that reducing levels
of VEGF-A (and likely other ligands) alone is not sufficient to
Figure 2. Cellular activity of apratoxins (HCT116 cells). (a) SAR by
immunoblot analysis for RTK levels (MET) after 24 h. Comparison
of apratoxin A and S4 (1e) effects on (b) cell viability (48 h, n = 4) and
(c) VEGF-A secretion (12 h, n = 3). Error bars indicate SD. (d) Dose-
dependent cell cycle effects of 1e determined by DNA content analysis,
demonstrating induction of G1 arrest (24 h).
inhibit proliferation and that receptor depletion is a necessity.
This result also raises the possibility that the apratoxins'
substrate selectivity with respect to inhibition of secretion
may be tuned through structural modifications, which is a
subject of ongoing research in our laboratory.
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Figure 3. Apratoxin S4 (1e) inhibits cotranslational processing in vitro.
Products of in vitro translation reactions (rabbit reticulocyte, amino
acids, [35S]methionine, canine pancreatic microsomal membranes,
mRNA template, and apratoxin 1e) were separated by SDS-PAGE to
autoradiographically detect effects on translation and glycosylation of α-
factor and signal peptide cleavage of β-lactamase. When PDGFR-β
cDNA was used as a template, in vitro transcription/translation was
carried out using T7 TNT Quick Master Mix and glycosylation
determined by SDS-PAGE followed by autoradiography.
Figure 4. In vivo activity of apratoxin S4 (1e) using a HCT116
xenograft mouse model. (a) Efficacy studies (daily ip). Subcutaneous
tumor-bearing mice were injected with 1e (n = 8) or DMSO vehicle (n =
10), and tumor volumes were monitored over time. Error bars indicate
SEM. (b) At the end of the efficacy studies, tumors were analyzed by
immunoblot analysis for receptor levels. (c) Levels of VEGFR2 were
analogously measured in liver tissue of vehicle- vs 1e-treated mice.
We then tested our prioritized compound (1e) on the cell
cycle to determine if its effects parallel those of apratoxin A.
Consistent with data for apratoxin A,29 dose-dependent DNA
content analysis of HCT116 cells upon 24 h of treatment
revealed that 1e increases the population of cells in G1 phase
concomitant with decrease of cells in S phase, starting at
subnanomolar concentrations (0.32 nM) with strongest effect
at 3.2 nM (Figure 2d), which is the concentration range for the
inhibition of VEGF-A and receptor levels and also near the IC50
for cell viability (Table 1). Next, we confirmed that the
inhibition of the secretory pathway by apratoxin S4 (1e) occurs
at the level of cotranslational translocation from the cytoplasm
to the endoplasmic reticlum.13 We used in vitro translation
experiments in the presence of microsomal membranes to
demonstrate the inhibition by 1e of cotranslational processing
events characteristic for secretory molecules, specifically glyco-
sylation (using α-factor mRNA substrate) and signal peptide
cleavage (using β-lactamase mRNA substrate). Inhibition of
cotranslational processing in a dose-dependent manner is
consistent with the mode of action that we had reported for
apratoxin A (Figure 3).13 We also used a human receptor cDNA
(PDGFR-β) and proved by coupled in vitro transcription/
translation that 1e inhibits processing of this cancer-associated
RTK (Figure 3).
Initial in vivo studies aimed at determining the maximum
tolerated dose (MTD) already suggested that 1e is much better
tolerated in vivo than apratoxin A, which is consistent with our
hypothesis that the conjugated system may be a liability in vivo.
While high doses (g0.375 mg/kg) of apratoxin A led to
irreversible toxicity, that is, continued weight loss and eventual
death even when ip injection was discontinued, mice treated
with apratoxin S4 (1e) quickly recovered when high-dose
injections were stopped. Using the same low dose of 0.25
mg/kg of 1e (daily ip for over 2 weeks) used for apratoxin A to
treat HCT116 tumor-bearing nu/nu mice (Figure S3 in the
Supporting Information), the antitumor effect was much
stronger (Figure 4a) without any evident toxicity based on
lack of weight loss and histological assessment of kidney and
liver tissue. To determine if the mechanism of action in vivo
correlates with our in vitro data, we measured receptor levels in
the tiny residual tumor tissue and found that VEGFR2 and
PDGFR-β levels were depleted (Figure 4b). In contrast, the
liver appeared less affected, as we did not observe a significant
reduction of VEGFR2 in this tissue of the same mice
(Figure 4c), which may indicate preferential uptake of 1e by
the tumor and/or enhanced dependency of tumor on the
secretory pathway due to more rapid turnover and resynthesis
of receptor molecules.
In conclusion, apratoxin S4 (1e) is the first viable candidate
of the apratoxin family that shows the requisite tumor
selectivity and increased antitumor activity and potency.
Thus, we have largely separated anticancer activity from
nonselective toxicity, indicating for the first time that inhibi-
tion of cotranslational translocation is a promising novel
mechanism for cancer therapy, particularly to treat cancers
that are characterized by aberrant autocrine loops such as
colorectal cancer. Furthermore, our SAR studies suggest that
through structural modification we may be able to tune
apratoxin selectivity toward certain substrates destined for
the secretory pathway, which may be exploited to provide
tailored therapy and also more selective chemical tools to
study the secretory pathway.
’ ASSOCIATED CONTENT
S
Supporting Information. Synthetic procedures, NMR
b
data and spectra, descriptions of biochemical assays, cell cycle
analysis, and in vivo experiments as well as additional figures,
schemes, and tables. This material is available free of charge via
the Internet at http://pubs.acs.org.
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LETTER
’ AUTHOR INFORMATION
(11) Matthew, S.; Schupp, P. J.; Luesch, H. Apratoxin E, a cytotoxic
peptolide from a Guamanian collection of the marine cyanobacterium
Lyngbya bouillonii. J. Nat. Prod. 2008, 71, 1113–1116.
(12) Tidgewell, K.; Engene, N.; Byrum, T.; Media, J.; Doi, T.;
Valeriote, F. A.; Gerwick, W. H. Evolved diversification of a modular
natural product pathway: Apratoxins F and G, two cytotoxic cyclic
depsipeptides from a Palmyra collection of Lyngbya bouillonii. Chem-
BioChem 2010, 11, 1458–1466.
(13) Liu, Y.; Law, B. K.; Luesch, H. Apratoxin A reversibly inhibits
the secretory pathway by preventing cotranslational translocation. Mol.
Pharmacol. 2009, 76, 91–104.
(14) Pleiotropic mechanisms on HSP90 client proteins have been
proposed for the apratoxins, although many of these observations may
be rationalized by inhibition of the secretory pathway:Shen, S.; Zhang,
P.; Lovchik, M. A.; Li, Y.; Tang, L.; Chen, Z.; Zeng, R.; Ma, D.; Yuan, J.;
Yu, Q. Cyclodepsipeptide toxin promotes the degradation of Hsp90
client proteins through chaperone-mediated autophagy. J. Cell. Biol.
2009, 185, 629–639.
(15) Ruan, W.-J.; Lai, M.-D. Autocrine stimulation in colorectal
carcinoma (CRC): Positive autocrine loops in human colorectal carci-
noma and applicable significance of blocking the loops. Med. Oncol.
2004, 21, 1–7.
(16) Chen, J.; Forsyth, C. J. Total synthesis of apratoxin A. J. Am.
Chem. Soc. 2003, 125, 8734–8735.
(17) Ma, D.; Zou, B.; Cai, G.; Hu, X.; Liu, J. O. Total synthesis of the
cyclodepsipeptide apratoxin A and its analogues and assessment of their
biological activities. Chem.—Eur. J. 2006, 12, 7615–7626.
(18) Doi, T.; Numajiri, Y.; Takahashi, T.; Takagi, M.; Shin-ya, K.
Solid-phase total synthesis of (ꢀ)-apratoxin A and its analogues and
their biological evaluation. Chem. Asian J. 2011, 6, 180–188.
(19) Doi, T.; Numajiri, Y.; Munakata, A.; Takahashi, T. Total
synthesis of apratoxin A. Org. Lett. 2006, 8, 531–534.
Corresponding Author
*E-mail: luesch@cop.ufl.edu.
Author Contributions
†These authors contributed equally to this work.
Funding Sources
This work was supported by the University of Florida Research
Opportunity Fund and the Bankhead-Coley Cancer Research
Program, Grant 1BG07.
’ ACKNOWLEDGMENT
We thank Prof. B. Law for initial help with tumor implantation.
’ ABBREVIATIONS
MET, met proto-oncogene (hepatocyte growth factor receptor);
PDGFR, platelet-derived growth factor receptor; RTK, receptor
tyrosine kinase; SAR, structureꢀactivity relationship; VEGF, vascu-
lar endothelial growth factor; VEGFR, VEGF receptor; Me, methyl;
Bu, butyl; Pr, propyl; PMB, p-methoxybenzyl; Troc, 2,2,2-trichlor-
oethoxycarbonyl; DMAP, 4-(dimethylamino)pyridine; DDQ, 2,3-
dichloro-4,5-dicyanobenzoquinone; Fmoc, 9-fluorenylmethoxycar-
bonyl; Trt, triphenylmethyl; HATU, O-(7-azabenzotriazol-1-yl)-N,
N,N0,N0-tetramethyluronium hexafluorophosphate; DIEA, diisopro-
pylethylamine; Tf, trifluoromethylsulfonyl
’ REFERENCES
(1) Baselga, J. Targeting tyrosine kinases in cancer: the second wave.
Science 2006, 312, 1175–1178.
(20) Numajiri, Y.; Takahashi, T.; Doi, T. Total synthesis of (ꢀ)-
apratoxin A, 34-epimer, and its oxazoline analogue. Chem. Asian J. 2009,
4, 111–125.
(21) Xu, Z.; Chen, Z.; Ye, T. Synthesis of polyketide segment of
apratoxin A. Tetrahedron: Asymmetry 2004, 15, 355–363.
(22) Roush, W. R.; Ando, K.; Powers, D. B.; Palkowitz, A. D.;
Halterman, R. L. Asymmetric synthesis using diisopropyl tartrate
modified (E)- and (Z)-crotylboronates: Preparation of the chiral
crotylboronates and reactions with achiral aldehydes. J. Am. Chem. Soc.
1990, 112, 6339–6348.
(23) Inanaga, J.; Hirata, K.; Saeki, H.; Katsuki, T.; Yamaguchi, M. A
rapid esterification by means of mixed anhydride and its application to
large-ring lactonization. Bull. Chem. Soc. Jpn. 1979, 52, 1989–1993.
(24) Travis, B. R.; Narayan, R. S.; Borhan, B. Osmium tetroxide-
promoted catalytic oxidative cleavage of olefins: an organometallic
ozonolysis. J. Am. Chem. Soc. 2002, 124, 3824–3825.
(25) You, S.-L.; Razavi, H.; Kelly, J. W. A biomimetic synthesis of
thiazolines using hexaphenyloxodiphosphonium trifluoromethanesulfo-
nate. Angew. Chem., Int. Ed. 2003, 42, 83–85.
(26) Carmeliet, P.; Jain, R. K. Angiogenesis in cancer and other
diseases. Nature 2000, 407, 249–257.
(27) Folkman, J. Angiogenesis in cancer, vascular, rheumatoid and
other disease. Nat. Med. 1995, 1, 27–31.
(28) Kerbel, R. S. Tumor angiogenesis: Past, present and the near
future. Carcinogenesis 2000, 21, 505–515.
(29) Luesch, H.; Chanda, S. K.; Raya, R. M.; DeJesus, P. D.; Orth,
A. P.; Walker, J. R.; Izpisꢀua Belmonte, J. C.; Schultz, P. G. A functional
genomics approach to the mode of action of apratoxin A. Nat. Chem.
Biol. 2006, 2, 158–167.
(2) Lurje, G.; Lenz, H.-J. EGFR signaling and drug discovery.
Oncology 2009, 77, 400–410.
(3) Chow, L. Q.; Eckhardt, S. G. Sunitinib: From rational design to
clinical efficacy. J. Clin. Oncol. 2007, 25, 884–96.
(4) Kling, J. Bundling next-generation cancer therapies for synergy.
Nat. Biotechnol. 2006, 24, 871–872.
(5) Martinelli, E.; Troiani, T.; Morgillo, F.; Rodolico, G.; Vitagliano,
D.; Morelli, M. P.; Tuccillo, C.; Vecchione, L.; Capasso, A.; Orditura, M.;
De Vita, F.; Eckhardt, S. G.; Santoro, M.; Berrino, L.; Cardiello, F.
Synergistic antitumor activity of sorafenib in combination with epider-
mal growth factor receptor inhibitors in colorectal and lung cancer cells.
Clin. Cancer Res. 2010, 16, 4990–5001.
(6) Hurwitz, H.; Fehrenbacher, L.; Novotny, W.; Cartwright, T.;
Hainsworth, J.; Heim, W.; Berlin, J.; Baron, A.; Griffing, S.; Holmgren,
E.; Ferrara, N.; Fyfe, G.; Rogers, B.; Ross, R.; Kabbinavar, F. Bevacizu-
mab plus irinotecan, fluorouracil, and leucovorin for metastatic color-
ectal cancer. N. Engl. J. Med. 2004, 350, 2335–2342.
(7) Koutras, A. K.; Starakis, I.; Kyriakopoulou, U.; Katsaounis, P.;
Nikolakopoulos, A.; Kalofonos, H. P. Targeted therapy in colorectal
cancer: Current status and future challenges. Curr. Med. Chem. 2011,
18, 1599–1612.
(8) Luesch, H.; Yoshida, W. Y.; Moore, R. E.; Paul, V. J.; Corbett,
T. H. Total structure determination of apratoxin A, a potent novel
cytotoxin from the marine cyanobacterium Lyngbya majuscula. J. Am.
Chem. Soc. 2001, 123, 5418–5423.
(9) Luesch, H.; Yoshida, W. Y.; Moore, R. E.; Paul, V. J.; Corbett,
T. H. New apratoxins of marine cyanobacterial origin from Guam and
Palau. Bioorg. Med. Chem. 2002, 10, 1973–1978.
(10) Gutierrez, M.; Suyama, T. L.; Engene, N.; Wingerd, J. S.;
Matainaho, T.; Gerwick, W. H. Apratoxin D, a potent cytotoxic
cyclodepsipeptide from Papua New Guinea collections of the marine
cyanobacteria Lyngbya majuscula and Lyngbya sordida. J. Nat. Prod. 2008,
71, 1099–1103.
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