Rare Streptomyces N-formyl amino-salicylamides inhibit oncogenic K-Ras

Article abstract of DOI:10.1021/ol502376e

During a search for inhibitors of oncogenic K-Ras, we detected two known and two new examples of the rare neoantimycin structure class from a liquid cultivation of Streptomyces orinoci, and reassigned/assigned structures to all based on detailed spectroscopic analysis and microscale C₃ Marfey's and C₃ Mosher chemical degradation/derivatization/analysis. SAR investigations inclusive of the biosynthetically related antimycins and respirantin, and synthetic benzoxazolone, documented a unique N-formyl amino-salicylamide pharmacophore as a potent inhibitor of oncogenic K-Ras.

Full text of DOI:10.1021/ol502376e

Letter
pubs.acs.org/OrgLett
Rare Streptomyces N-Formyl Amino-salicylamides Inhibit Oncogenic
K‑Ras
Angela A. Salim,† Kwang-Jin Cho,‡ Lingxiao Tan,‡ Michelle Quezada,† Ernest Lacey,§ John F. Hancock,‡
and Robert J. Capon*,†
†Institute for Molecular Bioscience, The University of Queensland, St. Lucia, QLD 4072, Australia
‡Integrative Biology and Pharmacology, The University of Texas Medical School, Houston, Texas 77030, United States,
§Microbial Screening Technologies Pty. Ltd., Building C 28-54 Percival Road, Smithfield, NSW 2164, Australia
S
* Supporting Information
ABSTRACT: During a search for inhibitors of oncogenic K-Ras, we detected two
known and two new examples of the rare neoantimycin structure class from a liquid
cultivation of Streptomyces orinoci, and reassigned/assigned structures to all based on
detailed spectroscopic analysis and microscale C3 Marfey’s and C3 Mosher chemical
degradation/derivatization/analysis. SAR investigations inclusive of the biosyntheti-
cally related antimycins and respirantin, and synthetic benzoxazolone, documented a
unique N-formyl amino-salicylamide pharmacophore as a potent inhibitor of
oncogenic K-Ras.
as GTPases, which are ubiquitously expressed as three
isoforms (H-Ras, N-Ras, and K-Ras), act as key molecular
R
switches that regulate cell growth, proliferation, and differ-
entiation.1 Constitutively activated K-Ras is involved in 90% of
pancreatic, 45% of colorectal, and 35% of lung carcinomas.2 Ras
signaling mechanisms have been researched extensively, and
studies have shown that preventing Ras plasma membrane (PM)
localization blocks all oncogenic activity.3 Although an attractive
anticancer target, the development of potent and selective
inhibitors of Ras protein PM localization with clinical application
has proven both challenging and elusive.
To address this challenge, we employed a high-throughput,
high-content assay to screen a library of microbial extracts,
establishing Streptomyces orinoci (MST-AS4461) as a source of
secondary metabolites that inhibit Ras protein PM localization.4
Bioassay-guided fractionation of an 8 L liquid fermentation
yielded the known secondary metabolites neoantimycin (1) and
neoantimycin F (2), along with two new exemplars, neo-
antimycin G (3) and neoantimycin H (4) (Figure 1), as
exceptionally potent (IC50 3−10 nM) inhibitors of K-Ras PM
localization. Planar structures were assigned to 1−4 on the basis
of detailed spectroscopic analysis, with microscale C3 Marfey’s
and C3 Mosher analyses permitting assignment of absolute
configurations, including revisions to 15 and 2.6 To support
structure−activity relationship (SAR) investigations into the K-
Ras modulatory properties of neoantimycins, we obtained the
biosynthetically related microbial metabolites, antimycin A2a
(5), antimycin A4a (6), and respirantin (7), and prepared the
novel benzoxazolone analog 8 (Figure 1). SAR studies
established that the natural products 1−7 were all potent
inhibitors of K-Ras PM localization and correlated this activity
with the presence of an N-formyl amino-salicylamide moiety. Of
note, this latter functionality is restricted in the natural products
literature to the neoantimycins, antimycins, and respirantin.
Figure 1. Neoantimycins and analogs (available to this study).
What follows is an account of our chemical and biological studies
into 1−8.
HRESI(+)MS measurements supported by analysis of NMR
(DMSO-d6 and CDCl3) data (Supporting Information (SI),
Tables S1a-b and S2a-b) permitted assignment of planar
structures to 1 (C36H46N2O12) and 2 (C35H44N2O12), consistent
with those previously ascribed to neoantimycin5,7 and neo-
antimycin F.6 Comparison of [α]D measurements for the
cometabolites 1 (+58.7) and 2 (+55.2), with that published for
neoantimycin (+58.3),7b supported the proposition that these
Received: August 11, 2014
Published: September 19, 2014
© 2014 American Chemical Society
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Letter
compounds belong to a common antipodal series. Comparable
HRESI(+)MS and NMR analyses (SI, Tables S3 and S4)
permitted assignment of planar structures to 3 (C37H48N2O12)
and 4 (C36H44N2O12), consistent with two new neoantimycin
homologues allocated the trivial names neoantimycin G and H
respectively. All the cometabolites 1−4 incorporate an aromatic
and two aliphatic hydroxycarboxylic acid residues, embedded in a
macrolactone ring featuring a threonine bearing a pendant N-
formyl aminosalicylic acid residue. Identification and assembly of
these residues were greatly assisted by diagnostic COSY and
HMBC correlations (Figure 2). In advance of chemical
Figure 3. Reported neoantimycin-like metabolites (9−15).
were inferred rather than proven. In summary, despite a history
extending back several decades, inclusive of chemical, spectro-
scopic, biosynthetic, and pharmacological investigations across
nine metabolites (1−2 and 9−15), knowledge of the neo-
antimycin structures (in particular relative and absolute
configuration) remains incomplete. In furthering our inves-
tigations into the chemistry and pharmacology of 1−4, we
applied a rapid microscale analytical methodology capable of
unambiguously assigning absolute configurations across the full
array of chiral centers within the neoantimycins (and related
classes).
Figure 2. Diagnostic 2D NMR (DMSO-d6) correlations for 1−4.
To assign the threonine configuration in 1−4 we employed a
C3 Marfey’s analysis. Microscale (50 μg) acid hydrolysates from
1−4 were derivatized with the L enantiomer of Marfey’s reagent
(L-FDAA), as were authentic standards of L-Thr and L-allo-Thr.
To prepare chromatographically equivalent surrogates for L-
FDAA derivatives of D-Thr and D-allo-Thr, authentic standards of
L-Thr and L-allo-Thr were derivatized with the D enantiomer of
Marfey’s reagent (D-FDAA). Subsequent C3 Marfey’s analyses
(HPLC-DAD-ESIMS) confirmed that 1−4 incorporate an L-Thr
residue (SI Figure S8).
In addition to L-Thr, the microscale acid hydrolysis of 1 yielded
(S)-2-hydroxyisovaleric acid (16) and (2S,3S)-2-hydroxy-3-
methylvaleric acid (17) (Figure 4).7b By contrast, acid hydrolysis
of 2−4 yielded one or both of these carboxylic acids, or their
enantiomers. To assign absolute configurations to these residues
in 2−4, a sample of the acid hydrolysate from 1 was derivatized
with the R enantiomer of Mosher’s reagent to yield authentic
standards of the (R)-MTPA esters 16a and 17a, while a duplicate
degradation, derivatization, and analytical studies designed to
assign absolute configurations to 1−4, it is useful to briefly review
the history of the neoantimycin scaffold.
The neoantimycins are a rare class of microbial natural
product, biosynthetically related to the ring-contracted anti-
mycins (e.g., 5−6) and ring-expanded respirantin (e.g., 7).8
Isolated in 1967 from a South American soil isolate of
Streptomyces (formerly Streptoverticillium) orinoci,7c the partial
configuration of neoantimycin was assigned in 1969 by
preparative-scale (1 g) degradation that yielded methyl (S)-2-
hydroxyisovalerate and methyl (2S,3S)-2-hydroxy-3-methyl-
valerate.7b At that time an L-Thr configuration was also asserted,
but not proven.7b A subsequent 1998 study used NMR to
(incorrectly, see below) attribute a 3S,4S configuration to the 3,4-
dihydroxy-2,2-dimethyl-5-phenylvaleric acid (DDPVA) residue
in neoantimycin.5 Only a handful of neoantimycin-like
metabolites have since been reported in the scientific literature
(Figure 3). Planar structures for SW-163A (9) and SW-163B
(10) were reported in 20019 from a Japanese soil Streptomyces sp.
(SNA15986), while the planar structures for prunustatin A (11),
and JBIR-04 (12) and JBIR-05 (13), were reported in 200510 and
200711 respectively from the Okinawan soil Streptomyces
violaceoniger (4521-SVS3), as down-regulators of the molecular
chaperone GRP-78. The absolute configurations of 9 and 11
were subsequently determined in 200712 by chemical degrada-
tion and derivatization. Neoantimycins D (14), E (15), and F (2)
were reported in 20136 using a genome mining approach on S.
orinoci, although configurational assignments made at that time
Figure 4. Hydrolysis products and Mosher esters from 1−4.
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Letter
1
sample was derivatized with (S)-MTPA to yield authentic
standards of the (S)-MTPA esters 16b and 17b (the latter being
chromatographic surrogates for the (R)-MTPA esters of the
enantiomers of 16 and 17 respectively) (Figure 4). Both sets of
standards were subjected to C3 Mosher analysis, confirming
resolution of all four diastereomeric MTPA esters (Figure 5A).
(C36H42N2O12). Key differences in the H NMR (DMSO-d6)
data for 8 versus 4 included the absence of formamide
(NHCHO) and phenolic (ArOH) resonances and changes in
the chemical shifts for the aminosalicylic acid protons H-5 (Δδ
−0.76), H-6 (Δδ +0.32), and H-7 (Δδ −0.68) (SI Table S6). As
neoantimycins bearing benzoxazolones have not been described
previously, the formation of 8 represented a valuable SAR
opportunity.
To advance SAR investigations further, we sourced and
characterized authentic samples of the biosynthetically related
antimycins A2a (5) and A4a (6), and respirantin (7) (Figure 1, SI
Figures S10−S11). The antimycin structure class was first
reported in 1949 (as an inseparable complex),13 with the planar
structure for antimycin A1 assigned in 196114 and the absolute
configuration in 1972.15 With several dozen antimycins reported
to date, selected antimycins have been attributed antifungal,13
insecticidal,16 nematocidal,17 and piscicidal18 activity, as well as
exhibiting binding to cytochrome c oxidoreductase to inhibit the
electron-transfer activity of ubiquinol in the mitochondrial
respiratory chain19 and to Bcl-2 to induce apoptotic cell death.20
By comparison, literature accounts of respirantin (7) are limited
and include its initial 1993 discovery as an insecticidal antibiotic
from a Japanese soil Streptomyces sp.21 and its 2007 rediscovery
(along with kitastatin) as a cytotoxic anticancer agent from an
Alaskan soil Kitasatospora sp.22
Historically, the assignment of absolute configurations to
antimycins and respirantin has proven problematic as in the case
for neoantimycins (see above) and would clearly benefit from a
more efficient and reliable methodology. Significantly, the C3
Marfey’s and C3 Mosher methodology described above meet all
these criteria and are equally applicable to neoantimycins,
antimycins, and respirantin.
We employed quantitative confocal imaging to measure the
ability of 1−8 to mislocalize oncogenic mutant K-Ras (mGFP-K-
RasG12 V) from the PM of intact Madin−Darby canine kidney
(MDCK) cells.4 These experiments established 1−7 as excep-
tionally potent inhibitors of K-Ras PM localization (Table 1), as
Figure 5. Single ion extracts (SIE) from C3 Mosher HPLC-ESIMS
chromatograms of analytes prepared from (A) 1 [(i) m/z 333 [M−H]−
(16a−b), (ii) m/z 347 [M−H]− (17a−b), (iii) m/z 437 [M + H]+
(18a−b)]; (B) 2 [(i) 333 [M−H]− (16a) and (ii) m/z 437 [M + H]+
(18a)]; (C) 3, [(i) m/z 347 [M−H]− (17a) and (ii) m/z 437 [M + H]+
(18a)]; and (D) 4 [(i) m/z 333 [M−H]− (16a) and (ii) m/z 347 [M−
H]− (17a)].
Subsequent derivatization of acid hydrolysates from 2−4 with
(R)-MTPA followed by C3 Mosher analysis confirmed that 2
incorporates two residues of 16 (Figure 5B), 3 incorporates two
residues of 17 (Figure 5C), and 4 incorporate one residue each of
16 and 17 (Figure 5D).
Table 1. K-Ras PM Mislocalization and MTT Cytotoxicity
Assay Results for 1−8
K-Ras PM mislocalization
MTT cytotoxicity
Alkaline hydrolysis of 1 had been reported in 1975 to yield the
DDPVA lactone 18,7a for which a 1998 study applied NOE
measurements to assign a 3S,4S configuration,5 enantiomeric
with the 3R,4R lactone produced during a 2007 alkaline
hydrolysis of SW-163A (9).12 Given this unexpected stereo-
complexity, we elected to use alkaline hydrolysis to independ-
ently investigate the absolute configuration of the DDPVA
residues in 1−4. Alkaline hydrolysis of 1 (5 mg) yielded the
lactone 18, which was derivatized to yield authentic samples of
the (R)-MTPA ester 18a and the (S)-MTPA ester 18b. The 1H
NMR (CDCl3) data for 18a−b recovered from 1 proved
identical with esters reported from 9 (SI Table S5),12 with a
Mosher NMR (δS − δR) analysis (Figure 4) establishing a 3R,4R
configuration (Figure 1), revising the 1998 3S,4S assignment.5
Application of a microscale (50 μg) alkaline hydrolysis and C3
Mosher analysis revealed a common 3R,4R configuration about
the DDPVA residue in 2 and 3 (Figure 5B and 5C).
SW620 IC50 SW620 Ad300 IC50
a
b
Emax
IC50 (nM)
(μM)
(μM)
FR
1
2
3
4
5
6
7
8
0.60 0.03
0.56 0.01
0.62 0.01
0.60 0.02
0.78 0.02
0.79 0.02
0.78 0.02
inactive
2.9 0.7
9.9 0.2
7.9 0.5
6.4 0.5
7.9 0.3
3.7 0.3
0.7 0.1
inactive
0.06
0.16
0.04
0.07
0.15
0.12
0.01
>30
0.23
0.61
0.26
0.19
0.32
0.44
0.03
c
3.8
3.8
6.5
2.7
2.1
3.7
3.0
a
Emax quantifies the maximum extent of mislocalization of K-Ras from
b
the PM to the endomembrane. FR: fold resistance is calculated as the
c
IC50 ratio for SW620 Ad300 versus SW620. Not tested due to limited
amount of compound.
compared to the reported inhibitory properties of spiro-
oxanthromicin (IC50 27 μM)23 and staurosporine (IC50 0.42
nM).4 These results were particularly informative as they indicate
that the N-formyl amino-salicylic acid residue, common to 1−7
(but not 8), and restricted in the natural products literature to the
neoantimycins, antimycins and respirantin, correlates with K-Ras
To complete our configurational investigations, a sample of 1
was subjected to Dess-Martin oxidation to yield a product
identical with 4 (SI Figure S9), confirming both the structure and
absolute configuration of 4 (Figure 1). An unexpected byproduct
of Dess-Martin oxidation was identified as the benzoxazolone 8
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Organic Letters
Letter
(7) (a) Benedetti, E.; Ganis, P.; Bombieri, G.; Caglioti, L.; Germain, G.
Acta Crystallogr., Sect. B 1975, B31, 1097. (b) Caglioti, L.; Misiti, D.;
Mondelli, R.; Selva, A.; Arcamone, F.; Cassinelli, G. Tetrahedron 1969,
25, 2193. (c) Cassinelli, G.; Grein, A.; Orezzi, P.; Pennella, P.;
Sanfilippo, A. Arch. Mikrobiol. 1967, 55, 358.
mislocalization. Furthermore, we determined that selected
neoantimycins, antimycins, and respirantin exhibited increased
cytotoxicity toward human lung cancer cells that feature a
mutated oncogenic K-Ras (e.g., A549) versus those that are not
K-Ras mutated (e.g., H522) (1, 50-fold; 5, >26-fold; and 7, >33-
fold).
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Finally, as up-regulation of ABC transporters such as P-
glycoprotein (P-gp) in cancers can lead to accelerated drug efflux
and multidrug resistance (MDR), we assayed 1−7 against the
colon cancer cell line SW620 and its P-gp overexpressing
daughter cell line SW620 Ad300. In control studies we confirmed
that SW620 Ad300 cells were 22-fold (FR = 22) less sensitive to
doxorubicin than SW620 cells and that coadministration with the
P-gp inhibitor verapamil quenched this efflux effect (FR = 2.4).
By comparison, 1−7 exhibited comparable cytotoxicity toward
both SW620 and SW620 Ad300 cell lines (FR = 2.1−6.5),
confirming that they were largely unaffected by P-gp mediated-
MDR (Table 1).
In conclusion, this study explored the rare neoantimycin,
antimycin, and respirantin structure classes, developing new
microanalytical methodology for assigning absolute configu-
rations, revising structures for known, and discovering and
assigning structures to new neoantimycins. Significantly, this
study discovered a new class of low nM K-Ras inhibitor that is not
subject to P-gp mediated drug efflux.
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ASSOCIATED CONTENT
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(20) Tzung, S.-P.; Kim, K. M.; Basanez, G.; Giedt, C. D.; Simon, J.;
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(23) Salim, A. A.; Xiao, X.; Cho, K.-J.; Piggott, A. M.; Lacey, E.;
Hancock, J. F.; Capon, R. J. Org. Biomol. Chem. 2014, 12, 4872.
S
* Supporting Information
These data include general experimental procedures, NMR
spectroscopic data, C3 Marfey’s and C3 Mosher methods, and K-
Ras and MTT cytotoxicity assays. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: r.capon@uq.edu.au.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank S. E. Bates and R. W. Robey (NIH, Bethesda, MD) for
providing SW620 and SW620 Ad300, A. M. Piggott (UQ) for
HRMS and NMR spectroscopic support, and D. Vuong and A. E.
Lacey (MST) for assistance with the fermentation and
metabolite purification. This research was funded in part by
The University of Queensland, the Institute for Molecular
Bioscience, the Cancer Prevention and Research Institute of
Texas (RP130059), and the Australian Research Council
(DP120100183 and LP120100088).
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