Zelkovamycin is an OXPHOS Inhibitory Member of the Argyrin Natural Product Family

Article abstract of DOI:10.1002/chem.202001577

Natural products (NPs) are an important inspirational source for developing drugs and chemical probes. In 1999, the group of ōmura reported the constitutional elucidation of zelkovamycin. Although largely unrecognized so far, this NP displays structural s

Full text of DOI:10.1002/chem.202001577

Accepted Article
Accepted Article
Title: Zelkovamycin is an OXPHOS inhibitory member of the argyrin
natural product family
Authors: Daniel Krahn, Geronimo Heilmann, Felix C. E. Vogel,
Chrisovalantis Papadopoulos, Susanne Zweerink, Farnusch
Kaschani, Hemmo Meyer, Alexander Roesch, and Markus
Kaiser
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To be cited as: Chem. Eur. J. 10.1002/chem.202001577
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01/2020

10.1002/chem.202001577
Chemistry - A European Journal
Zelkovamycin is an OXPHOS inhibitory member
of the argyrin natural product family
Daniel Krahn[a], Geronimo Heilmann[a], Felix C. E. Vogel[c, †], Chrisovalantis Papadopoulos[b],
Susanne Zweerink[a, ǁ], Farnusch Kaschani[a], Hemmo Meyer[b],
Alexander Roesch[c], Markus Kaiser[a, *]
[a]
[b]
[c]
Chemische Biologie, Universität Duisburg-Essen, ZMB, Fakultät für Biologie,
Universitätsstr. 2, 45117 Essen, Germany
Molekularbiologie I, Universität Duisburg-Essen, ZMB, Fakultät für Biologie,
Universitätsstr. 2, 45117 Essen, Germany
Department of Dermatology, University hospital Essen, West German Cancer Center,
University Duisburg-Essen and the German Cancer Consortium (DKTK)
†
Current address: Division of Tumor Metabolism and Microenvironment, German
Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg,
Germany
ǁ
Current address: University of Cologne, Faculty of Medicine and University Hospital of
Cologne, Department of Gastroenterology and Hepatology, Kerpener Str. 62, 50937
Cologne, Germany
*
corresponding author
1
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10.1002/chem.202001577
Chemistry - A European Journal
Abstract
Natural products (NPs) are an important inspirational source for developing drugs and
chemical probes. In 1999, the group of Ōmura reported the constitutional elucidation of
Zelkovamycin. Although largely unrecognized so far, this NP displays structural similarities as
well as differences to the argyrin NP family, a class of peptidic NPs with promising anticancer
activities and diverse mode-of-action at the molecular level. By a combination of structure
elucidation experiments, the first total synthesis of Zelkovamycin and bioassays, we
determined the configurational and revised the previously proposed molecular structure of
Zelkovamycin. The full structure assignment proves Zelkovamycin as an additional member
of the argyrins with however unique OXPHOS inhibitory properties. Zelkovamycin may
therefore not only serve as a new starting point for chemical inhibitors of the OXPHOS system,
but also guide customized argyrin NP isolation and biosynthesis studies.
2
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Main Text
Natural products (NPs) represent an exceptional source of inspirations in drug discovery and
frequently serve as valuable starting points in chemical probe synthesis.1 To harness the full
potential of NPs, a profound characterization of their chemical structure as well as inherent
bioactivities is however essential.2
In 1999, the group of Ōmura reported the isolation of the NP Zelkovamycin as a
moderately potent antibacterial cycloheptapeptide from a fermentation broth of Streptomyces
sp. K96-0670 (Fig. 1a).3 In 2002, the argyrins, a class of currently fifteen cycloheptapeptide
NPs, were discovered which besides antibiotic properties display very potent and promising
anticancer activities (Fig. 1b).4 Although largely unrecognized to date, Zelkovamycin obviously
displays pronounced structural similarities with the argyrin NP family, suggesting that also
Zelkovamycin may harbor so far unexplored bioactivities (Fig. 1a and Fig. 1b).
In addition to the lack of knowledge of its bioactivities, the exact molecular structure of
Zelkovamycin is however still elusive. The Ōmura group reported Zelkovamycin as a
cycloheptapeptide built up in the given order from the amino acids glycine (Gly), 2-amino
butyric acid (Abu), (Z)-dehydrobutyric acid ((Z)-Dhb), sarcosine (Sar), an alanine-thiazole
amino acid (AlaThz), 7-methoxy tryptophan ((7-MeO)Trp) and 2-methyl dehydro threonine ((2-
Me)Thr), a so far unprecedented amino acid in a peptide NP (Fig. 1a); the stereochemistry
of the different amino acids however was not reported. Moreover, despite the striking structural
similarities between Zelkovamycin and the argyrins, there are also distinct differences: Most
obviously, argyrins typically feature a 4-methoxy tryptophan ((4-MeO)Trp) or an unsubstituted
Trp, but never a (7-MeO)Trp residue, as reported for Zelkovamycin.4a-c Moreover, the position
of the (4-MeO)Trp residue in the argyrins is occupied by the unusual (2-Me)Thr moiety in
Zelkovamycin, while the (7-MeO)Trp residue of Zelkovamycin is at the site of an unmodified
Trp residue in the argyrins. Therefore, more detailed structural studies that enable
stereochemical assignment and a confirmation of its overall structure are required.
3
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Chemical synthesis is a well-established strategy to unambiguously elucidate the
molecular structure of a NP, including cyclopeptides.5 So far, chemical synthesis of
Zelkovamycin has not yet been achieved which contrasts with the argyrin NP family for which
a few total syntheses have already been established.6 In order to unambiguously establish the
molecular structure of Zelkovamycin and to characterize its biological activities, we describe
in this study a set of structure elucidation experiments, a total synthesis and bioassays that i)
resulted in a structural revision and full stereochemical assignment of Zelkovamycin, ii)
characterized Zelkovamycin as a member of the argyrin NP family, and iii) revealed
Zelkovamycin as an inhibitor of oxidative phosphorylation (OXPHOS).
We started our investigations by re-analyzing Zelkovamycin’s overall amino acid
sequence. With a small sample of isolated Zelkovamycin at hand, we performed an MS2
experiment and annotated the resulting spectrum with the software mMass using either the
originally reported amino acid sequence (AlaThz|(MeO)Trp|(2-Me)ΔThr|Gly|Abu|Dhb|Sar;
cyclic) or a more argyrin-like sequence where the (MeO)Trp group was placed on the position
of the (2-Me)Thr moiety (AlaThz|(2-Me)ΔThr|(MeO)Trp|Gly|Abu|Dhb|Sar; cyclic).7 As the
published sequence enabled assignment of 66% vs. 57% of the total intensity (Supporting
Fig. 1 and Supporting File 1) and most of the unassigned peaks were intense b2-b4 ions
containing the motif AlaThz|(MeO)Trp and Sar|AlaThz|(MeO)Trp (Δ mass smaller than 0.001
ppm; Supporting Table 1), this analysis strongly supported the reported sequence
connectivity (Fig. 1c).
Nevertheless, these experiments did not allow to differentiate between (4-MeO)Trp
and (7-MeO)Trp residues due to their identical m/z value or to define the stereochemistry of
the different amino acids. We thus performed a LC-MS-based Marfey’s analysis after HCl
hydrolysis of which enabled detection and stereochemical assignment of the amino acids Gly,
(D)-Abu, Sar and (D)-AlaThz (Fig. 1c and Supporting Fig. 2 & 3); of note, the stereochemical
assignments were thereby verified by direct comparison with corresponding chemically
synthesized Marfey’s modified amino acids (Supporting Fig. 3 & 4). The other amino acids
4
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of Zelkovamycin however could not be detected, neither as a Marfey derivative nor in an
unmodified form. Detection of the (MeO)Trp and Z-Dhb residue is hampered by their limited
chemical stability during hydrolysis conditions.8 The failed identification of a Marfey’s-modified
(2-Me)Thr residue however might be caused by its low reactivity due to its quaternary sp3-
center at the -position. For defining the so far missing exact constitution and configuration of
the (MeO)Trp residue, we thus resorted to a milder hydrolysis protocol and treated isolated
Zelkovamycin with 3 N TsOH in presence of 2% thioglycolic acid at 110 °C for 18 h.8
Subsequent LC-MS analysis of this hydrolysate in conjunction with a co-injection experiments
with chemically synthesized (L)-(4-MeO)Trp or (L)-(7-MeO)Trp revealed the presence of a (4-
MeO)Trp residue (as found in all other argyrins known so far, Supporting Fig. 5). A
subsequent Marfey’s analysis, using also a chemically synthesized Marf-(L)-(4-MeO)Trp as a
control, then demonstrated that the (4-MeO)Trp moiety has the (L)-configuration (Supporting
Fig. 6).
Altogether, these experiments thus led to a refined and partially revised proposal for
the chemical structure of Zelkovamycin in which all amino acids displayed ‘argyrin-like’
features (constitution and stereochemistry, Fig. 1d). Unfortunately, the stereochemistry of the
(2-Me)Thr residue however remained elusive. To obtain a full assignment and proof of the
chemical structure of Zelkovamycin and to provide further material for subsequent biological
assays, we thus arbitrarily assigned a (L)-configuration to this amino acid, inter alia because
the argyrins feature an (L)-amino acid on this position (Fig 1d). We then embarked for a total
synthesis using a flexible, modular synthesis approach. Accordingly, Zelkovamycin was
formally divided into three peptidic fragments (fragments A – C) with comparable structural
complexity and with a C-terminal amino acid resistant to racemization during fragment
coupling conditions (Fig. 2). As we suspected that the carbonyl moiety of the (2-Me)Thr
residue might cause problems during the synthesis, we decided to install it at the last stage of
the synthesis via oxidation from the corresponding alcohol.
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For synthesis of fragment A, we first generated Boc-(D)-AlaThz-OMe via the Arndt
methodology (Supporting Fig. 7).9 Cleavage of the Boc protecting group with 4 M HCl in
dioxane, followed by a coupling of Boc-Sar-OH using EDC/HOBt activation and another Boc
deprotection step then finalized fragment A (Fig. 3a). Fragment B was obtained from H-(D)-
Dab-OH that was Boc-protected, followed by an EDC/HOBt-mediated coupling with H-Thr-
OMe (Fig. 3b). This dipeptide was then converted into Boc-(D)-Dab-(Z)-Dhb-OH via an
EDC/Cu(I)Cl-mediated water elimination using a modified protocol from the Albericio group,10
followed by a methyl ester hydrolysis with LiOH. For the synthesis of fragment C, the
generation of the (2-Me)Thr and (4-MeO)Trp building block was first required. As the
retrosynthetic plan involved a late-stage oxidation of the hydroxyl residue of the (2-Me)Thr
moiety, the stereochemistry at this site can be freely chosen. We therefore decided to
synthesize allo-(L)-(2-Me)Thr-OMe due to an available synthesis protocol from the Goodman
group that was slightly adapted to our needs (Supporting Fig. 8).11 The synthesis of the
required (L)-Boc-(4-MeO)Trp-OH building block was carried out according to a Pd-catalyzed
heteroannulation approach from the Zhu group (Supporting Fig. 9).12 With these compounds
in hands, we then synthesized fragment C by first coupling Boc-(L)-(4-MeO)Trp-OH with allo-
(L)-(2-Me)Thr-OMe (Fig. 3c) via pre-treatment of allo-(L)-H-(2-Me)Thr-OMe with trimethyl silyl
chloride, followed by a treatment with HATU/HOAt. Saponification of the methyl ester with
LiOH, a subsequent coupling with H-Gly-OMe and Boc-deprotection with TFA in DCM then
completed the synthesis of fragment C. Assembly of the different fragments into Zelkovamycin
started with fragment A (H-Sar-(D)-AlaThz-OMe) that was coupled to fragment B (Boc-(D)-
Abu-(Z)-Dhb-OH) with EDC/HOBt (Fig. 4). The C-terminal ester of the resulting linear
polypeptide was hydrolyzed and this intermediate was coupled to fragment C ((L)-H-(4-
MeO)Trp-allo-(L)-(2-Me)Thr-Gly-OH) featuring the same coupling conditions. Cyclization was
then achieved by a sequence of reactions consisting of methyl ester hydrolysis, acidic
cleavage of the N-terminal Boc-protecting group and EDC/HOBt-mediated intramolecular
peptide coupling under high dilution conditions. After Dess-Martin periodinane oxidation, the
desired final product 1 was obtained. A comparison of the spectral data of synthesized
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Zelkovamycin with those reported for isolated Zelkovamycin as well as MS2 fragmentation,
LC-MS co-injection and NMR co-mixture experiments with isolated Zelkovamycin revealed
that our structure assignment, including the (L)-stereochemistry of the (2-Me)Thr residue,
was correct (Supporting Fig. 1, 10 and 11).3 Zelkovamycin thus has the chemical structure
shown in Fig. 1d.
We next started to evaluate the bioactivity of Zelkovamycin in a HeLa cell culture and
noted an apparent color change of the medium pH indicator (Phenol red) due to an unexpected
extracellular acidification after 48 h treatment with 20 µM Zelkovamycin (Fig. 5a). We could
mimic these effects by an alternative addition of 2.5 µM Carbonyl cyanide m-chlorophenyl
hydrazine (CCCP) or 1 µM Antimycin A (AMA), both inhibitors of oxidative phosphorylation
(OXPHOS) in mitochondria.13 As extracellular acidification may be caused by increased
secretion of lactate,14 we measured the medium lactate concentrations after 48 h treatment
with 20 µM Zelkovamycin and found increased levels in HeLa and an even stronger rise in
SH-SY5Y cells in a time-dependent manner (Fig. 5b).
These experiments thus indicate a Zelkovamycin-induced metabolic switch towards a
more glycolytic phenotype. To corroborate this with an unbiased approach, we performed a
full proteome analysis of HeLa cells after a 16 h treatment with 20 µM Zelkovamycin. The
comparison of the resulting label-free quantification (LFQ) profile with the corresponding
DMSO treatment resulted in the identification of 2424 proteins of which 101 were significantly
less and 5 more abundant after Zelkovamycin treatment (Supporting Table 2). Intriguingly,
60 (59%) of the less abundant proteins are annotated to be localized in mitochondria or linked
to mitochondrial function (Fig. 5c). A gene ontology (GO) and Kyoto Encyclopedia of Genes
and Genomes (KEGG) enrichment analysis of these proteins furthermore indicates that most
of these proteins are associated with ATP biosynthesis, suggesting that Zelkovamycin affects
mitochondrial function in eukaryotic cells (Fig 5d).14, 15
All performed assays so far thus indicate that long-term Zelkovamycin treatment leads
to an impairment of mitochondrial function. To deduce if Zelkovamycin however also directly
7
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affects mitochondria, we next tested its impact on cellular OXPHOS levels that can be
displayed by real-time cell measurements of the mitochondrial oxygen consumption rate
(OCR, ‘seahorse assay’). Zelkovamycin application to HeLa cells and the more OXPHOS-
dependent SH-SY5Y and WM3734 melanoma cells resulted in an immediate concentration-
dependent OCR reduction (Fig. 6a and Supporting Fig. 12).16 Zelkovamycin has a unique
chemical structure which is distinct from the related argyrins by the presence of the (2-Me)Thr
and the position of the (4-MeO)Trp moiety in the macrocycle. We therefore wondered if the
observed OXPHOS effects are Zelkovamycin-specific and depend on these unusual moieties.
Accordingly, we repeated the OCR measurements with Argryin B as an argryin representative
with a potential mitochondrial target (elongation factor G) and two additional Zelkovamycin
analogues 13 ((2-Me)Thr  (2-Me)Thr) and 14 ((4-MeO)Trp  Trp, Fig. 6b).4g-h In contrast
to Zelkovamycin, none of them displayed significant OXPHOS reduction at the tested 10 µM
concentration (Fig. 6c) nor led to higher lactate levels in cell medium (Supporting Fig. 13).
We thus showed that Zelkovamycin is a direct OXPHOS inhibitor. As impairment of
mitochondrial function should also lead to reduced cell viability, we tested the effect of
Zelkovamycin on 293T, HepG2, HeLa Kyoto, HeLa, HCT116 cells that were only weakly
affected after 48 h treatment, and SH-SY5Y neuroblastoma cells, for which cell viability was
reduced by more than 50% already at 20 µM Zelkovamycin (Supporting Fig. 14). This finding
is in accordance with the different metabolic needs of SH-SY5Y cells that gain their cellular
energy mostly by mitochondrial OXPHOS, thereby also explaining the observed higher lactate
levels (Fig. 5b), whereas the other five cancer cells rely on cytosol-located glycolysis.17
Accordingly, Zelkovamycin treatment of HeLa cells in a glucose-deprived and thus glycolysis-
restrained cell culture medium (4.5 mM instead of 25 mM glucose) resulted in a > 100-fold
reduction of cell viability (from an IC50 > 200 µM in glucose-rich medium to 0.5 µM in glucose-
deprived medium, Fig. 6d). Of note, SH-SY5Y cells, in accordance with their stronger
dependence on OXPHOS, displayed an IC50 of 19.5 µM in glucose-rich medium while they did
not grow at all on glucose-deprived medium. Finally, cell viability after 72 h of the OXPHOS-
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dependent melanoma cell line WM3734 was decreased by 75% at 10 µM Zelkovamycin or
60% at 5 µM Zelkovamycin, while no effect was observed on MeWo melanoma control cells
that can metabolize glutamine as a mitochondrial-alternative energy source (Fig. 6e).16
Overall, our studies therefore clarified and revised the chemical structure of
Zelkovamycin, demonstrating that it is a so far unrecognized member of the argyrin NP family.
Moreover, we established its first chemical synthesis, thereby allowing the future chemical
synthesis of customized analogues, e.g. for structure-activity relationship studies. Finally, we
demonstrated that Zelkovamycin is an OXPHOS inhibitor which may offer possibilities for
developing compounds with potential chemical biology or clinical use.18 We anticipate that
these findings demonstrate Zelkovamycin as a promising argyrin NP for future research, e.g.
of its direct molecular target or unusual biosynthesis.4c, 19
Conflict of Interest
There are no conflicts to declare.
Acknowledgement
Financial support by the CRC1093 (H.M. and M.K.) is greatly acknowledged.
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Figure 1. Structural similarities of Zelkovamycin and the argyrins and workflow for structure
elucidation. (a) Chemical structure of Zelkovamycin as published in the original structure
elucidation study3. The numbered bullet points indicate positions in the molecule where the
connectivity or stereochemistry was uncertain. Comparison of this structure with Argyrin B (b)
as a representative member of the argyrin natural product family reveal substantial structural
similarities but also differences that needed to be clarified for a full assignment and
confirmation of the chemical structure of Zelkovamycin.4a-c (c) Overview of the different
experiments that were carried out to define the structure and stereochemistry of Zelkovamycin.
(d) Revised structure of Zelkovamycin (1) with elucidated stereochemistry as deduced from
the above experiments. The configuration of the (2-Me)Thr residue (green color) could
however not be established and was therefore arbitrarily set to (L) to define a target structure
for the subsequent total synthesis. Later chemical synthesis then confirmed that this proposed
structure is the correct chemical structure of Zelkovamycin.
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Figure 2. Overview on the envisaged synthesis route to Zelkovamycin. Zelkovamycin was
retrosynthetically divided into three fragments A – C with comparable structural complexity
and a C-terminal amino acid insusceptible to racemization upon activation during peptide
couplings.
14
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Figure 3. Synthesis of the three fragments A (a), B (b) and C (c) for assembly of the
Zelkovamycin core ring system.
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Figure 4. Assembly of the fragments to the final natural product Zelkovamycin (1).
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Figure 5. Zelkovamycin induces lactate reflux and lower abundances of mitochondrial
proteins. (a) Zelkovamycin leads to extracellular medium acidification, which can be mimicked
17
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by the OXPHOS inhibitors CCCP and AMA.13 HeLa cells were treated with indicated
compounds for 48 h. Acidification was measured by absorbance (Abs) of phenol red (550 nm,
425 nm = reference). Error bars indicate standard deviation from three replicates and
significance was tested by student’s t-test. ** indicates a p-value < 0.01. (b) Zelkovamycin
increases extracellular medium lactate levels in a time-dependent manner. Lactate levels of
culture medium (DMEM: +10 % FCS and 1 % Penicillin/Streptomycin) were measured by an
(L)-Lactate Assay Kit after cells (upper panel: HeLa, lower panel: SH-SY5Y) were grown for
48 h and an aliquot of 100 µL was analyzed at the indicated time points. Error bars indicate
standard deviation from three replicates and significance was tested by student’s t-test. * and
** indicates a p-value < 0.05 or 0.01, respectively. (c) Zelkovamycin treatment results in lower
protein abundance of mitochondrial proteins after label-free MS-based proteome analysis.
HeLa cells were treated with 20 µM Zelkovamycin for 16 h and applied to MS-based proteome
analysis. The black line indicates significantly (permutation-based FDR = 0.01, s0 = 0.1, n =
6) regulated proteins (squares). Y-axis indicates the –log p-value, while x-axis indicates the
protein abundance difference in log2-fold change (LFQ). Proteins on the upper left of the
significance line are down-regulated, while proteins on the upper right of the significance line
are up-regulated. Proteins colored in green are annotated to be localized in mitochondria or
interact with mitochondrial function. Blue colored proteins are related with ATP biosynthesis.
(d) Enrichment analysis of all significantly down-regulated proteins. For enrichment analysis,
Gene Names were annotated by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and
Genomes (KEGG) terms. P-value was calculated by Benjamini-Hochberg-FDR. Enrichment
factor indicates increased occurrence of GO or KEGG term compared to approximate
occurrence. Displayed are top ten enriched GO or KEGG terms.
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10.1002/chem.202001577
Chemistry - A European Journal
Figure 6. Zelkovamycin inhibits OXPHOS in different cell lines. (a) Zelkovamycin inhibits OCR
in a concentration-dependent manner. OCR of HeLa cells was measured after application of
Zelkovamycin (black arrow). To depict RRC, FCCP was applied (yellow arrow). Error bars
indicate standard deviation from 6 - 14 replicates. (b) Chemical structure of two Zelkovamycin
derivatives (13 & 14) used for structure-activity relationship studies. Red colored moieties
indicate structural differences to Zelkovamycin. (c) Argyrin B or Zelkovamycin derivatives 13
& 14 do not affect OCR. OCR of HeLa cells was measured after application of the compounds
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10.1002/chem.202001577
Chemistry - A European Journal
(black arrow). To depict RRC, FCCP was applied (yellow arrow). Error bars indicate standard
deviation from 6 – 14 replicates. (d) Cell viability of SH-SY5Y and HeLa cells at high or low
glucose concentrations determined by a MTT assay in the concentration range of 0.25 - 100
µM Zelkovamycin and normalized to DMSO control. Error bars indicate standard deviation
from four replicates. (e) Cell viability of the OXPHOS-dependent melanoma WM3734 and
non-susceptible MeWo melanoma cell line determined by a MTT assay and normalized to
DMSO. Error bars indicate standard deviation from four replicates and significance was tested
by student’s t-test. **** indicates a p-value < 0.0001.
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10.1002/chem.202001577
Chemistry - A European Journal
Table of Contents
Zelkovamycin is an argyrin NP. By a combination of structural elucidation methodologies,
total synthesis and bioassays, the exact molecular structure of Zelkovamycin was assigned
and revised and its OXHPOS inhibitory properties revealed.
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