Photoactivated Colibactin Probes Induce Cellular DNA Damage

Article abstract of DOI:10.1002/anie.201812326

Colibactin is a small molecule produced by certain bacterial species of the human microbiota that harbour the pks genomic island. Pks⁺ bacteria induce a genotoxic phenotype in eukaryotic cells and have been linked with colorectal cancer progression. Colibactin is produced in a benign, prodrug form which, prior to export, is enzymatically matured by the producing bacteria to its active form. Although the complete structure of colibactin has not been determined, key structural features have been described including an electrophilic cyclopropane motif, which is believed to alkylate DNA. To investigate the influence of the putative “warhead” and the prodrug strategy on genotoxicity, a series of photolabile colibactin probes were prepared that upon irradiation induced a pks⁺ like phenotype in HeLa cells. Furthermore, results from DNA cross-linking and imaging studies of clickable analogues enforce the hypothesis that colibactin effects its genotoxicity by directly targeting DNA.

Full text of DOI:10.1002/anie.201812326

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Accepted Article
Title: Photoactivated Colibactin Probes Induce Cellular DNA Damage
Authors: Lindon William Kirk Moodie, Madlen Hubert, Xin Zhou,
Michael F Albers, Richard Lundmark, Sjoerd Wanrooij, and
Christian Hedberg
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201812326
Angew. Chem. 10.1002/ange.201812326
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10.1002/anie.201812326
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Photoactivated Colibactin Probes Induce Cellular DNA Damage
Lindon W. K. Moodie,* Madlen Hubert, Xin Zhou, Michael F. Albers, Richard Lundmark, Sjoerd
Wanrooij and Christian Hedberg*
Abstract: Colibactin is a small molecule produced by certain
A
O
O
Inactive
Precolibactin
bacterial species of the human microbiota that harbour the pks
genomic island. Pks+ bacteria induce a genotoxic phenotype in
eukaryotic cells and have been linked with colorectal cancer
progression. Colibactin is produced in a benign prodrug form which,
prior to export, is enzymatically matured by the producing bacteria to
its active form. Although the complete structure of colibactin has not
been determined, key structural features have been described
including an electrophilic cyclopropane motif, which is believed to
alkylate DNA. To investigate the influence of the putative “warhead”
and the prodrug strategy on genotoxicity, we prepared a series of
photolabile colibactin probes that upon irradiation induced a pks+ like
phenotype in HeLa cells. Furthermore, results from DNA cross-
linking and imaging studies of clickable analogues enforce the
hypothesis that colibactin effects its genotoxicity by directly targeting
DNA.
DNA damage
NH2
O
H
N
ClbP
C13H27
N
Colibactin
H3N Colibactin
H
N-myristoyl-D-Asn
B
O
HO
HO
R1
NH2
O
O
O
H
O
N
N
N
N
C13H27
N
H
H
S
S
O
O
NH
A
O
O
1, R1 = H
2, R1 = A
O
N
N
R2
N
S
S
B
C
NH2
O
H
HO
Whilst co-evolution between humans and their resident
microbes has yielded beneficial relationships, shifts in
commensal microbial populations are linked with a number of
disease states.[1] However, these correlations are not
necessarily indicative of causation and it is important to
understand the interactions driving these phenotypes at the
molecular level.[2] In 2006, Nougayréde et al reported that
certain commensal species of Escherichia coli and
Enterobacteriaceae induced DNA damage and genomic
instability during infection of eukaryotic cells.[3] This phenotype
was attributed to the pks genomic island that produces the small
molecule colibactin. Carriage of pks containing E. coli strains is
overrepresented in biopsies from patients suffering colorectal
cancer (CRC), inflammatory bowel disease and familial
adenomatous polyposis.[4] Furthermore, pks+ E. coli enhance in
N
C13H27
N
H
O
HN
O
NH
O
N
O
3, R2 = B
4, R2 = C
O
S
SH
HO
O
S
S
N
N
HO
NH2
HN
O
N
O
H
O
O
N
O
H
C13H27
N
N
H
O
5
Figure 1. (A) Enzymatic prodrug mechanism of colibactin genotoxicity. (B)
Precolibactins of the lactam (1-2), pyridone (3-4) and macrocycle (5) classes.
5]
vivo tumour growth in mice that are predisposed to CRC.[4a,
Colibactin is produced in the bacterial cytoplasm in an inactive
prodrug-like form (precolibactin) (Figure 1A). After export into
the periplasmic space,[6] the precolibactin is cleaved by the
peptidase ClbP to yield the benign N-myristoyl-D-asparagine[7]
and the active genotoxic colibactin “warhead”. It has been
proposed that this prodrug mechanism, in combination with the
production of the protective protein ClbS,[8] reduces autotoxicity.
Given these factors, colibactin has been the subject of several
reviews.[9]
To date, the isolation of a colibactin metabolite produced by
the full complement of biosynthetic genes required for in vivo
genotoxicity has proven elusive. Furthermore, no de-acylated
active colibactins have been isolated, presumably due to low
production and instability. ClbP mutant E. coli strains have
allowed the isolation of more stable pre-colibactins. However,
evidence suggests that ClbP deletion alters the metabolite
profile.[10] Three classes of precolibactins have been reported:
lactams (1,2),[11] pyridones (3,4)[12] and the macrocycle 5 (Figure
1B).[13] The lactam type class is of significant interest due to their
spirocyclopropane containing heterocycle, a motif reminiscent of
known DNA and protein alkylating molecules.[14] As
precolibactins are not genotoxic, it has been proposed that, after
deacylation by ClbP and cyclization to form an a,b-unsaturated
iminium species, the electrophilic cyclopropane is activated
towards an endogenous nucleophile to affect its genotoxicity
(Figure 2A).[11a, 11b] Recent studies have shown that precolibactin
1[11a] and synthetic colibactin analogues[15] can crosslink (to
some extent) and alkylate purified DNA, and nucleobase-
colibactin adducts have been identified by mass spectrometry.[16]
However, although the pks gene cluster has been investigated in
infection models, studies of homogenous, activated colibactins
Dr. L. W. K. Moodie, Dr. M. F. Albers, Prof. Dr. C. Hedberg
Department of Chemistry
Umeå University
90187 Umeå, Sweden
E-mail: Lindon.moodie@umu.se
E-mail: Christian.hedberg@umu.se
Dr. M. Hubert, Prof. Dr. R. Lundmark
Integrative Medical Biology
Umeå University
90187 Umeå, Sweden
Dr. X. Zhou, Prof. Dr. S. Wanrooij, Prof. Dr. R. Lundmark
Medical Biochemistry and Biophysics
Umeå University
90187 Umeå, Sweden
Supporting information for this article is given via a link at the end of
the document
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10.1002/anie.201812326
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(isolated or synthetic) in a cellular context have not been
reported. Accordingly, we aimed to determine if an active
colibactin analogue featuring the putative cyclopropane-
H
O
H
:DNA
N
N
+H+
containing warhead, such as 6, could induce
a
cellular
ClbP
1
phenotype similar to that reported for pks+ bacterial infection.
Given the suspected instability of colibactins towards isolation,
we opted to use probes where the N-terminal amine is masked
by a photolabile group (nitroveratryloxycarbonyl, NVOC) (Figure
2).[17] Analogous to the work of Tietze et al,[18] probes could be
activated via photolysis and then added to the relevant biological
system for evaluation. This approach would avoid the enzymatic
maturation strategy used by the producing bacteria and,
importantly, provide proof of concept that genotoxicity can be
affected conditionally by alternate means of activation. During
the progress of our studies, the synthesis of cyclized colibactin
analogues was reported.[15a] However, compound 6 has yet to
be synthesised, or isolated via cleavage of its corresponding
precolibactin 1 using recombinant enzymes.[10, 11b] Accordingly,
probes 7 and 8 were synthesised (See SI), allowing comparison
of colibactin and precolibactin type motifs depending on if
photolysis is employed before treatment of cells. In addition, the
geminal dimethyl group of 8 allows us to elucidate the influence
of the cyclopropane motif.
Pathway A
NH
6
NH
O
DNA
DNA:
DNA crosslinking
hv
Pathway B
MeO
MeO
NO2
H
O
O
N
O
NH
O
masked warhead
photo-labile
NVOC group
7
O
NVOCHN
NH
8
O
O
O
NVOCHN
HN
NVOCHN
HN
NH
NH
O
O
O
O
9
10
Figure 2. Proposed mechanism of DNA crosslinking.[11a, 19] Probes 7-10
featuring the photolabile NVOC group, hv indicates irradiation.
To investigate the behavior of 7 and 8 in cell culture, we
examined three phenotypes that have been reported in pks+ E.
coli infection of HeLa cells: megalocytosis, cell cycle arrest at
the G2/M transition and histone H2AX phosphorylation.[3] Probes
7 and 8 (50 µM) were activated by irradiation at 365 nm
(activated and non-activated compounds denoted as +hv and –
hv, respectively) and then added to HeLa cells. Cell morphology
evaluation revealed that cells treated with 7(+hv) for 24 h
displayed the megalocytosis phenotype: cellular and nuclear
enlargement and the absence of mitosis (Figure 3A).
Megalocytosis was not observed for NVOC protected amines 7(-
hv) and 8(-hv), and 8(+hv), even after extended incubation
(Figure S2). Flow cytometry was used to monitor the effect of 7
and 8 on the cell cycle. After 72 h, only unsynchronized cells
treated with 7(+hv) arrested at the G2/M transition (4n DNA
content and negligible cell division) (Figure 3B). Cells
synchronized in the G1/S phase and treated with 7 and 8 (both
+/-hv) were monitored over 48 h. Cells dosed with 7(+hv) lagged
in the S phase (6 and 10 h) before halting at the G2/M transition.
7(-hv) also induced cell cycle delay (6 h), however the normal
cell cycle was restored after 10 h. Cells were also monitored for
H2AX phosphorylation, a sensitive marker for DNA double
strand breaks,[20] via immunostaining and fluorescence
microscopy. Only cells treated with 7(+hv) exhibited an increase
in gH2AX foci after 24 h (Figure 3C,S4). Consistent with cell
cycle studies, non-irradiated 7 induced H2AX activation after 6
h, which returned to background levels after 24 h (Figure S4).
These results show for the first time that an activated synthetic
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colibactin analogue containing the putative warhead can induce
these phenotypic responses observed during pks+ bacterial
infection. Precolibactin type structure, 7(-hv), elicited only a
transient response in terms of delaying cell cycle progression
and inducing gH2AX foci. Although this effect could be attributed
to NVOC hydrolysis, the stability of this group in cellular
environments has been noted.[21] The attenuated activity of 7(-
hv), along with the lack of observed genotoxicity of 7(+hv) at low
concentrations, suggests that cellular DNA repair mechanisms
can manage low-levels of colibactin induced DNA damage;
consistent with findings that low doses of pks+ E. coli induce
reversible activation of the DNA damage response in CHO cells,
and that cell lines deficient in the nonhomologous end-joining
DNA repair pathway were highly susceptible to cell death.[22]
These results, and the benign nature of dimethyl analogue 8,
support the hypotheses that colibactin genotoxicity is augmented
by enzymatic N-deacylation, and the cyclopropane group is vital
for genotoxicity. Whilst our work was under review, it was
reported that synthetic colibactin analogues induced gH2AX foci
but not 53BP1 activation in cell culture; implying that while
model colibactins are
a valuable tool they do not fully
recapitulate the phenotype of pks+ E. coli infection.[15b]
Figure 3. (A) Megalocytosis phenotype. Cells incubated with 7 or 8 (both +/-
hν) (Giemsa stain). Scale bar, 40 μm (B) Cell cycle progression. Cells were
either synchronized in G1/S phase (Synchron) or left unsynchronized
(Unsynchron). After incubation with probes, DNA content was analyzed by
flow
cytometry.
(C)
Representative
confocal
images
showing
immunofluorescence of γ-H2AX and DNA (DAPI). Cells were treated with
compounds for 24 h. Scale bar, 10 μm. For A-C: HeLa cells treated with 50 µM
of compound, non-treated cells used as negative control. No significant effects
observed for 8(-hv) (Figure S3).
We next aimed to validate DNA as a cellular target.
Linearized pBR322 plasmid DNA was incubated with either 7 or
8 (pretreated with or without irradiation before DNA incubation:
+hv and –hv, respectively) and DNA crosslinking was evaluated
by agarose gel electrophoresis.[15a] After 4 h, only 7(+hv)
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induced concentration dependent interstrand crosslinking in vitro
(Figure 4A). Increased incubation times led to significant duplex
DNA degradation, highlighting the instability of these adducts.
attenuated reactivity of the warhead towards
nucleotide 1,4-addition requires further examination.
a second
Inspired by previous work on DNA targeting molecules,[24]
we used alkyne-containing analogues 9 and 10 that, after click
conjugation with an azide-functionalized fluorophore, are
capable of reporting cellular localization. Phenotype and DNA
crosslinking studies of these compounds were conducted to
verify their suitability as probes (Figures S5-S7). HeLa cells
were incubated with either 9 or 10 (+/-hv, 50 µM) for 24 h and,
after gH2AX immunostaining, were treated with a click cocktail
containing CuSO4, TBTA, TCEP and Alexa Fluor 647 azide. The
subcellular distribution of the click derived fluorescent signal was
examined by confocal fluorescence microscopy. Only cells
treated with 9(+hv) showed intense fluorescence, which
localized with the gH2AX signal within nuclei (DAPI) (Figure 5).
Accumulation in the nucleoli and minor cytoplasmic staining was
observed, which has also been noted during studies of platinum
based DNA targeting probes.[24d, 24e]
Considering that 7(-hv) provoked
a
mild, reversible pks+
phenotype, and 1 is reported to weakly crosslink DNA,[11a] it was
surprising that no significant DNA crosslinking was observed for
7(-hv); perhaps due to DNA-adduct instability under the assay
conditions. DNA melting curve analysis showed a concentration
dependent increase in Tm (temperature where duplex DNA is
50% dissociated) only for DNA incubated with 7(+hv), which is
consistent with the cross-linking assays (Fig 4B).
Figure 4. (A) DNA crosslinking assay. Linearized pBR322 plasmid incubated
with 7 and 8 (both +/-hν) at concentrations shown (4h, 37°C). After NaOH
induced strand separation, samples were run on 1% agarose Tris-Borate-
EDTA (TBE) gel (75V, 1 h). A non-denatured sample and Cisplatin (CP) were
used as positive controls. (B) DNA Melting curve analysis. Probes were
incubated with DNA (4h, 37°C). Curves show the negative first derivative of
fluorescence (F) with respect to temperature (T).
Figure 5. Click labeling of HeLa cells after treatment with 9 or 10 (both +/-hν).
Cells incubated with probe (50 μM) for 24 h, fixed, permeabilized and
immunostained for γ-H2AX. Alexa Fluor 647 azide was conjugated to the
alkyne motif via a CuSO4-catalyzed click reaction. DNA stained with DAPI.
Scale bar, 10 μm. No significant effects observed for 10(-hv) (Figure S8).
The ability of 7(+hv) to moderately crosslink DNA in a
cyclopropane dependent manner is consistent with the proposed
mechanism of genotoxicity where, after cyclopropane opening
by DNA, tautomerization leads to a species that behaves as a
Michael acceptor for a second nucleophilic attack by DNA
(Figure 2).[11a, 11b] This is supported by a recent study showing
that infection of cultured human cells with +pks bacteria results
in cross-linking of genomic DNA and, furthermore, that the
addition of extracellular DNA reduced this effect.[23] Healy et al
recently reported that colibactin analogues featuring the
unsaturated imine found in 6 only alkylated purified DNA.[15a]
Subsequent work using optimized assay conditions revealed
these compounds are also capable of DNA crosslinking,
however the adducts are more labile than those induced by pks+
E. coli.[15b] It is of note that these compounds were more potent
than 7(+hv), which can be attributed to the DNA binding
properties of the thiazole and amine containing side chains.
Taken together, this suggests that colibactin structure can
govern the nature of DNA damage. Determining whether this
reactivity is due to differing DNA-colibactin interactions or
Our crosslinking and cellular localization studies provide
strong evidence that activated colibactin molecules 7 and 9
featuring the lactam warhead directly target nuclear DNA.
Reactivity with members of the proteome is also possible,[25] and
activity based protein-profiling studies will aim to address this.
Colibactin represents an enigmatic example of a genotoxic
metabolite derived from our resident bacteria that has been
linked to both colorectal cancer and inflammatory bowel disease.
Our studies with light activated probes 7-10 show that colibactin
analogues containing the activated warhead motif are capable of
inducing phenotypes associated with pks+ E. coli infection. We
note that, given its low potency and lack of more complex
structural features resulting from the full complement of
biosynthetic genes in the pks cluster, compound 6 is not
responsible for genotoxicity induced by +pks bacteria. However,
our work does link an active colibactin analogue with cellular
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10.1002/anie.201812326
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23]
Future work will aim at determining the nature of colibactin –
DNA interactions using MS and CD-based techniques.[26] The
current study is also proof of principle that alternative prodrug
activation manifolds can be used to elicit DNA damage. Whilst
this work provides a compelling link between molecular structure
and cellular genotoxicity, the story of colibactin is far from
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Acknowledgements
[15]
This work is supported by the Knut and Alice Wallenberg
Foundation (SW and CH (KAW 2013.0187)), the Kempe
Foundation
(LM),
The
Swedish
Cancer
Foundation
[16]
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(CAN2014/746) (RL and MH) and The Hagbergs Foundation
(RL and MH). We acknowledge the Biochemical Imaging Center
at Umeå University and the National Microscopy Infrastructure,
NMI (VR-RFI 2016-00968) for microscopy assistance. We thank
Dr Tomas Kindahl and Dr Magnus Engqvist for optical rotation
measurements, Dr Thomas Kieselbach for mass spectrometry,
and Dr Anna Lena Chabes (Umeå University, Sweden) for
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Keywords: Colibactin • Microbiome • Photochemistry • DNA
[22]
Damage • Click Chemistry
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10.1002/anie.201812326
Angewandte Chemie International Edition
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Photoactivated colibactin probes
provoke a cellular phenotype that is
consistent with the DNA damage
observed during infection with
colibactin producing bacteria. DNA
cross-linking and cellular localisation
studies support the hypothesis that
the colibactin warhead targets nuclear
DNA.
Lindon W. K. Moodie,* Madlen Hubert,
Xin Zhou, Michael F. Albers, Richard
Lundmark, Sjoerd Wanrooij and
Christian Hedberg*
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Photoactivated Colibactin Probes
Induce Cellular DNA Damage
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