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The colibactin warhead crosslinks DNA.

Vizcaino MI, Crawford JM - Nat Chem (2015)

Bottom Line: Guided by metabolomic analyses, here we employ a combination of NMR spectroscopy and bioinformatics-guided isotopic labelling studies to characterize the colibactin warhead, an unprecedented substituted spirobicyclic structure.The warhead crosslinks duplex DNA in vitro, providing direct experimental evidence for colibactin's DNA-damaging activity.The data support unexpected models for both colibactin biosynthesis and its mode of action.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Chemistry, Yale University, New Haven, Connecticut 06510, USA [2] Chemical Biology Institute, Yale University, West Haven, Connecticut 06516, USA.

ABSTRACT
Members of the human microbiota are increasingly being correlated to human health and disease states, but the majority of the underlying microbial metabolites that regulate host-microbe interactions remain largely unexplored. Select strains of Escherichia coli present in the human colon have been linked to the initiation of inflammation-induced colorectal cancer through an unknown small-molecule-mediated process. The responsible non-ribosomal peptide-polyketide hybrid pathway encodes 'colibactin', which belongs to a largely uncharacterized family of small molecules. Genotoxic small molecules from this pathway that are capable of initiating cancer formation have remained elusive due to their high instability. Guided by metabolomic analyses, here we employ a combination of NMR spectroscopy and bioinformatics-guided isotopic labelling studies to characterize the colibactin warhead, an unprecedented substituted spirobicyclic structure. The warhead crosslinks duplex DNA in vitro, providing direct experimental evidence for colibactin's DNA-damaging activity. The data support unexpected models for both colibactin biosynthesis and its mode of action.

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The colibactin warhead crosslinks DNA(a) DNA alkylation by the warhead is hypothesized to occur through a homo-Michael addition reaction, followed by a (b) pseudo-intramolecular Michael addition reaction, generating a DNA interstrand crosslink. (c) DNA crosslinking was observed using an EcoRI-linearized pBR322 plasmid in the presence of 27 (0.5-1.0 mM) with or without reducing agents. DMSO (-) and 15 controls did not lead to detectable activity. Reactions were performed at 37 °C for 20 h (Gel 1). Under these conditions, positive control mitomycin C + DTT caused substantial DNA degradation. Consequently, experiment was repeated with a shorter incubation time and reduced temperature for mitomycin C + DTT (2 h, 20 °C), while the DMSO (-) control and 27 were incubated at 37 °C for 20 h with and without β-ME (Gel 2). mit C, mitomycin C; DTT, dithiothreitol; β-ME, β-mercaptoethanol; I, single-stranded DNA; II, cross-linked DNA.
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Figure 4: The colibactin warhead crosslinks DNA(a) DNA alkylation by the warhead is hypothesized to occur through a homo-Michael addition reaction, followed by a (b) pseudo-intramolecular Michael addition reaction, generating a DNA interstrand crosslink. (c) DNA crosslinking was observed using an EcoRI-linearized pBR322 plasmid in the presence of 27 (0.5-1.0 mM) with or without reducing agents. DMSO (-) and 15 controls did not lead to detectable activity. Reactions were performed at 37 °C for 20 h (Gel 1). Under these conditions, positive control mitomycin C + DTT caused substantial DNA degradation. Consequently, experiment was repeated with a shorter incubation time and reduced temperature for mitomycin C + DTT (2 h, 20 °C), while the DMSO (-) control and 27 were incubated at 37 °C for 20 h with and without β-ME (Gel 2). mit C, mitomycin C; DTT, dithiothreitol; β-ME, β-mercaptoethanol; I, single-stranded DNA; II, cross-linked DNA.

Mentions: The unprecedented structural features illuminated in our characterized shunt precolibactin 27 and predicted advanced precolibactin 32 served as a chemical guide for theoretical insights into colibactin’s modes of action. The colibactin warhead shares the structural hallmarks of “cyclopropane trigger compounds”32 with the requisite labile ring-strained spirocyclopropyl substituent for nucleophile-induced ring opening and irreversible covalent binding to its targets. Additionally, the proposed ClbP-catalyzed cleavage of precolibactin 32 would liberate the warhead with its N-terminal primary amine and predicted C-terminal thiazolinyl-thiazole tail (Fig. 3c, Fig. 4a). These flanking features are consistent with DNA/RNA being colibactin’s primary targets. Terminal amines are common in small molecules that bind DNA and RNA, such as the classically studied bleomycins, and participate in electrostatic interactions with the macromolecules’ phosphate moieties33. Additionally, the thiazolinyl-thiazole tail of related phleomycins contributes to their partial intercalative mode of DNA binding34. In contrast to the bleomycins’ and phleomycins’ ability to induce DNA double-strand breaks, the colibactin warhead is reminiscent of the duocarmycin family of DNA alkylators35. In an analogous reaction, the colibactin warhead could alkylate its targets via a homo-Michael addition reaction (Fig. 4a). NMR solution studies demonstrate that 2-hydroxypyrroles with acyl-substituents in the 3-position, such as the proposed alkylated-intermediate, strongly favor their keto-tautomeric forms36, as shown boxed in Figure 4a. Intriguingly, this suggests that upon alkylation, colibactin could present a second Michael acceptor, expanding its covalent functional utilities relative to the duocarmycins. In the case of DNA alkylation, a pseudo-intramolecular Michael addition reaction could generate DNA interstrand crosslinks (Fig. 4b).


The colibactin warhead crosslinks DNA.

Vizcaino MI, Crawford JM - Nat Chem (2015)

The colibactin warhead crosslinks DNA(a) DNA alkylation by the warhead is hypothesized to occur through a homo-Michael addition reaction, followed by a (b) pseudo-intramolecular Michael addition reaction, generating a DNA interstrand crosslink. (c) DNA crosslinking was observed using an EcoRI-linearized pBR322 plasmid in the presence of 27 (0.5-1.0 mM) with or without reducing agents. DMSO (-) and 15 controls did not lead to detectable activity. Reactions were performed at 37 °C for 20 h (Gel 1). Under these conditions, positive control mitomycin C + DTT caused substantial DNA degradation. Consequently, experiment was repeated with a shorter incubation time and reduced temperature for mitomycin C + DTT (2 h, 20 °C), while the DMSO (-) control and 27 were incubated at 37 °C for 20 h with and without β-ME (Gel 2). mit C, mitomycin C; DTT, dithiothreitol; β-ME, β-mercaptoethanol; I, single-stranded DNA; II, cross-linked DNA.
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Related In: Results  -  Collection

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Figure 4: The colibactin warhead crosslinks DNA(a) DNA alkylation by the warhead is hypothesized to occur through a homo-Michael addition reaction, followed by a (b) pseudo-intramolecular Michael addition reaction, generating a DNA interstrand crosslink. (c) DNA crosslinking was observed using an EcoRI-linearized pBR322 plasmid in the presence of 27 (0.5-1.0 mM) with or without reducing agents. DMSO (-) and 15 controls did not lead to detectable activity. Reactions were performed at 37 °C for 20 h (Gel 1). Under these conditions, positive control mitomycin C + DTT caused substantial DNA degradation. Consequently, experiment was repeated with a shorter incubation time and reduced temperature for mitomycin C + DTT (2 h, 20 °C), while the DMSO (-) control and 27 were incubated at 37 °C for 20 h with and without β-ME (Gel 2). mit C, mitomycin C; DTT, dithiothreitol; β-ME, β-mercaptoethanol; I, single-stranded DNA; II, cross-linked DNA.
Mentions: The unprecedented structural features illuminated in our characterized shunt precolibactin 27 and predicted advanced precolibactin 32 served as a chemical guide for theoretical insights into colibactin’s modes of action. The colibactin warhead shares the structural hallmarks of “cyclopropane trigger compounds”32 with the requisite labile ring-strained spirocyclopropyl substituent for nucleophile-induced ring opening and irreversible covalent binding to its targets. Additionally, the proposed ClbP-catalyzed cleavage of precolibactin 32 would liberate the warhead with its N-terminal primary amine and predicted C-terminal thiazolinyl-thiazole tail (Fig. 3c, Fig. 4a). These flanking features are consistent with DNA/RNA being colibactin’s primary targets. Terminal amines are common in small molecules that bind DNA and RNA, such as the classically studied bleomycins, and participate in electrostatic interactions with the macromolecules’ phosphate moieties33. Additionally, the thiazolinyl-thiazole tail of related phleomycins contributes to their partial intercalative mode of DNA binding34. In contrast to the bleomycins’ and phleomycins’ ability to induce DNA double-strand breaks, the colibactin warhead is reminiscent of the duocarmycin family of DNA alkylators35. In an analogous reaction, the colibactin warhead could alkylate its targets via a homo-Michael addition reaction (Fig. 4a). NMR solution studies demonstrate that 2-hydroxypyrroles with acyl-substituents in the 3-position, such as the proposed alkylated-intermediate, strongly favor their keto-tautomeric forms36, as shown boxed in Figure 4a. Intriguingly, this suggests that upon alkylation, colibactin could present a second Michael acceptor, expanding its covalent functional utilities relative to the duocarmycins. In the case of DNA alkylation, a pseudo-intramolecular Michael addition reaction could generate DNA interstrand crosslinks (Fig. 4b).

Bottom Line: Guided by metabolomic analyses, here we employ a combination of NMR spectroscopy and bioinformatics-guided isotopic labelling studies to characterize the colibactin warhead, an unprecedented substituted spirobicyclic structure.The warhead crosslinks duplex DNA in vitro, providing direct experimental evidence for colibactin's DNA-damaging activity.The data support unexpected models for both colibactin biosynthesis and its mode of action.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Chemistry, Yale University, New Haven, Connecticut 06510, USA [2] Chemical Biology Institute, Yale University, West Haven, Connecticut 06516, USA.

ABSTRACT
Members of the human microbiota are increasingly being correlated to human health and disease states, but the majority of the underlying microbial metabolites that regulate host-microbe interactions remain largely unexplored. Select strains of Escherichia coli present in the human colon have been linked to the initiation of inflammation-induced colorectal cancer through an unknown small-molecule-mediated process. The responsible non-ribosomal peptide-polyketide hybrid pathway encodes 'colibactin', which belongs to a largely uncharacterized family of small molecules. Genotoxic small molecules from this pathway that are capable of initiating cancer formation have remained elusive due to their high instability. Guided by metabolomic analyses, here we employ a combination of NMR spectroscopy and bioinformatics-guided isotopic labelling studies to characterize the colibactin warhead, an unprecedented substituted spirobicyclic structure. The warhead crosslinks duplex DNA in vitro, providing direct experimental evidence for colibactin's DNA-damaging activity. The data support unexpected models for both colibactin biosynthesis and its mode of action.

Show MeSH
Related in: MedlinePlus