Mechanism of Action of KL-50, a Candidate Imidazotetrazine for the Treatment of Drug-Resistant Brain Cancers.

Aberrant DNA repair is a hallmark of cancer, and many tumors display reduced DNA repair capacities that sensitize them to genotoxins. Here, we demonstrate that the differential DNA repair capacities of healthy and transformed tissue may be exploited to obtain highly selective chemotherapies. We show that the novel N3-(2-fluoroethyl)imidazotetrazine "KL-50" is a selective toxin toward tumors that lack the DNA repair protein O6-methylguanine-DNA-methyltransferase (MGMT), which reverses the formation of O6-alkylguanine lesions. We establish that KL-50 generates DNA interstrand cross-links (ICLs) by a multistep process comprising DNA alkylation to generate an O6-(2-fluoroethyl)guanine (O6FEtG) lesion, slow unimolecular displacement of fluoride to form an N1,O6-ethanoguanine (N1,O6EtG) intermediate, and ring-opening by the adjacent cytidine. The slow rate of N1,O6EtG formation allows healthy cells expressing MGMT to reverse the initial O6FEtG lesion before it evolves to N1,O6EtG, thereby suppressing the formation of toxic DNA-MGMT cross-links and reducing the amount of DNA ICLs generated in healthy cells. In contrast, O6-(2-chloroethyl)guanine lesions produced by agents such as lomustine and the N3-(2-chloroethyl)imidazotetrazine mitozolomide rapidly evolve to N1,O6EtG, resulting in the formation of DNA-MGMT cross-links and DNA ICLs in healthy tissue. These studies suggest that careful consideration of the rates of chemical DNA modification and biochemical DNA repair may lead to the identification of other tumor-specific genotoxic agents.

The Fanconi anemia pathway repairs colibactin-induced DNA interstrand cross-links.

Colibactin is a secondary metabolite produced by bacteria present in the human gut and is implicated in the progression of colorectal cancer and inflammatory bowel disease. This genotoxin alkylates deoxyadenosines on opposite strands of host cell DNA to produce DNA interstrand cross-links (ICLs) that block DNA replication. While cells have evolved multiple mechanisms to resolve ("unhook") ICLs encountered by the replication machinery, little is known about which of these pathways promote resistance to colibactin-induced ICLs. Here, we use Xenopus egg extracts to investigate replication-coupled repair of plasmids engineered to contain site-specific colibactin-ICLs. We show that replication fork stalling at a colibactin-ICL leads to replisome disassembly and activation of the Fanconi anemia ICL repair pathway, which unhooks the colibactin-ICL through nucleolytic incisions. These incisions generate a DNA double-strand break intermediate in one sister chromatid, which can be repaired by homologous recombination, and a monoadduct ("ICL remnant") in the other. Our data indicate that translesion synthesis past the colibactin-ICL remnant depends on Polη and a Polκ-REV1-Polζ polymerase complex. Although translesion synthesis past colibactin-induced DNA damage is frequently error-free, it can introduce T>N point mutations that partially recapitulate the mutation signature associated with colibactin exposure in vivo. Taken together, our work provides a biochemical framework for understanding how cells tolerate a naturally-occurring and clinically-relevant ICL.

The microbial genotoxin colibactin exacerbates mismatch repair mutations in colorectal tumors.

Certain Enterobacteriaceae strains contain a 54-kb biosynthetic gene cluster referred to as "pks" encoding the biosynthesis of a secondary metabolite, colibactin. Colibactin-producing E. coli promote colorectal cancer (CRC) in preclinical models, and in vitro induce a specific mutational signature that is also detected in human CRC genomes. Yet, how colibactin exposure affects the mutational landscape of CRC in vivo remains unclear. Here we show that colibactin-producing E. coli-driven colonic tumors in mice have a significantly higher SBS burden and a larger percentage of these mutations can be attributed to a signature associated with mismatch repair deficiency (MMRd; SBS15), compared to tumors developed in the presence of colibactin-deficient E. coli. We found that the synthetic colibactin 742 but not an inactive analog 746 causes DNA damage and induces transcriptional activation of p53 and senescence signaling pathways in non-transformed human colonic epithelial cells. In MMRd colon cancer cells (HCT 116), chronic exposure to 742 resulted in the upregulation of BRCA1, Fanconi anemia, and MMR signaling pathways as revealed by global transcriptomic analysis. This was accompanied by increased T>N single-base substitutions (SBS) attributed to the proposed pks+E. coli signature (SBS88), reactive oxygen species (SBS17), and mismatch-repair deficiency (SBS44). A significant co-occurrence between MMRd SBS44 and pks-associated SBS88 signature was observed in a large cohort of human CRC patients (n=2,945), and significantly more SBS44 mutations were found when SBS88 was also detected. Collectively, these findings reveal the host response mechanisms underlying colibactin genotoxic activity and suggest that colibactin may exacerbate MMRd-associated mutations.

Structure Elucidation of Secondary Metabolites: Current Frontiers and Lingering Pitfalls.

Analytical methods allow for the structure determination of submilligram quantities of complex secondary metabolites. This has been driven in large part by advances in NMR spectroscopic capabilities, including access to high-field magnets equipped with cryogenic probes. Experimental NMR spectroscopy may now be complemented by remarkably accurate carbon-13 NMR calculations using state-of-the-art DFT software packages. Additionally, microED analysis stands to have a profound effect on structure elucidation by providing X-ray-like images of microcrystalline samples of analytes. Nonetheless, lingering pitfalls in structure elucidation remain, particularly for isolates that are unstable or highly oxidized. In this Account, we discuss three projects from our laboratory that highlight nonoverlapping challenges to the field, with implications for chemical, synthetic, and mechanism of action studies. We first discuss the lomaiviticins, complex unsaturated polyketide natural products disclosed in 2001. The original structures were derived from NMR, HRMS, UV-vis, and IR analysis. Owing to the synthetic challenges presented by their structures and the absence of X-ray crystallographic data, the structure assignments remained untested for nearly two decades. In 2021, the Nelson group at Caltech carried out microED analysis of (-)-lomaiviticin C, leading to the startling discovery that the original structure assignment of the lomaiviticins was incorrect. Acquisition of higher-field (800 MHz 1H, cold probe) NMR data as well as DFT calculations provided insights into the basis for the original misassignment and lent further support to the new structure identified by microED. Reanalysis of the 2001 data set reveals that the two structure assignments are nearly indistinguishable, underscoring the limitations of NMR-based characterization. We then discuss the structure elucidation of colibactin, a complex, nonisolable microbiome metabolite implicated in colorectal cancer. The colibactin biosynthetic gene cluster was detected in 2006, but owing to colibactin's instability and low levels of production, it could not be isolated or characterized. We used a combination of chemical synthesis, mechanism of action studies, and biosynthetic analysis to identify the substructures in colibactin. These studies, coupled with isotope labeling and tandem MS analysis of colibactin-derived DNA interstrand cross-links, ultimately led to a structure assignment for the metabolite. We then discuss the ocimicides, plant secondary metabolites that were studied as agents against drug-resistant P. falciparum. We synthesized the core structure of the ocimicides and found significant discrepancies between our experimental NMR spectroscopic data and that reported for the natural products. We determined the theoretical carbon-13 NMR shifts for 32 diastereomers of the ocimicides. These studies indicated that a revision of the connectivity of the metabolites is likely needed. We end with some thoughts on the frontiers of secondary metabolite structure determination. As modern NMR computational methods are straightforward to execute, we advocate for their systematic use in validating the assignments of novel secondary metabolites.

Commensal microbiota from patients with inflammatory bowel disease produce genotoxic metabolites.

Microbiota-derived metabolites that elicit DNA damage can contribute to colorectal cancer (CRC). However, the full spectrum of genotoxic chemicals produced by indigenous gut microbes remains to be defined. We established a pipeline to systematically evaluate the genotoxicity of an extensive collection of gut commensals from inflammatory bowel disease patients. We identified isolates from divergent phylogenies whose metabolites caused DNA damage and discovered a distinctive family of genotoxins-termed the indolimines-produced by the CRC-associated species Morganella morganii. A non-indolimine-producing M. morganii mutant lacked genotoxicity and failed to exacerbate colon tumorigenesis in mice. These studies reveal the existence of a previously unexplored universe of genotoxic small molecules from the microbiome that may affect host biology in homeostasis and disease.

Stereocontrolled Synthesis of the Fully Glycosylated Monomeric Unit of Lomaiviticin A.

We describe a stereocontrolled synthesis of 3, the fully glycosylated monomeric unit of the dimeric cytotoxic bacterial metabolite (-)-lomaiviticin A (2). A novel strategy involving convergent, site- and stereoselective coupling of the ß,γ-unsaturated ketone 6 and the naphthyl bromide 7 (92%, 15:1 diastereomeric ratio (dr)), followed by radical-based annulation and silyl ether cleavage, provided the tetracycle 5 (57% overall), which contains the carbon skeleton of the aglycon of 3. The ß-linked 2,4,6-trideoxy-4-aminoglycoside l-pyrrolosamine was installed in 73% yield and with 15:1 ß:α selectivity using a modified Koenigs-Knorr glycosylation. The diazo substituent was introduced via direct diazo transfer to an electron-rich benzoindene (4 → 27). The α-linked 2,6-dideoxyglycoside l-oleandrose was introduced by gold-catalyzed activation of an o-alkynyl glycosylbenzoate (75%, >20:1 α:ß selectivity). A carefully orchestrated endgame sequence then provided efficient access to 3. Cell viability studies indicated that monomer 3 is not cytotoxic at concentrations up to 1 µM, providing conclusive evidence that the dimeric structure of (-)-lomaiviticin A (2) is required for cytotoxic effects. The preparation of 3 provides a foundation to complete the synthesis of (-)-lomaiviticin A (2) itself.

Mechanism-based design of agents that selectively target drug-resistant glioma.

Approximately half of glioblastoma and more than two-thirds of grade II and III glioma tumors lack the DNA repair protein O6-methylguanine methyl transferase (MGMT). MGMT-deficient tumors respond initially to the DNA methylation agent temozolomide (TMZ) but frequently acquire resistance through loss of the mismatch repair (MMR) pathway. We report the development of agents that overcome this resistance mechanism by inducing MMR-independent cell killing selectively in MGMT-silenced tumors. These agents deposit a dynamic DNA lesion that can be reversed by MGMT but slowly evolves into an interstrand cross-link in MGMT-deficient settings, resulting in MMR-independent cell death with low toxicity in vitro and in vivo. This discovery may lead to new treatments for gliomas and may represent a new paradigm for designing chemotherapeutics that exploit specific DNA repair defects.

Chemoproteomic Profiling by Cysteine Fluoroalkylation Reveals Myrocin G as an Inhibitor of the Nonhomologous End Joining DNA Repair Pathway.

Chemoproteomic profiling of cysteines has emerged as a powerful method for screening the proteome-wide targets of cysteine-reactive fragments, drugs, and natural products. Herein, we report the development and an in-depth evaluation of a tetrafluoroalkyl benziodoxole (TFBX) as a cysteine-selective chemoproteomic probe. We show that this probe features numerous key improvements compared to the traditionally used cysteine-reactive probes, including a superior target occupancy, faster labeling kinetics, and broader proteomic coverage, thus enabling profiling of cysteines directly in live cells. In addition, the fluorine "signature" of probe 7 constitutes an additional advantage resulting in a more confident adduct-amino acid site assignment in mass-spectrometry-based identification workflows. We demonstrate the utility of our new probe for proteome-wide target profiling by identifying the cellular targets of (-)-myrocin G, an antiproliferative fungal natural product with a to-date unknown mechanism of action. We show that this natural product and a simplified analogue target the X-ray repair cross-complementing protein 5 (XRCC5), an ATP-dependent DNA helicase that primes DNA repair machinery for nonhomologous end joining (NHEJ) upon DNA double-strand breaks, making them the first reported inhibitors of this biomedically highly important protein. We further demonstrate that myrocins disrupt the interaction of XRCC5 with DNA leading to sensitization of cancer cells to the chemotherapeutic agent etoposide as well as UV-light-induced DNA damage. Altogether, our next-generation cysteine-reactive probe enables broader and deeper profiling of the cysteinome, rendering it a highly attractive tool for elucidation of targets of electrophilic small molecules.

Probing Microbiome Genotoxicity: A Stable Colibactin Provides Insight into Structure-Activity Relationships and Facilitates Mechanism of Action Studies.

Colibactin is a genotoxic metabolite produced by commensal-pathogenic members of the human microbiome that possess the clb (aka pks) biosynthetic gene cluster. clb+ bacteria induce tumorigenesis in models of intestinal inflammation and have been causally linked to oncogenesis in humans. While colibactin is believed underlie these effects, it has not been possible to study the molecule directly due to its instability. Herein, we report the synthesis and biological studies of colibactin 742 (4), a stable colibactin derivative. We show that colibactin 742 (4) induces DNA interstrand-cross-links, activation of the Fanconi Anemia DNA repair pathway, and G2/M arrest in a manner similar to clb+E. coli. The linear precursor 9, which mimics the biosynthetic precursor to colibactin, also recapitulates the bacterial phenotype. In the course of this work, we discovered a novel cyclization pathway that was previously undetected in MS-based studies of colibactin, suggesting a refinement to the natural product structure and its mode of DNA binding. Colibactin 742 (4) and its precursor 9 will allow researchers to study colibactin's genotoxic effects independent of the producing organism for the first time.

Synthesis and Biological Evaluation of (2S,2'S)-Lomaiviticin A.

(-)-Lomaiviticin A (1) is a genotoxic C2-symmetric metabolite that arises from the formal dimerization of two bis(glycosylated) diazotetrahydrobenzo[b]fluorenes. Here we present a synthesis of the monomer 17 and its coupling to form (2S,2'S)-lomaiviticin A (4), an unnatural diastereomer of 1. (2S,2'S)-Lomaiviticin A (4) is significantly less genotoxic, a result we attribute to changes in the orientation of the diazofluorene and carbohydrate residues, relative to 1. These data bring the importance of the configuration of the conjoining bond to light and place the total synthesis of 1 itself within reach.

Employing chemical synthesis to study the structure and function of colibactin, a "dark matter" metabolite.

Covering: 2015 to 2020 The field of natural products is dominated by a discovery paradigm that follows the sequence: isolation, structure elucidation, chemical synthesis, and then elucidation of mechanism of action and structure-activity relationships. Although this discovery paradigm has proven successful in the past, researchers have amassed enough evidence to conclude that the vast majority of nature's secondary metabolites - biosynthetic "dark matter" - cannot be identified and studied by this approach. Many biosynthetic gene clusters (BGCs) are expressed at low levels, or not at all, and in some instances a molecule's instability to fermentation or isolation prevents detection entirely. Here, we discuss an alternative approach to natural product identification that addresses these challenges by enlisting synthetic chemistry to prepare putative natural product fragments and structures as guided by biosynthetic insight. We demonstrate the utility of this approach through our structure elucidation of colibactin, an unisolable genotoxin produced by pathogenic bacteria in the human gut.

Structure and bioactivity of colibactin.

Colibactin is a secondary metabolite produced by certain strains of bacteria found in the human gut. The presence of colibactin-producing bacteria has been correlated to colorectal cancer in humans. Colibactin was first discovered in 2006, but because it is produced in small quantities and is unstable, it has yet to be isolated from bacterial cultures. Here we summarize advances in the field since ~2017 that have led to the identification of the structure of colibactin as a heterodimer containing two DNA-reactive electrophilic cyclopropane residues. Colibactin has been shown to form interstrand cross-links by alkylation of adenine residues on opposing strands of DNA. The structure of colibactin contains two thiazole rings separated by a two-carbon linker that is thought to exist as an α-aminoketone following completion of the biosynthetic pathway. However, synthetic studies have now established that this α-aminoketone is unstable toward aerobic oxidation; the resulting oxidation products are in turn unstable toward nucleophilic cleavage under mild conditions. These data provide a simple molecular-level explanation for colibactin's instability and potentially also explain the observation that cell-to-cell contact is required for genotoxic effects.

Depurination of Colibactin-Derived Interstrand Cross-Links.

Colibactin is a genotoxic gut microbiome metabolite long suspected of playing an etiological role in colorectal cancer. Evidence suggests that colibactin forms DNA interstrand cross-links (ICLs) in eukaryotic cells and activates ICL repair pathways, leading to the production of ICL-dependent DNA double-strand breaks (DSBs). Here we show that colibactin ICLs can evolve directly to DNA DSBs. Using the topology of supercoiled plasmid DNA as a proxy for alkylation adduct stability, we find that colibactin-derived ICLs are unstable toward depurination and elimination of the 3' phosphate. This ICL degradation pathway leads progressively to single strand breaks (SSBs) and subsequently DSBs. The spontaneous conversion of ICLs to DSBs is consistent with the finding that nonhomologous end joining repair-deficient cells are sensitized to colibactin-producing bacteria. The results herein refine our understanding of colibactin-derived DNA damage and underscore the complexities underlying the DSB phenotype.

Synthesis and reactivity of precolibactin 886.

The clb gene cluster encodes the biosynthesis of metabolites known as precolibactins and colibactins. The clb pathway is found in gut commensal Escherichia coli, and clb metabolites are thought to initiate colorectal cancer via DNA crosslinking. Here we report confirmation of the structural assignment of the complex clb product precolibactin 886 via a biomimetic synthetic pathway. We show that an α-ketoimine linear precursor undergoes spontaneous cyclization to precolibactin 886 on HPLC purification. Studies of this α-ketoimine and the related α-dicarbonyl revealed that these compounds are unexpectedly susceptible to nucleophilic cleavage under mildly basic conditions. This cleavage pathway forms other known clb metabolites or biosynthetic intermediates and explains the difficulties in isolating fully mature biosynthetic products. This cleavage also accounts for a recently identified colibactin-adenine adduct. The colibactin peptidase ClbP deacylates synthetic precolibactin 886 to form a non-genotoxic pyridone, which suggests precolibactin 886 lies off the path of the major biosynthetic route.

Structure elucidation of colibactin and its DNA cross-links.

Colibactin is a complex secondary metabolite produced by some genotoxic gut Escherichia coli strains. The presence of colibactin-producing bacteria correlates with the frequency and severity of colorectal cancer in humans. However, because colibactin has not been isolated or structurally characterized, studying the physiological effects of colibactin-producing bacteria in the human gut has been difficult. We used a combination of genetics, isotope labeling, tandem mass spectrometry, and chemical synthesis to deduce the structure of colibactin. Our structural assignment accounts for all known biosynthetic and cell biology data and suggests roles for the final unaccounted enzymes in the colibactin gene cluster.

Model Colibactins Exhibit Human Cell Genotoxicity in the Absence of Host Bacteria.

Colibactins are genotoxic secondary metabolites produced in select Enterobacteriaceae, which induce downstream DNA double-strand breaks (DSBs) in human cell lines and are thought to promote the formation of colorectal tumors. Although key structural and functional features of colibactins have been elucidated, the full molecular mechanisms regulating these phenotypes remain unknown. Here, we demonstrate that free model colibactins induce DSBs in human cell cultures and do not require delivery by host bacteria. Through domain-targeted editing, we demonstrate that a subset of native colibactins generated from observed module skipping in the nonribosomal peptide synthetase-polyketide synthase (NRPS-PKS) biosynthetic assembly line share DNA alkylation phenotypes with the model colibactins in vitro. However, module skipping eliminates the strong DNA interstrand cross-links formed by the wild-type pathway in cell culture. This product diversification during the modular NRPS-PKS biosynthesis produces a family of metabolites with varying observed mechanisms of action (DNA alkylation versus cross-linking) in cell culture. The presence of membranes separating human cells from model colibactins attenuated genotoxicity, suggesting that membrane diffusion limits colibactin activity and could account for the reported bacterium-human cell-to-cell contact phenotype. Additionally, extracellular supplementation of the colibactin resistance protein ClbS was able to intercept colibactins in an Escherichia coli-human cell transient infection model. Our studies demonstrate that free model colibactins recapitulate cellular phenotypes associated with module-skipped products in the native colibactin pathway and define specific protein domains that are required for efficient DNA interstrand cross-linking in the native pathway.

Synthesis of Myrocin G, the Putative Active Form of the Myrocin Antitumor Antibiotics.

The antiproliferative antimicrobial fungal metabolites known as the myrocins have been proposed to cross-link DNA by double nucleotide addition. However, the nature of the DNA-reactive species is ambiguous, as myrocins have been isolated as functionally distinct 5-hydroxy-γ-lactone and diosphenol isomers. Based on literature precedent, we hypothesized that the diosphenol 7 (assigned here the trivial name myrocin G) is the biologically active form of the representative isolate (+)-myrocin C (1). To probe this, we developed a short enantioselective route to 7. A powerful fragment-coupling reaction that forms the central ring of the target in 38% yield and in a single step was developed. In support of our hypothesis, 7 was efficiently transformed to the bis(sulfide) 6, a product previously isolated from reactions of 1 with excess benzenethiol. This work provides the first direct access to the diosphenol 7, sets the stage for elucidating the mode of interaction of the myrocins with DNA, and provides a foundation for the synthesis of other pimarane diterpenes.

Characterization of Natural Colibactin-Nucleobase Adducts by Tandem Mass Spectrometry and Isotopic Labeling. Support for DNA Alkylation by Cyclopropane Ring Opening.

Colibactins are genotoxic secondary metabolites whose biosynthesis is encoded in the clb gene cluster harbored by certain strains of gut commensal Escherichia coli. Using synthetic colibactin analogues, we previously provided evidence that colibactins alkylate DNA by addition of a nucleotide to an electrophilic cyclopropane intermediate. However, natural colibactin-nucleobase adducts have not been identified, to the best of our knowledge. Here we present the first identification of such adducts, derived from treatment of pUC19 DNA with clb + E. coli. Previous biosynthetic studies established cysteine and methionine as building blocks in colibactin biosynthesis; accordingly, we used cysteine (Δ cysE) and methionine (Δ metA) auxotrophic strains cultured in media supplemented with l-[U-13C]Cys or l-[U-13C]Met to facilitate the identification of nucleobases bound to colibactins. Using MS2 and MS3 analysis, in conjunction with the known oxidative instability of colibactin cyclopropane-opened products, we were able to characterize adenine adducts derived from cyclopropane ring opening. This study provides the first reported detection of nucleobase adducts derived from clb + E. coli and lends support to our earlier model suggesting DNA alkylation by addition of a nucleotide to an electrophilic cyclopropane.

ClbS Is a Cyclopropane Hydrolase That Confers Colibactin Resistance.

Certain commensal Escherichia coli contain the clb biosynthetic gene cluster that codes for small molecule prodrugs known as precolibactins. Precolibactins are converted to colibactins by N-deacylation; the latter are postulated to be genotoxic and to contribute to colorectal cancer formation. Though advances toward elucidating (pre)colibactin biosynthesis have been made, the functions and mechanisms of several clb gene products remain poorly understood. Here we report the 2.1 Å X-ray structure and molecular function of ClbS, a gene product that confers resistance to colibactin toxicity in host bacteria and which has been shown to be important for bacterial viability. The structure harbors a potential colibactin binding site and shares similarity to known hydrolases. In vitro studies using a synthetic colibactin analog and ClbS or an active site residue mutant reveal cyclopropane hydrolase activity that converts the electrophilic cyclopropane of the colibactins into an innocuous hydrolysis product. As the cyclopropane has been shown to be essential for genotoxic effects in vitro, this ClbS-catalyzed ring-opening provides a means for the bacteria to circumvent self-induced genotoxicity. Our study provides a molecular-level view of the first reported cyclopropane hydrolase and support for a specific mechanistic role of this enzyme in colibactin resistance.