A red light-triggered chemical tool for sequence-specific alkylation of G-quadruplex and I-motif DNA.

The importance of non-canonical DNA structures such as G-quadruplexes (G4) and intercalating-motifs (iMs) in the fine regulation of a variety of cellular processes has been recently demonstrated. As the crucial roles of these structures are being unravelled, it is becoming more and more important to develop tools that allow targeting these structures with the highest possible specificity. While targeting methodologies have been reported for G4s, this is not the case for iMs, as evidenced by the limited number of specific ligands able to bind the latter and the total absence of selective alkylating agents for their covalent targeting. Furthermore, strategies for the sequence-specific covalent targeting of G4s and iMs have not been reported thus far. Herein, we describe a simple methodology to achieve sequence-specific covalent targeting of G4 and iM DNA structures based on the combination of (i) a peptide nucleic acid (PNA) recognizing a specific sequence of interest, (ii) a pro-reactive moiety enabling a controlled alkylation reaction, and (iii) a G4 or iM ligand orienting the alkylating warhead to the reactive residues. This multi-component system allows for the targeting of specific G4 or iM sequences of interest in the presence of competing DNA sequences and under biologically relevant conditions.

The complexity and regulation of repair of alkylation damage to nucleic acids.

DNA damaging agents have been a cornerstone of cancer therapy for nearly a century. The discovery of many of these chemicals, particularly the alkylating agents, are deeply entwined with the development of poisonous materials originally intended for use in warfare. Over the last decades, their anti-proliferative effects have focused on the specific mechanisms by which they damage DNA, and the factors involved in the repair of such damage. Due to the variety of aberrant adducts created even for the simplest alkylating agents, numerous pathways of repair are engaged as a defense against this damage. More recent work has underscored the role of RNA damage in the cellular response to these agents, although the understanding of their role in relation to established DNA repair pathways is still in its infancy. In this review, we discuss the chemistry of alkylating agents, the numerous ways in which they damage nucleic acids, as well as the specific DNA and RNA repair pathways which are engaged to counter their effects.

Regulation of DNA Alkylation Damage Repair: Lessons and Therapeutic Opportunities.

Alkylation chemotherapy is one of the most widely used systemic therapies for cancer. While somewhat effective, clinical responses and toxicities of these agents are highly variable. A major contributing factor for this variability is the numerous distinct lesions that are created upon alkylation damage. These adducts activate multiple repair pathways. There is mounting evidence that the individual pathways function cooperatively, suggesting that coordinated regulation of alkylation repair is critical to prevent toxicity. Furthermore, some alkylating agents produce adducts that overlap with newly discovered methylation marks, making it difficult to distinguish between bona fide damaged bases and so-called 'epigenetic' adducts. Here, we discuss new efforts aimed at deciphering the mechanisms that regulate these repair pathways, emphasizing their implications for cancer chemotherapy.

Alkylated epidermal creatine kinase as a biomarker for sulfur mustard exposure: comparison to adducts of albumin and DNA in an in vivo rat study.

Sulfur mustard (SM) is a chemical warfare agent which use is banned under international law and that has been used recently in Northern Iraq and Syria by the so-called Islamic State. SM induces the alkylation of endogenous proteins like albumin and hemoglobin thus forming covalent adducts that are targeted by bioanalytical methods for the verification of systemic poisoning. We herein report a novel biomarker, namely creatine kinase (CK) B-type, suitable as a local biomarker for SM exposure on the skin. Human and rat skin were proven to contain CK B-type by Western blot analysis. Following exposure to SM ex vivo, the CK-adduct was extracted from homogenates by immunomagnetic separation and proteolyzed afterwards. The cysteine residue Cys282 was found to be alkylated by the SM-specific hydroxyethylthioethyl (HETE)-moiety detected as the biomarker tetrapeptide TC(-HETE)PS. A selective and sensitive micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (µLC-ESI MS/HRMS) method was developed to monitor local CK-adducts in an in vivo study with rats percutaneously exposed to SM. CK-adduct formation was compared to already established DNA- and systemic albumin biomarkers. CK- and DNA-adducts were successfully detected in biopsies of exposed rat skin as well as albumin-adducts in plasma. Relative biomarker concentrations make the CK-adduct highly appropriate as a local dermal biomarker. In summary, CK or rather Cys282 in CK B-type was identified as a new, additional dermal target of local SM exposures. To our knowledge, it is also the first time that HETE-albumin adducts, and HETE-DNA adducts were monitored simultaneously in an in vivo animal study.

Alkylation of rabbit muscle creatine kinase surface methionine residues inhibits enzyme activity in vitro.

Creatine kinase (CK) catalyzes the formation of phosphocreatine from adenosine triphosphate (ATP) and creatine. The highly reactive free cysteine residue in the active site of the enzyme (Cys283) is considered essential for the enzymatic activity. In previous studies we demonstrated that Cys283 is targeted by the alkylating chemical warfare agent sulfur mustard (SM) yielding a thioether with a hydroxyethylthioethyl (HETE)-moiety. In the present study, the effect of SM on rabbit muscle CK (rmCK) activity was investigated with special focus on the alkylation of Cys283 and of reactive methionine (Met) residues. For investigation of SM-alkylated amino acids in rmCK, micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry measurements were performed using the Orbitrap technology. The treatment of rmCK with SM resulted in a decrease of enzyme activity. However, this decrease did only weakly correlate to the modification of Cys283 but was conclusive for the formation of Met70-HETE and Met179-HETE. In contrast, the activity of mutants of rmCK produced by side-directed mutagenesis that contained substitutions of the respective Met residues (Met70Ala, Met179Leu, and Met70Ala/Met179Leu) was highly resistant against SM. Our results point to a critical role of the surface exposed Met70 and Met179 residues for CK activity.

Mechanistic Studies of Cytochrome P450 3A4 Time-Dependent Inhibition Using Two Cysteine-Targeting Electrophiles.

Experiments designed to identify the mechanism of cytochrome P450 inactivation are critical to drug discovery. Small molecules irreversibly inhibit P450 enzymatic activity via two primary mechanisms: apoprotein adduct formation or heme modification. Understanding the interplay between chemical structures of reactive electrophiles and the impact on CYP3A4 structure and function can ultimately provide insights into drug design to minimize P450 inactivation. In a previous study, raloxifene and N-(1-pyrene) iodoacetamide (PIA) alkylated CYP3A4 in vitro; however, only raloxifene influenced enzyme activity. Here, two alkylating agents with cysteine selectivity, PIA and pyrene maleimide (PM), were used to investigate this apparent compound-dependent disconnect between CYP3A4 protein alkylation and activity loss. The compound's effect on 1) enzymatic activity, 2) carbon monoxide (CO) binding capacity, 3) intact heme content, and 4) protein conformation were measured. Results showed that PM had a large time-dependent loss of enzyme activity, whereas PIA did not. The differential effect on enzymatic activity between PM and PIA was mirrored in the CO binding data. Despite disruption of CO binding, neither compound affected the heme concentrations, inferring there was no destruction or alkylation of the heme. Lastly, differential scanning fluorescence showed PM-treated CYP3A4 caused a shift in the onset temperature required to induce protein aggregation, which was not observed for CYP3A4 treated with PIA. In conclusion, alkylation of CYP3A4 apoprotein can have a variable impact on catalytic activity, CO binding, and protein conformation that may be compound-dependent. These results highlight the need for careful interpretation of experimental results aimed at characterizing the nature of P450 enzyme inactivation. SIGNIFICANCE STATEMENT: Understanding the mechanism of CYP3A4 time-dependent inhibition is critical to drug discovery. In this study, we use two cysteine-targeting electrophiles to probe how subtle variation in inhibitor structure may impact the mechanism of CYP3A4 time-dependent inhibition and confound interpretation of traditional diagnostic experiments. Ultimately, this simplified system was used to reveal insights into CYP3A4 biochemical behavior. The insights may have implications that aid in understanding the susceptibility of CYP enzymes to the effects of electrophilic intermediates generated via bioactivation.

Glutamine deficiency induces DNA alkylation damage and sensitizes cancer cells to alkylating agents through inhibition of ALKBH enzymes.

Driven by oncogenic signaling, glutamine addiction exhibited by cancer cells often leads to severe glutamine depletion in solid tumors. Despite this nutritional environment that tumor cells often experience, the effect of glutamine deficiency on cellular responses to DNA damage and chemotherapeutic treatment remains unclear. Here, we show that glutamine deficiency, through the reduction of alpha-ketoglutarate, inhibits the AlkB homolog (ALKBH) enzymes activity and induces DNA alkylation damage. As a result, glutamine deprivation or glutaminase inhibitor treatment triggers DNA damage accumulation independent of cell death. In addition, low glutamine-induced DNA damage is abolished in ALKBH deficient cells. Importantly, we show that glutaminase inhibitors, 6-Diazo-5-oxo-L-norleucine (DON) or CB-839, hypersensitize cancer cells to alkylating agents both in vitro and in vivo. Together, the crosstalk between glutamine metabolism and the DNA repair pathway identified in this study highlights a potential role of metabolic stress in genomic instability and therapeutic response in cancer.

Heteroexpression of Mycobacterium leprae hypothetical protein ML0190 provides protection against DNA-alkylating agent methyl methanesulfonate.

Repair of DNA alkylation damage is essential for maintaining genome integrity and Fe(II)/2-oxoglutarate(2OG)-dependent dioxygenase family of enzymes play crucial role in repairing some of the alkylation damages. Alkylation repair protein-B (AlkB) of Escherichia coli belongs to Fe(II)/2OG-dependent dioxygenase family and carries out DNA dealkylation repair. We report here identification of a hypothetical Mycobacterium leprae protein (accession no. ML0190) from the genomic database and show that this 615-bp open reading frame encodes a protein with sequence and structural similarity to Fe(II)/2OG-dependent dioxygenase AlkB. We identified mRNA transcript of this gene in the M. leprae infected clinical skin biopsy samples isolated from the leprosy patients. Heterologous expression of ML0190 in methyl methane sulfonate (MMS) sensitive and DNA repair deficient strain of Saccharomyces cerevisiae and Escherichia coli resulted in resistance to alkylating agent MM. The results of the present study imply that Mycobacterium leprae ML0190 is involved in protecting the bacterial genome from DNA alkylation damage.

A New Alkylation of Aryl Alcohols by Boron Trifluoride Etherate.

The ethylation of aryl alcohols by an ethyl moiety of boron trifluoride etherate is described. The reaction proceeded cleanly and afforded good yields of the corresponding aryl ethyl ethers. It tolerated the presence of functional groups such as aryl, alkyl, halogens, nitro, nitrile, and amino. However, the presence of amino or nitro groups ortho to a hydroxyl group of an aryl compound drastically reduced the yields of the anticipated products due to the chelation of the aforementioned functional groups with boron trifluoride etherate. A nitrogen atom in the aromatic ring system, as exemplified by hydroxypyridine and 8-hydroxyquinoline, completely inhibited the reaction. Resorcinol, hydroquinone, and aryl alcohols with aldehyde functions decomposed under the reaction conditions.

Use of Phenoxyaniline Analogues To Generate Biochemical Insights into the Interactio n of Polybrominated Diphenyl Ether with CYP2B Enzymes.

Human hepatic cytochromes P450 (CYP) are integral to xenobiotic metabolism. CYP2B6 is a major catalyst of biotransformation of environmental toxicants, including polybrominated diphenyl ethers (PBDEs). CYP2B substrates tend to contain halogen atoms, but the biochemical basis for this selectivity and for species specific determinants of metabolism has not been identified. Spectral binding titrations and inhibition studies were performed to investigate interactions of rat CYP2B1, rabbit CYP2B4, and CYP2B6 with a series of phenoxyaniline (POA) congeners that are analogues of PBDEs. For most congeners, there was a <3-fold difference between the spectral binding constants (KS) and IC50 values. In contrast, large discrepancies between these values were observed for POA and 3-chloro-4-phenoxyaniline. CYP2B1 was the enzyme most sensitive to POA congeners, so the Val-363 residue from that enzyme was introduced into CYP2B4 or CYP2B6. This substitution partially altered the protein-ligand interaction profiles to make them more similar to that of CYP2B1. Addition of cytochrome P450 oxidoreductase (POR) to titrations of CYP2B6 with POA or 2'4'5'TCPOA decreased the affinity of both ligands for the enzyme. Addition of cytochrome b5 to a recombinant enzyme system containing POR and CYP2B6 increased the POA IC50 value and decreased the 2'4'5'TCPOA IC50 value. Overall, the inconsistency between KS and IC50 values for POA versus 2'4'5'TCPOA is largely due to the effects of redox partner binding. These results provide insight into the biochemical basis of binding of diphenyl ethers to human CYP2B6 and changes in CYP2B6-mediated metabolism that are dependent on POA congener and redox partner identity.

Mutagenic potential of nitrogen mustard-induced formamidopyrimidine DNA adduct: Contribution of the non-canonical α-anomer.

Nitrogen mustards (NMs) are DNA-alkylating compounds that represent the earliest anticancer drugs. However, clinical use of NMs is limited because of their own mutagenic properties. The mechanisms of NM-induced mutagenesis remain unclear. The major product of DNA alkylation by NMs is a cationic NM-N7-dG adduct that can yield the imidazole ring-fragmented lesion, N5-NM-substituted formamidopyrimidine (NM-Fapy-dG). Characterization of this adduct is complicated because it adopts different conformations, including both a canonical ß- and an unnatural α-anomeric configuration. Although formation of NM-Fapy-dG in cellular DNA has been demonstrated, its potential role in NM-induced mutagenesis is unknown. Here, we created site-specifically modified single-stranded vectors for replication in primate (COS7) or Escherichia coli cells. In COS7 cells, NM-Fapy-dG caused targeted mutations, predominantly G → T transversions, with overall frequencies of ∼11-12%. These frequencies were ∼2-fold higher than that induced by 8-oxo-dG adduct. Replication in E. coli was essentially error-free. To elucidate the mechanisms of bypass of NM-Fapy-dG, we performed replication assays in vitro with a high-fidelity DNA polymerase, Saccharomyces cerevisiae polymerase (pol) δ. It was found that pol δ could catalyze high-fidelity synthesis past NM-Fapy-dG, but only on a template subpopulation, presumably containing the ß-anomeric adduct. Consistent with the low mutagenic potential of the ß-anomer in vitro, the mutation frequency was significantly reduced when conditions for vector preparation were modified to favor this configuration. Collectively, these data implicate the α-anomer as a major contributor to NM-Fapy-dG-induced mutagenesis in primate cells.

Profiling the Nucleobase and Structure Selectivity of Anticancer Drugs and other DNA Alkylating Agents by RNA Sequencing.

Drugs that covalently modify DNA are components of most chemotherapy regimens, often serving as first-line treatments. Classically, the reactivity and selectivity of DNA alkylating agents has been determined in vitro with short oligonucleotides. A statistically sound analysis of sequence preferences of alkylating agents is untenable with serial analysis methods because of the combinatorial explosion of sequence possibilities. Next-generation sequencing (NGS) is ideally suited for the broad characterization of sequence or structure selectivities because it analyzes many sequences at once. Herein, NGS is used to report on the chemoselectivity of alkylating agents on RNA and this technology is applied to the previously uncharacterized alkylating agent trimethylsilyl diazomethane.

Marine Natural Product Honaucin A Attenuates Inflammation by Activating the Nrf2-ARE Pathway.

The cyanobacterial marine natural product honaucin A inhibits mammalian innate inflammation in vitro and in vivo. To decipher its mechanism of action, RNA sequencing was used to evaluate differences in gene expression of cultured macrophages following honaucin A treatment. This analysis led to the hypothesis that honaucin A exerts its anti-inflammatory activity through activation of the cytoprotective nuclear erythroid 2-related factor 2 (Nrf2)-antioxidant response element/electrophile response element (ARE/EpRE) signaling pathway. Activation of this pathway by honaucin A in cultured human MCF7 cells was confirmed using an Nrf2 luciferase reporter assay. In vitro alkylation experiments with the natural product and N-acetyl-l-cysteine suggest that honaucin A activates this pathway through covalent interaction with the sulfhydryl residues of the cytosolic repressor protein Keap1. Honaucin A presents a potential therapeutic lead for diseases with an inflammatory component modulated by Nrf2-ARE.

Adverse outcome pathway development from protein alkylation to liver fibrosis.

In modern toxicology, substantial efforts are undertaken to develop alternative solutions for in vivo toxicity testing. The adverse outcome pathway (AOP) concept could facilitate knowledge-based safety assessment of chemicals that does not rely exclusively on in vivo toxicity testing. The construction of an AOP is based on understanding toxicological processes at different levels of biological organisation. Here, we present the developed AOP for liver fibrosis and demonstrate a linkage between hepatic injury caused by chemical protein alkylation and the formation of liver fibrosis, supported by coherent and consistent scientific data. This long-term process, in which inflammation, tissue destruction, and repair occur simultaneously, results from the complex interplay between various hepatic cell types, receptors, and signalling pathways. Due to the complexity of the process, an adequate liver fibrosis cell model for in vitro evaluation of a chemical's fibrogenic potential is not yet available. Liver fibrosis poses an important human health issue that is also relevant for regulatory purposes. An AOP described with enough mechanistic detail might support chemical risk assessment by indicating early markers for downstream events and thus facilitating the development of an in vitro testing strategy. With this work, we demonstrate how the AOP framework can support the assembly and coherent display of distributed mechanistic information from the literature to support the use of alternative approaches for prediction of toxicity. This AOP was developed according to the guidance document on developing and assessing AOPs and its supplement, the users' handbook, issued by the Organisation for Economic Co-operation and Development.

Cytotoxicity of electrophilic iron(II)-clathrochelates in human promyelocytic leukemia cell line.

We observed that electrophilic iron(II)-clathrochelates exhibit significant cytotoxicity in human promyelocytic leukemia cells (IC50=6.5±4.6µM), which correlates with the enhancement of intracellular oxidative stress (17-fold increase with respect to the cells treated with the solvent only). Based on in vitro studies we suggested that this effect is caused by alkylation of glutathione leading to inhibition of the cellular antioxidative system and by catalytic generation of reactive oxygen species by products of the alkylation reaction.

Role of Cys³6°² in the function and regulation of the cardiac ryanodine receptor.

The cardiac Ca²âº release channel [ryanodine receptor type 2 (RyR2)] is modulated by thiol reactive agents, but the molecular basis of RyR2 modulation by thiol reagents is poorly understood. Cys³6³5 in the skeletal muscle RyR1 is one of the most hyper-reactive thiols and is important for the redox and calmodulin (CaM) regulation of the RyR1 channel. However, little is known about the role of the corresponding cysteine residue in RyR2 (Cys³6°²) in the function and regulation of the RyR2 channel. In the present study, we assessed the impact of mutating Cys³6°² (C³6°²A) on store overload-induced Ca²âº release (SOICR) and the regulation of RyR2 by thiol reagents and CaM. We found that the C³6°²A mutation suppressed SOICR by raising the activation threshold and delayed the termination of Ca²âº release by reducing the termination threshold. As a result, C³6°²A markedly increased the fractional Ca²âº release. Furthermore, the C³6°²A mutation diminished the inhibitory effect of N-ethylmaleimide on Ca²âº release, but it had no effect on the stimulatory action of 4,4'-dithiodipyridine (DTDP) on Ca²âº release. In addition, Cys³6°² mutations (C³6°²A or C³6°²R) did not abolish the effect of CaM on Ca²âº-release termination. Therefore, RyR2-Cys³6°² is a major site mediating the action of thiol alkylating agent N-ethylmaleimide, but not the action of the oxidant DTDP. Our data also indicate that residue Cys³6°² plays an important role in the activation and termination of Ca²âº release, but it is not essential for CaM regulation of RyR2.

Aag DNA glycosylase promotes alkylation-induced tissue damage mediated by Parp1.

Alkylating agents comprise a major class of front-line cancer chemotherapeutic compounds, and while these agents effectively kill tumor cells, they also damage healthy tissues. Although base excision repair (BER) is essential in repairing DNA alkylation damage, under certain conditions, initiation of BER can be detrimental. Here we illustrate that the alkyladenine DNA glycosylase (AAG) mediates alkylation-induced tissue damage and whole-animal lethality following exposure to alkylating agents. Aag-dependent tissue damage, as observed in cerebellar granule cells, splenocytes, thymocytes, bone marrow cells, pancreatic ß-cells, and retinal photoreceptor cells, was detected in wild-type mice, exacerbated in Aag transgenic mice, and completely suppressed in Aag⁻/⁻ mice. Additional genetic experiments dissected the effects of modulating both BER and Parp1 on alkylation sensitivity in mice and determined that Aag acts upstream of Parp1 in alkylation-induced tissue damage; in fact, cytotoxicity in WT and Aag transgenic mice was abrogated in the absence of Parp1. These results provide in vivo evidence that Aag-initiated BER may play a critical role in determining the side-effects of alkylating agent chemotherapies and that Parp1 plays a crucial role in Aag-mediated tissue damage.

First-in-human, phase I/IIa clinical study of the peptidase potentiated alkylator melflufen administered every three weeks to patients with advanced solid tumor malignancies.

PURPOSE: Melflufen (melphalan flufenamide, previously designated J1) is an optimized and targeted derivative of melphalan, hydrolyzed by aminopeptidases overexpressed in tumor cells resulting in selective release and trapping of melphalan, and enhanced activity in preclinical models. METHODS: This was a prospective, single-armed, open-label, first-in-human, dose-finding phase I/IIa study in 45 adult patients with advanced and progressive solid tumors without standard treatment options. Most common tumor types were ovarian carcinoma (n = 20) and non-small-cell lung cancer (NSCLC, n = 11). RESULTS: In the dose-escalating phase I part of the study, seven patients were treated with increasing fixed doses of melflufen (25-130 mg) Q3W. In the subsequent phase IIa part, 38 patients received in total 115 cycles of therapy at doses of 30-75 mg. No dose-limiting toxicities (DLTs) were observed at 25 and 50 mg; at higher doses DLTs were reversible neutropenias and thrombocytopenias, particularly evident in heavily pretreated patients, and the recommended phase II dose (RPTD) was set to 50 mg. Response Evaluation Criteria In Solid Tumors (RECIST) evaluation after 3 cycles of therapy (27 patients) showed partial response in one (ovarian cancer), and stable disease in 18 patients. One NSCLC patient received nine cycles of melflufen and progressed after 7 months of therapy. CONCLUSIONS: In conclusion, melflufen can safely be given to cancer patients, and the toxicity profile was as expected for alkylating agents; RPTD is 50 mg Q3W. Reversible and manageable bone marrow suppression was identified as a DLT. Clinical activity is suggested in ovarian cancer, but modest activity in treatment of refractory NSCLC.

Bis-3-chloropiperidines containing bridging lysine linkers: Influence of side chain structure on DNA alkylating activity.

A series of bis-3-chloropiperidines containing lysine linkers was synthesised as DNA alkylating model compounds by using a bidirectional synthetic strategy. These novel piperidine mustard based agents have been evaluated for their alkylating properties towards nucleic acids and were shown to alkylate and cleave DNA with strong preference for guanine residues. Our studies reveal that the introduction of aromatic groups in the side chain of the lysine linker has an impact on DNA alkylating activity. Analysis by ESI mass spectrometry enabled the verification of the reactive aziridinium ion formation. Overall, the results confirm our recently proposed reaction mechanism of bis-3-chloropiperidines.

Quantitative proteomics and dynamic imaging reveal that G3BP-mediated stress granule assembly is poly(ADP-ribose)-dependent following exposure to MNNG-induced DNA alkylation.

Poly(ADP-ribose) (pADPr) is a heterogenic molecule synthesised from NAD by poly(ADP-ribose) polymerases (PARPs). Many cellular functions from genome integrity surveillance, cell cycle progression and DNA repair to apoptosis are affected by pADPr through its network of associated proteins. Using quantitative proteomics, we established a temporal map of pADPr-associated complexes upon genotoxic stress. Results suggested a strong pADPr association to many proteins involved in stress granule formation, notably the ras-GAP SH3-binding protein G3BP, as well as in the later phases of alkylation-stress-induced responses. Further investigation with dynamic imaging clearly demonstrated a pADPr-dependent initiation of stress granule assembly originating from the nucleus. The co-transfection of G3BP with poly(ADP-ribose) glycohydrolase (PARG) indicates that pADPr is involved in modulating the nuclear translocation of G3BP. Moreover, a peptide pADPr blot assay of G3BP revealed that pADPr binds to the glycine-arginine-rich domain of G3BP. Thereafter, we established a comprehensive G3BP interactome in the presence of pADPr. Our findings establish a novel function for pADPr in the formation of G3BP-induced stress granules upon genotoxic stress.