DNA Interstrand Cross-link Formation Research Articles

Lonidamine Increases the Cytotoxic Effect of 1-[(4-Amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-chloroethyl)-3-nitrosourea via Energy Inhibition, Disrupting Redox Homeostasis, and Downregulating MGMT Expression in Human Lung Cancer Cell Line.

Lung cancer ranks as the second most diagnosed cancer and the leading cause of cancer-related deaths worldwide. Novel chemotherapeutic strategies are crucial to efficiently target tumor cells while minimizing toxicity to normal cells. In this study, we proposed a combination strategy using energy blocker lonidamine (LND) and cytotoxic drug nimustine (ACNU, 1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-chloroethyl)-3-nitrosourea) to enhance the killing of a human lung cancer cell line and investigated the potential chemo-sensitizing mechanism of LND. LND was found to remarkably increase the cytotoxicity of ACNU to A549 and H1299 cells without significantly affecting normal lung BEAS2B cells. The combination of LND and ACNU also produced significant effects on cell apoptosis, colony formation, cell migration, and invasion assays compared to single drug treatment. Mechanistically, LND decreased intracellular ATP levels by inhibiting glycolysis and inducing mitochondrial dysfunction. Furthermore, the combination of LND and ACNU could intensify cellular oxidative stress, decrease cellular GSH contents, and increase reactive oxygen species (ROS) production. Notably, LND alone dramatically downregulated the expression of DNA repair protein MGMT (O6-methylguanine-DNA methyltransferase), enhancing DNA interstrand cross-link formation induced by ACNU. Overall, LND represents a potential chemo-sensitizer to enhance ACNU therapy through energy inhibition, disrupting redox homeostasis and downregulating MGMT expression in human lung cancer cell line under preclinical and clinical background.

Energy Blocker Lonidamine Reverses Nimustine Resistance in Human Glioblastoma Cells through Energy Blockade, Redox Homeostasis Disruption, and O6-Methylguanine-DNA Methyltransferase Downregulation: In Vitro and In Vivo Validation.

Tumor resistance seriously hinders the clinical application of chloroethylnitrosoureas (CENUs), such as O6-methylguanine-DNA methylguanine (MGMT), which can repair O6-alkyl lesions, thereby inhibiting the formation of cytotoxic DNA interstrand cross-links (ICLs). Metabolic differences between tumor and normal cells provide a biochemical basis for novel therapeutic strategies aimed at selectively inhibiting tumor energy metabolism. In this study, the energy blocker lonidamine (LND) was selected as a chemo-sensitizer of nimustine (ACNU) to explore its potential effects and underlying mechanisms in human glioblastoma in vitro and in vivo. A series of cell-level studies showed that LND significantly increased the cytotoxic effects of ACNU on glioblastoma cells. Furthermore, LND plus ACNU enhanced the energy deficiency by inhibiting glycolysis and mitochondrial function. Notably, LND almost completely downregulated MGMT expression by inducing intracellular acidification. The number of lethal DNA ICLs produced by ACNU increased after the LND pretreatment. The combination of LND and ACNU aggravated cellular oxidative stress. In resistant SF763 mouse tumor xenografts, LND plus ACNU significantly inhibited tumor growth with fewer side effects than ACNU alone. Finally, we proposed a new "HMAGOMR" chemo-sensitizing mechanism through which LND may act as a potential chemo-sensitizer to reverse ACNU resistance in glioblastoma: moderate inhibition of hexokinase (HK) activity (H); mitochondrial dysfunction (M); suppressing adenosine triphosphate (ATP)-dependent drug efflux (A); changing redox homeostasis to inhibit GSH-mediated drug inactivation (G) and increasing intracellular oxidative stress (O); downregulating MGMT expression through intracellular acidification (M); and partial inhibition of energy-dependent DNA repair (R).

Development of 4,4′-dibromobinaphthalene analogues with potent photo-inducible DNA cross-linking capability and cytotoxicity towards breast MDA-MB 468 cancer cells

Photoinduced DNA cross-linking process showed advantages of high spatio-temporal resolution and control. We have designed, synthesized, and characterized several 4,4′-dibromo binaphthalene analogues (1a-f) that can be activated by 350 nm irradiation to induce various DNA damage, including DNA interstrand cross-links (ICL) formation, strand cleavages, and alkaline labile DNA lesions. The degree and types of DNA damage induced by these compounds depend on the leaving groups of the substrates, pH value of the buffer solution, and DNA sequences. The DNA ICL products were produced from the carbocations formed via the oxidation of free radicals photo-generated from 1a-f. Most of these compounds alone exhibited minimum cytotoxicity towards cancer cells while 350 nm irradiation greatly improved their anticancer effects (up to 40-fold enhancement) because of photo-induced cellular DNA damage. This work provides guidance for further design of photo-inducible DNA cross-linking agents as potent photo-activated anticancer prodrugs with good control over toxicity and selectivity.

Abstract 2638: Preclinical development of ADCT-212, a PSMA-targeted antibody-drug conjugate employing the pyrrolobenzodiazepine dimer SG2000 for PSMA-expressing cancers

Abstract Prostate-specific membrane antigen (PSMA) is a membrane-bound glutamate carboxypeptidase that is highly expressed in nearly all prostate cancers with the highest expression in metastatic castration-resistant prostate cancer. Moreover, PSMA is expressed in the neovasculature that supplies most non-prostatic solid tumors, including carcinomas of the lung, colon, breast, kidney, liver, and pancreas. ADCT-212 is an antibody-drug conjugate composed of the human IgG1 antibody 2A10 directed against human PSMA, site-specifically conjugated using GlycoConnectTM technology to PL1801, which contains HydraspaceTM, a valine-alanine cleavable linker and the pyrrolobenzodiazepine (PBD) dimer warhead SG2000 (drug-antibody ratio of ~1.8). The purpose of this study was to characterize the in vitro and in vivo anti-tumor activity of ADCT-212 in human cancer cell lines and xenograft models and to determine its tolerability and pharmacokinetic (PK) in the rat. In vitro, ADCT-212 demonstrated potent cytotoxicity in a panel of PSMA-positive prostate cancer cell lines, whereas its activity was greatly reduced in PSMA-negative cell lines. ADCT-212 was efficiently internalized by PSMA-expressing LNCaP cells. Trafficking to the lysosomes started as early as 30 minutes and peaked at 1-2 hours post treatment. In line with the mechanism of action of the PBD dimer, ADCT-212 produced DNA interstrand cross-links (ICLs) that peaked by 12 hours and persisted for up to 36 hours post-treatment. In contrast, the peak of DNA ICLs formation for SG2000, the PBD dimer warhead alone, was observed immediately after 2-hour incubation, while a non-targeted PBD-ADC did not yield any appreciable DNA ICLs. Moreover, ADCT-212 showed indirect bystander killing activity in PSMA-negative PC3 cells incubated with conditioned medium from ADCT-212-treated LNCaP cells. In vivo, ADCT-212 showed strong antitumor activity against CWR22Rv1 and LNCaP prostate cancer xenograft models. In the CWR22Rv1 model, a tumor with heterogeneous PSMA expression, ADCT-212 achieved dose-dependent antitumor activity when administered as single dose at 2, 4 or 6 mg/kg, which resulted in increased survival compared to the control animals. In the LNCaP model, a single dose of ADCT-212 at 5 or 10 mg/kg resulted in strong and specific antitumor activity. Conversely, in the PSMA-negative PC3 xenograft model, ADCT-212 did not show anti-tumor activity compared to the controls, highlighting its target-mediated antitumor activity. ADCT-212 was tolerated as a single 20 mg/kg dose in male rats, with exposure data being indicative of a linear PK profile with a half-life of approximately 12 days. In conclusion, ADCT-212 demonstrated potent and specific in vitro and in vivo anti-tumor activity while it was stable and well tolerated in the rat, warranting further development of ADCT-212 into the clinic. Citation Format: Francesca Zammarchi, Karin Havenith, Lolke de Haan, Ian Kirby, Narinder Janghra, Veronica Gil, Pedro Alves, Kristina Zaitseva, Meghann Kerr, Ben Leatherdale, Afroze Patel, Marie Thoelke, Shiran Huang, John A Hartley, Patrick H van Berkel. Preclinical development of ADCT-212, a PSMA-targeted antibody-drug conjugate employing the pyrrolobenzodiazepine dimer SG2000 for PSMA-expressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2638.

Comparative analysis of chlorambucil-induced DNA lesion formation and repair in a spectrum of different human cell systems.

Chlorambucil (CLB) belongs to the class of nitrogen mustards (NMs), which are highly reactive bifunctional alkylating agents and were the first chemotherapeutic agents developed. They form DNA interstrand crosslinks (ICLs), which cause a blockage of DNA strand separation, inhibiting essential processes in DNA metabolism like replication and transcription. In fast replicating cells, e.g., tumor cells, this can induce cell death. The upregulation of ICL repair is thought to be a key factor for the resistance of tumor cells to ICL-inducing cytostatic agents including NMs. To monitor induction and repair of CLB-induced ICLs, we adjusted the automated reversed fluorometric analysis of alkaline DNA unwinding assay (rFADU) for the detection of ICLs in adherent cells. For the detection of monoalkylated DNA bases we established an LC-MS/MS method. We performed a comparative analysis of adduct formation and removal in five human cell lines and in peripheral blood mononuclear cells (PBMCs) after treatment with CLB. Dose-dependent increases in adduct formation were observed, and suitable treatment concentrations were identified for each cell line, which were then used for monitoring the kinetics of adduct formation. We observed significant differences in the repair kinetics of the cell lines tested. For example, in A2780 cells, hTERT immortalized VH10 cells, and in PBMCs a time-dependent repair of the two main monoalkylated DNA-adducts was confirmed. Regarding ICLs, repair was observed in all cell systems except for PBMCs. In conclusion, LC-MS/MS analyses combined with the rFADU technique are powerful tools to study the molecular mechanisms of NM-induced DNA damage and repair. By applying these methods to a spectrum of human cell systems of different origin and transformation status, we obtained insight into the cell-type specific repair of different CLB-induced DNA lesions, which may help identify novel resistance mechanisms of tumors and define molecular targets for therapeutic interventions.

Light and hydrogen peroxide dual-responsive DNA interstrand crosslink precursors with potent cytotoxicity

Arylboronic acid/esters and phenyl selenides-based quinone methide (QM) precursors were reported to induce DNA interstrand crosslink (ICL) formation upon reaction with the inherently high concentrations of H2O2 in cancer cells. However, some normal cells (such as macrophages) also contain high-levels of H2O2, which may interfere with precursors’ selectivity. In order to enhance the spatiotemporal specificity by the photolysis, we developed photo- and H2O2– dual-responsive DNA ICL precursors 1–3, bearing a photo-responsive coumarin moiety and a H2O2 inducible phenyl selenide group. Precursors 1–3 are efficiently activated by photoirradiation and H2O2 to generate reactive QMs crosslinking DNA. Moreover, the reactivity of precursors can be modulated by the introduction of aromatic substituents (OMe, F), and the electron donating group (OMe) displays a more pronounced promoting effect on DNA ICL formation. A subsequent piperidine heat stability study confirmed that the formed QMs primarily alkylate dAs, dGs and dCs in DNA. Furthermore, 1–3 inhibit lung cancer cell (H1299) growth by inducing DNA damage and producing toxic reactive oxygen species (ROS) upon photolysis of released coumarin. This study illustrates the potent cytotoxicity achieved by novel photo/H2O2 dual-responsive QM precursors 1–3, affording a novel strategy for the development of inducible DNA interstrand cross-linkers.

CD25-targeted antibody-drug conjugate camidanlumab tesirine for relapsed or refractory classical Hodgkin lymphoma.

Classic Hodgkin lymphoma (cHL) accounts for more than 90% of HL in developed countries. Although the current combined modality therapy make it have a high cure rate, the prognosis for heavily pretreated patients with relapsed or refractory (R/R) cHL remains poor. A novel antibody-drug conjugate (ADC), named camidanlumab tesirine (ADCT-301, Cami), is currently being evaluated for its efficacy and safety in R/R cHL. The primary objective of this review is to examine the current pharmacological properties of camidanlumab tesirine as well as its clinical antitumor activity and safety. Camidanlumab tesirine comprises a human IgG1 anti-CD25 monoclonal antibody HuMax®-TAC, conjugated to a pyrrolobenzodiazepine dimer toxin. Once it bound to CD25-expressing cells, camidanlumab tesirine is internalized by cells and delivers SG3199, then SG3199 irreversibly binds to DNA and forms DNA interstrand crosslinks, ultimately leading to cell death. In the phase 1 study, patients with R/R cHL who received camidanlumab tesirine had an overall response rate (ORR) of 71% and a complete response rate (CRR) of 42%. Additionally, the recommended doses provided in R/R cHL were determined to be 30 and 45μg/kg. The pivotal phase 2 trial showed significant antitumor activity of camidanlumab tesirine in heavily pretreated R/R cHL patients who failed brentuximab vedotin and programmed death-1 blockade: ORR was 70.1% and CRR was 33.3%, and the median duration of response was 13.7months. Adverse events such as fatigue, maculopapular rash, and anemia were frequently observed following administration of camidanlumab tesirine. Moreover, camidanlumab tesirine may cause Guillain-Barré syndrome or polyradiculopathy.

Photo-Reactivity of Binaphthalene Triphenylphosphonium Salts: DNA Interstrand Cross-Link Formation and Substituent Effects.

Five novel 1,1'-binaphthalene analogues 1a-1e with triphenylphosphonium (TPP+) salts as a leaving group have been synthesized and characterized as photo-activatable DNA alkylating agents. Phototriggered release of the TPP+ group from 1a-1e generated naphthalenylmethyl-free radicals that were spontaneously transformed to the corresponding cations directly producing DNA interstrand cross-link (ICL) formation via alkylation. The substituents at position 4 not only affect the efficiency of ICL formation but also influence the reaction rate for DNA cross-linking. Groups with small or medium size favor ICL formation, while a bulky substituent (e.g., phenyl group) prevents DNA interstrand cross-linking. DNA alkylation by the naphthalenylmethyl cations photo-generated from 1a-1e occurs at dG, dC, and dA, while interstrand cross-linking took place with dG/dC base pairs. The TPP+ salts (1a-1e) are cations with both lipophilic and hydrophilic properties, which have great potential for biological applications.

Proliferating Stem Cells are Acutely Affected by DNA Damage Induced by Sulfur Mustard.

Sulfur mustard (SM), a chemical warfare agent, can form adducts with DNA, RNA, and proteins. Reactions with DNA lead to the formation of both DNA monoadducts and interstrand cross-links, resulting in DNA damage, and is an important component of SM toxicity. Our previous in vivo studies indicated that dividing cells such as hematopoietic stem cells and intestinal villi stem cells seemed to have increased sensitivity to SM. Therefore, to compare the sensitivity of somatic and stem cells to SM and to investigate the mechanism of SM cytotoxicity, we isolated human foreskin fibroblasts, reprogrammed them into pluripotent stem cells, and then compared the DNA damage repair pathways involved upon SM treatment. Our results indicated that proliferating stem cells were more sensitive to SM-induced DNA damage, and the damage mainly comprised single-stranded breaks. Furthermore, the pathways involved in DNA repair in stem cells and somatic cells were different.

Spatial and Temporal Resolution of the Oxygen-Independent Photoinduced DNA Interstrand Cross-Linking by a Nitroimidazole Derivative.

DNA damage is ubiquitous in nature and is at the basis of emergent treatments such as photodynamic therapy, which is based on the activation of highly oxidative reactive oxygen species by photosensitizing O2. However, hypoxia observed in solid tumors imposes the necessity to devise oxygen-independent modes of action able to induce DNA damage under a low oxygen concentration. The complexity of these DNA damage mechanisms in realistic environments grows exponentially when taking into account light absorption and subsequent excited-state population, photochemical and (photo)-redox reactions, the multiple species involved in different electronic states, noncovalent interactions, multiple reaction steps, and the large number of DNA reactive sites. This work tackles all the intricate reactivity of a photosensitizer based on a nitroimidazole derivative reacting toward DNA in solution under UV light exposition. This is performed through a combination of ground- and excited-state quantum chemistry, classical molecular dynamics, and hybrid QM/MM simulations to rationalize in detail the formation of DNA interstrand cross-links (ICLs) exerted by the noncanonical noncovalent photosensitizer. Unprecedented spatial and temporal resolution of these phenomena is achieved, revealing that the ICL is sequence-specific and that the fastest reactions take place at AT, GC, and GT steps involving either the opposite nucleobases or adjacent Watson–Crick base pairs. The N7 and O6 positions of guanine, the N7 and N3 sites of adenine, the N4 position of cytosine, and the O2 atom of thymine are deemed as the most nucleophile sites and are positively identified to participate in the ICL productions. This work provides a multiscale computational protocol to study DNA reactivity with noncovalent photosensitizers, and contributes to the understanding of therapies based on photoinduced DNA damage at molecular and electronic levels. In addition, we believe the depth understanding of these processes should assist the design of new photosensitizers considering their molecular size, electronic properties, and the observed regioselectivity toward nucleic acids.

Formation of bifunctional cross-linked products due to reaction of NAMI-A with DNA bases – a DFT study

It is reported that NAMI-A and other Ru-anticancer complexes preferably bind with the N7 site of guanine and can also form DNA inter-strand cross-links. Therefore, in order to understand the DNA cross-link formation capability of NAMI-A, we have investigated here the structure and energetics of the reactions of the GN7-NAMI-A (a monofunctional adduct of NAMI-A with the N7 site of guanine) with the N3, N7, and O6 sites of guanine; the N1, N3, and N7 sites of adenine; the O2 and N3 sites of cytosine; and the O2 and O4 sites of thymine, using the M06-2X functional of density functional theory. It is found that the GN7-NAMI-A can form stable cross-linked products at all the sites studied here except at the N3 site of cytosine and O2 site of thymine. The calculated reaction free energies and reaction enthalpies indicate that the N3 site of adenine (AN3) and N7 site of guanine (GN7) are most exothermic among all the studied reactions. This study shows that NAMI-A would favorably form the cross-linked products involving the N7 site of guanine at one side and the N7 site of guanine or the N3 site of adenine at the other side.

Development and biological evaluation of AzoBGNU: A novel hypoxia-activated DNA crosslinking prodrug with AGT-inhibitory activity

Chloroethylnitrosoureas (CENUs) are an important family of chemotherapies in clinical treatment of cancers, which exert antitumor activity by inducing the formation of DNA interstrand crosslinks (dG-dC ICLs). However, the drug resistance mediated by O6-alkylguanine-DNA alkyltransferase (AGT) and absence of tumor-targeting ability largely decrease the antitumor efficacy of CENUs. In this study, we synthesized an azobenzene-based hypoxia-activated combi-nitrosourea prodrug, AzoBGNU, and evaluated its hypoxic selectivity and antitumor activity. The prodrug was composed of a CENU pharmacophore and an O6-benzylguanine (O6-BG) analog moiety masked by a N,N-dimethyl-4-(phenyldiazenyl)aniline segment as a hypoxia-activated trigger, which was designed to be selectively reduced via azo bond break in hypoxic tumor microenvironment, accompanied with releasing of an O6-BG analog to inhibit AGT and a chloroethylating agent to induce dG-dC ICLs. AzoBGNU exhibited significantly increased cytotoxicity and apoptosis-inducing ability toward DU145 cells under hypoxia compared with normoxia, indicating the hypoxia-responsiveness as expected. Predominant higher cytotoxicity was observed in the cells treated by AzoBGNU than those by traditional CENU chemotherapy ACNU and its combination with O6-BG. The levels of dG-dC ICLs in DU145 cells induced by AzoBGNU was remarkably enhanced under hypoxia, which was approximately 6-fold higher than those in the AzoBGNU-treated groups under normoxia and those in the ACNU-treated groups. The results demonstrated that azobenzene-based combi-nitrosourea prodrug possessed desirable tumor-hypoxia targeting ability and eliminated chemoresistance compared with the conventional CENUs.

Formation and Repair of an Interstrand DNA Cross-Link Arising from a Common Endogenous Lesion.

Interstrand DNA cross-links (ICLs) are cytotoxic because they block the strand separation required for read-out and replication of the genetic information in duplex DNA. The unavoidable formation of ICLs in cellular DNA may contribute to aging, neurodegeneration, and cancer. Here, we describe the formation and properties of a structurally complex ICL derived from an apurinic/apyrimidinic (AP) site, which is one of the most common endogenous lesions in cellular DNA. The results characterize a cross-link arising from aza-Michael addition of the N2-amino group of a guanine residue to the electrophilic sugar remnant generated by spermine-mediated strand cleavage at an AP site in duplex DNA. An α,β-unsaturated iminium ion is the critical intermediate involved in ICL formation. Studies employing the bacteriophage φ29 polymerase provided evidence that this ICL can block critical DNA transactions that require strand separation. The results of biochemical studies suggest that this complex strand break/ICL might be repaired by a simple mechanism in which the 3'-exonuclease action of the enzyme apurinic/apyrimidinic endonuclease (APE1) unhooks the cross-link to initiate repair via the single-strand break repair pathway.

Ethanol-Induced Cell Damage Can Result in the Development of Oral Tumors.

Simple SummaryAlcohol consumption is linked to 26.4% of all lip and oral cavity cancer cases worldwide. Despite this clear causal relationship, the exact molecular mechanisms by which ethanol damages cells are still under investigation. It is well-established that the metabolism of ethanol plays an important role. Ethanol metabolism yields reactive metabolites that can directly damage the DNA. If the damage is repaired incorrectly, mutations can be fixed in the DNA sequence. Whenever mutations affect key regulatory genes, for instance cell cycle regulating genes, uncontrolled cell growth can be the consequence. Recently, global patterns of mutations have been identified. These so-called mutational signatures represent a fingerprint of the different mutational processes over time. Interestingly, there were ethanol-related signatures discovered that did not associate with ethanol metabolism. This finding highlights there might be other molecular effects of ethanol that are yet to be discovered.Alcohol consumption is an underestimated risk factor for the development of precancerous lesions in the oral cavity. Although alcohol is a well-accepted recreational drug, 26.4% of all lip and oral cavity cancers worldwide are related to heavy drinking. Molecular mechanisms underlying this carcinogenic effect of ethanol are still under investigation. An important damaging effect comes from the first metabolite of ethanol, being acetaldehyde. Concentrations of acetaldehyde detected in the oral cavity are relatively high due to the metabolization of ethanol by oral microbes. Acetaldehyde can directly damage the DNA by the formation of mutagenic DNA adducts and interstrand crosslinks. Additionally, ethanol is known to affect epigenetic methylation and acetylation patterns, which are important regulators of gene expression. Ethanol-induced hypomethylation can activate the expression of oncogenes which subsequently can result in malignant transformation. The recent identification of ethanol-related mutational signatures emphasizes the role of acetaldehyde in alcohol-associated carcinogenesis. However, not all signatures associated with alcohol intake also relate to acetaldehyde. This finding highlights that there might be other effects of ethanol yet to be discovered.

New Synthetic Analogs of Nitrogen Mustard DNA Interstrand Cross-Links and Their Use to Study Lesion Bypass by DNA Polymerases.

Nitrogen mustards are a widely used class of antitumor agents that exert their cytotoxic effects through the formation of DNA interstrand cross-links (ICLs). Despite being among the first antitumor agents used, the biological responses to NM ICLs remain only partially understood. We have previously reported the generation of NM ICL mimics by incorporation of ICL precursors into DNA using solid-phase synthesis at defined positions, followed by a double reductive amination reaction. However, the structure of these mimics deviated from the native NM ICLs. Using further development of our approach, we report a new class of NM ICL mimics that only differ from their native counterpart by substitution of dG with 7-deaza-dG at the ICL. Importantly, this approach allows for the synthesis of diverse NM ICLs, illustrated here with a mimic of the adduct formed by chlorambucil. We used the newly generated ICLs in reactions with replicative and translesion synthesis DNA polymerase to demonstrate their stability and utility for functional studies. These new NM ICLs will allow for the further characterization of the biological responses to this important class of antitumor agents.

Photoinduced DNA Interstrand Cross-Linking by 1,1'-Biphenyl Analogues: Substituents and Leaving Groups Combine to Determine the Efficiency of Cross-Linker.

Two series of 1,1'-biphenyl analogues with various leaving groups (L=OAc, OCH3 , OCHCH=CH2 , OCH2 Ph, SPh, SePh, and Ph3 P+ ) were synthesized. Their reactivity towards DNA and the reaction mechanism were investigated by determining DNA interstrand cross-link (ICL) efficiency, radical and carbocation formation, and the cross-linking reaction sites. All compounds induced DNA ICL formation upon 350 nm irradiation via a carbocation that was generated from oxidation of the corresponding free radicals. The ICL efficiency and the reaction rate strongly depended on the combined effect of the leaving group and the substituent. Among all compounds tested, the high ICL efficiency (30-43 %) and fast reaction rate were observed with compounds carrying a nitrophenyl group and acetate (2 a), ether (2 b and 2 c), or triphenylphosphonium salt (2 g) as leaving groups. Most compounds with a 4-methoxybenzene group showed similar DNA ICL efficiency (≈30 %) with a slow DNA cross-linking reaction rate. Both cation trapping and free radical trapping adducts were detected in the photo activation process of these compounds, which provided direct evidence for the proposed mechanism. Heat stability study in combination with sequence study suggested that these photo-generated benzyl cations alkylate DNA at dG, dA, and dC sites.

Near-Infrared Light Irradiation Induced Mild Hyperthermia Enhances Glutathione Depletion and DNA Interstrand Cross-Link Formation for Efficient Chemotherapy.

DNA alkylating agents generally kill tumor cells by covalently binding with DNA to form interstrand or intrastrand cross-links. However, in the case of cisplatin, only a few DNA adducts (<1%) are highly toxic irreparable interstrand cross-links. Furthermore, cisplatin is rapidly detoxified by high levels of intracellular thiols such as glutathione (GSH). Since the discovery of its mechanism of action, people have been looking for ways to directly and efficiently remove intracellular GSH and increase interstrand cross-links to improve drug efficacy and overcome resistance, but there has been little breakthrough. Herein, we hypothesized that the anticancer efficiency of cisplatin can be enhanced through iodo-thiol click chemistry mediated GSH depletion and increased formation of DNA interstrand cross-links via mild hyperthermia triggered by near-infrared (NIR) light. This was achieved by preparing an amphiphilic polymer with platinum(IV) (Pt(IV)) prodrugs and pendant iodine atoms (iodides). The polymer was further used to encapsulate IR780 and assembled into Pt-I-IR780 nanoparticles. Induction of mild hyperthermia (43 °C) at the tumor site by NIR light irradiation had three effects: (1) it accelerated the GSH-mediated reduction of Pt(IV) in the polymer main chain to platinum(II) (Pt(II)); (2) it boosted the iodo-thiol substitution click reaction between GSH and iodide, thereby attenuating the GSH-mediated detoxification of cisplatin; (3) it increased the proportion of highly toxic and irreparable Pt-DNA interstrand cross-links. Therefore, we find that mild hyperthermia induced via NIR irradiation can enhance the killing of cancer cells and reduce the tumor burden, thus delivering efficient chemotherapy.

Synthesis of Oligonucleotides containing the cis-Interstrand Crosslink Produced by Mitomycins in their Reaction with DNA.

Mitomycin C (MC) an antitumor drug and decarbamoylmitomycin C (DMC), a derivative of MC lacking the carbamoyl moiety, are DNA alkylating agents which can form DNA interstrand crosslinks (ICLs) between deoxyguanosine residues located on opposing DNA strands. MC forms primarily deoxyguanosine adducts with a 1"-R stereochemistry at the guanine-mitosene bond (1"-α, trans) whereas DMC forms mainly adducts with a 1"-S stereochemistry (1"-β, cis). The crosslinking reaction is diastereospecific: trans-crosslinks are formed exclusively at CpG sequences, while cis-crosslinks are formed only at GpC sequences. Until now, oligonucleotides containing 1"-β-deoxyguanosine adducts or ICL at a specific site could not be synthesized, thus limiting the investigation of the role played by the stereochemical configuration at C1'' in the toxicity of these compounds. Here, a novel biomimetic synthesis to access these substrates is presented. Structural proof of the adducted oligonucleotides and ICL were provided by enzymatic digestion to nucleosides, high resolution mass spectral analysis, CD spectroscopy and UV melting temperature studies. Finally, a virtual model of the 25-mer 1"-β ICL synthesized was created to explore the conformational space and structural features of the crosslinked duplex.

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.

Nitrogen Mustard Induces Formation of DNA-Histone Cross-Links in Nucleosome Core Particles.

Nitrogen mustards have long been used in cancer chemotherapy, and their cytotoxicity has traditionally been attributed to the formation of DNA interstrand cross-links and DNA monoalkylation. Recent studies have shown that exposure to nitrogen mustards also induces the formation of DNA-protein cross-links (DPCs) via bridging between N7 of a deoxyguanosine residue in the DNA and the side chain of a Cys residue in the protein. However, the formation of nitrogen mustard-induced DNA-histone cross-links has never been observed. Herein, we demonstrate that treating reconstituted nucleosome core particles (NCPs) with the nitrogen mustard mechlorethamine results in the formation of DNA-histone cross-links in addition to DNA monoalkylation and interstrand cross-link formation. The yields of these three types of DNA lesions in the NCPs decreased in the following order: DNA monoalkylation ≫ DNA interstrand cross-links > DNA-histone cross-links. Mechanistic studies involving tailless histones and competitive inhibition by a polyamine demonstrated that Lys residues in the N- and C-terminal tails of the histones were the predominant sites involved in DNA-histone cross-link formation. Given that NCPs are the fundamental repeating units of chromatin in eukaryotes, our findings suggest that nitrogen mustard-induced formation of DNA-histone cross-links may occur in living cells and that DPC formation may contribute to the cytotoxicity of nitrogen mustards.