Formation Of Interstrand Cross-links Research Articles

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.

Mitomycin C and its analog trigger cytotoxicity in MCF-7 and K562 cancer cells through the regulation of RAS and MAPK/ERK pathways

Mitomycin C (MC) is an anti-cancer drug which functions by forming interstrand crosslinks (ICLs) between opposing DNA strands. MC analog, 10-decarbamoyl mitomycin C (DMC), unlike MC, has stronger cytotoxic effects on cancer cells with TP53 mutation. We previously demonstrated that MC/DMC could activate p21WAF1/CIP1 in MCF-7 (TP53-proficient) and K562 (TP53 deficient) cells in a TP53-independent mode. We also found that MC/DMC regulate AKT activation in a TP53-dependent manner and that AKT deactivation is not associated with the activation of p21WAF1/CIP1 in response to MC/DMC treatment. RAS proteins are known players in the upstream mediated signaling of p21WAF1/CIP1 activation that leads to control of cell proliferation and cell death. Thus, this prompted us to investigate the effect of both drugs on the expression of RAS proteins and regulation of the MAPK/ERK signaling pathways in MCF-7 and K562 cancer cells. To accomplish this goal, we performed comparative label free proteomics profiling coupled to bioinformatics/complementary phosphoprotein arrays and Western blot validations of key signaling molecules. The MAPK/ERK pathway exhibited an overall downregulation upon MC/DMC treatment in MCF-7 cells but only DMC exhibited a mild downregulation of that same pathway in TP53 mutant K562 cells. Furthermore, treatment of MCF-7 and K562 cell lines with oligonucleotides containing the interstrand crosslinks (ICLs) formed by MC or DMC shows that both ICLs had a stronger effect on the downregulation of RAS protein expression in mutant TP53 K562 cells. We discuss the implication of this regulation of the MAPK/ERK pathway in relation to cellular TP53 status.

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).

Mechanism of AGT-Mediated Repair of dG-dC Cross-Links in the Drug Resistance to Chloroethylnitrosoureas: Molecular Docking, MD Simulation, and ONIOM (QM/MM) Investigation.

Chloroethylnitrosoureas (CENUs) are important chemotherapies applied in the treatment of cancer. They exert anticancer activity by inducing DNA interstrand cross-links (ICLs) via the formation of two O6-alkylguanine intermediates, O6-chloroethylguanine (O6-ClEtG) and N1,O6-ethanoguanine (N1,O6-EtG). However, O6-alkylguanine-DNA alkyltransferase (AGT), a DNA-repair enzyme, can restore the O6-alkylguanine damages and thereby obstruct the formation of ICLs (dG-dC cross-link). In this study, the inhibitory mechanism of ICL formation was investigated to elucidate the drug resistance of CENUs mediated by AGT in detail. Based on the structures of the substrate-enzyme complexes obtained from docking and MD simulations, two ONIOM (QM/MM) models with different sizes of the QM region were constructed. The model with a larger QM region, which included the substrate (O6-ClEtG or N1,O6-EtG), a water molecule, and five residues (Tyr114, Cys145, His146, Lys165, and Glu172) in the active pocket of AGT, accurately described the repairing reaction and generated the results coinciding with the experimental outcomes. The repair process consists of two sequential steps: hydrogen transfer to form a thiolate anion on Cys145 and alkyl transfer from the O6 site of guanine (the rate-limiting step). The repair of N1,O6-EtG was more favorable than that of O6-ClEtG from both kinetics and thermodynamics aspects. Moreover, the comparison of the repairing process with the formation of dG-dC cross-link and the inhibition of AGT by O6-benzylguanine (O6-BG) showed that the presence of AGT could effectively interrupt the formation of ICLs leading to drug resistance, and the inhibition of AGT by O6-BG that was energetically more favorable than the repair of O6-ClEtG could not prevent the repair of N1,O6-EtG. Therefore, it is necessary to completely eliminate AGT activity before CENUs medication to enhance the chemotherapeutic effectiveness. This work provides reasonable explanations for the supposed mechanism of AGT-mediated drug resistance of CENUs and will assist in the development of novel CENU chemotherapies and their medication strategies.

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 1568: Mechanistic studies of KL-50, a novel imidazotetrazine for the treatment of MGMT-/MMR- gliomas and glioblastoma

Abstract The DNA damage repair protein O6-methylguanine methyl transferase (MGMT) reverses O6-alkylguanine lesions via SN2 transfer of the alkyl lesion to an active site cysteine which restores DNA to its native state. MGMT is ubiquitously expressed in healthy tissue but is silenced by promoter hypermethylation in approximately half of glioblastomas and up to 75% of lower grade gliomas. As such, patients with MGMT deficient (MGMT-) tumors benefit from DNA alkylation agents such as temozolomide (TMZ), an imidazotetrazine that deposits methyl lesions at O6-guanine. However, the cytotoxicity of TMZ requires an intact DNA mismatch repair (MMR) pathway and acquired resistance to TMZ via MMR silencing negates the efficacy of the drug. We recently reported the synthesis, discovery, and in vivo evaluation of KL-50, an imidazotetrazine derivative that overcomes this resistance mechanism while maintaining high selectivity for MGMT- cells in vitro and in vivo. Our preliminary mechanistic data suggest that KL-50 induces cell death independent of MMR status via the deposition of 2-fluoroethyl lesions at O6-guanine to give an O6-(2-fluoroethyl)guanine (O6FEtG) lesion that evolves to a deadly DNA interstrand cross-link (ICL). We hypothesize that this occurs via a two-step pathway involving slow cyclization of the initial O6FEtG lesion to an electrophilic N1,O6-ethanoguanine intermediate and subsequent nucleophilic ring-opening by the base-paired cytidine residue to yield a guanine(N1)-cytidine(N3) ethyl ICL. Our data suggest the high therapeutic index of KL-50 derives from the relative rates of MGMT reversal of O6FEtG and its conversion to an ICL, specifically that MGMT proficient healthy cells can repair the O6FEtG before it evolves to the ICL. However, direct MGMT reversal of O6FEtG has not been reported, and the rates of ICL formation are not known. Additionally, the formation of the putative ICL has not been demonstrated. Herein, we demonstrate that MGMT can repair the O6FEtG lesion. Kinetics studies using well-defined, photocaged oligonucleotides containing a single O6FEtG lesion show that ICL formation occurs on the order of hours. Furthermore, addition of MGMT following photo-deprotection prevents the formation of ICLs via rapid reversal of O6FEtG. Finally, we have detected a mass corresponding to the putative ICL in pUC19 DNA treated with KL-50 via LCMS . These findings bolster our mechanistic model and suggest strategies that exploit the relative rates of DNA damage and repair may be extensible to other tumor types harboring specific DNA repair defects. Citation Format: Eric D. Huseman, Anna Lo, Olga Fedorova, Kingson Lin, Susan Gueble, Ranjini Sundaram, Anna M. Pyle, Ranjit S. Bindra, Seth Herzon. Mechanistic studies of KL-50, a novel imidazotetrazine for the treatment of MGMT-/MMR- gliomas and glioblastoma [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 1568.

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.

Efficient Synthesis of DNA Duplexes Containing Reduced Acetaldehyde Interstrand Cross-Links.

DNA interstrand cross-links (ICLs) prevent DNA replication and transcription and can lead to potentially lethal events, such as cancer or bone marrow failure. ICLs are typically repaired by proteins within the Fanconi Anemia (FA) pathway, although the details of the pathway are not fully established. Methods to generate DNA containing ICLs are key to furthering the understanding of DNA cross-link repair. A major route to ICL formation in vivo involves reaction of DNA with acetaldehyde, derived from ethanol metabolism. This reaction forms a three-carbon bridged ICL involving the amino groups of adjacent guanines in opposite strands of a duplex resulting in amino and imino functionalities. A stable reduced form of the ICL has applications in understanding the recognition and repair of these types of adducts. Previous routes to creating DNA duplexes containing these adducts have involved lengthy post-DNA synthesis chemistry followed by reduction of the imine. Here, an efficient and high-yielding approach to the reduced ICL using a novel N2-((R)-4-trifluoroacetamidobutan-2-yl)-2'-deoxyguanosine phosphoramidite is described. Following standard automated DNA synthesis and deprotection, the ICL is formed overnight in over 90% yield upon incubation at room temperature with a complementary oligodeoxyribonucleotide containing 2-fluoro-2'-deoxyinosine. The cross-linked duplex displayed a melting transition 25 °C higher than control sequences. Importantly, we show using the Xenopus egg extract system that an ICL synthesized by this method is repaired by the FA pathway. The simplicity and efficiency of this methodology for preparing reduced acetaldehyde ICLs will facilitate access to these DNA architectures for future studies on cross-link repair.

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.

Effects of Local Sequence, Reaction Conditions, and Various Additives on the Formation and Stability of Interstrand Cross-Links Derived from the Reaction of an Abasic Site with an Adenine Residue in Duplex DNA.

The experiments described here examined the effects of reaction conditions, various additives, and local sequence on the formation and stability interstrand cross-links (ICLs) derived from the reaction of an apurinic/apyrimidinic (AP) site with the exocyclic amino group of an adenine residue on the opposing strand in duplex DNA. Cross-link formation was observed in a range of different buffers, with faster formation rates observed at pH 5. Inclusion of the base excision repair enzyme alkyladenine DNA glycosylase (hAAG) which binds tightly to AP-containing duplexes decreased, but did not completely prevent, formation of the dA-AP ICL. Formation of the dA-AP ICL was not altered by the presence of the biological metal ion Mg2+ or the biological thiol, glutathione. Several organocatalysts of imine formation did not enhance the rate of dA-AP ICL formation. Duplex length did not have a large effect on dA-AP yield, so long as the melting temperature of the duplex was not significantly below the reaction temperature (the duplex must remain hybridized for efficient ICL formation). Formation of the dA-AP ICL was examined in over 40 different sequences that varied the neighboring and opposing bases at the cross-linking site. The results indicate that ICL formation can occur in a wide variety of sequence contexts under physiological conditions. Formation of the dA-AP ICL was strongly inhibited by the aldehyde-trapping agents methoxyamine and hydralazine, by NaBH3CN, by the intercalator ethidium bromide, and by the minor groove-binding agent netropsin. ICL formation was inhibited to some extent in bicarbonate and Tris buffers. The dA-AP ICL showed substantial inherent stability under a variety of conditions and was not a substrate for AP-processing enzymes APE1 or Endo IV. Finally, we characterized cross-link formation in a small (11 bp) stem-loop (hairpin) structure and in DNA-RNA hybrid duplexes.

Formation Mechanism of Inter-Crosslink in DNA by Nitrogen Oxides Pollutants through A Diazonium Intermediate.

Outdoor air pollution is a mixture of multiple atmospheric pollutants, among which nitrogen oxide (NOx) stands out due to its association with several diseases. NOx reactivity can conduct to DNA damage as severe as interstrand crosslinks (ICL) formation, that in turn is able to block DNA replication and transcription. Experimental studies have suggested that the ICL formation due to NOx is realized through a diazonium intermediate (DI). In this work, we have modeled the DI structure, including a DNA double-strand composed of two base pairs GC/CG, being diazotized as one of the guanine nucleotides. The structural stability of DNA with DI lesion was essayed through 500 ns molecular dynamics simulations. It was found that the DNA structure of the oligonucleotide is stable when the DI is present since the loss of a Guanine–Cytosine hydrogen bond is replaced by the presence of two cation-π interactions. Additionally, we have studied the mechanism of formation of a crosslink between the two guanine nucleobases from the modeled DI by carrying out DFT calculations at the M06-L/DNP+ level of theory. Our results show that the mechanism is thermodynamically favored by a strong stabilization of the ICL product, and the process is kinetically viable since its limiting stage is accessible.

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.

Potential Signaling Regulations of Stereisomeric DNA Interstrand Crosslinks Produced by Mitomycins in K562 Cells

Mitomycin C (MC) is a DNA alkylating agent broadly used in chemotherapy. MC and Decarbamoyl mitomycin C (DMC), a synthetic MC derivative which lacks the carbamoyl at the C10 position, can form interstrand crosslinks (ICLs) between the exocyclic amines of opposing deoxyguanosine moieties. These ICLs are responsible for the cytotoxicity of mitomycins towards cancer cells since they prevent replication of highly proliferating cells. MC and DMC can crosslink DNA in both 5’‐CpG and 5’‐GpC sequence contexts, and the crosslinking is diastereospecific: At 5’‐CpG, mitomycins produce trans‐ICLs whereas cis‐ICLs are found at 5’‐GpC steps. The major ICL formed by Mitomycin C in culture cells is the trans‐ICL whereas decarbamoylmitomycin C yields the stereoisomeric cis‐ICL preferentially. Our overarching goal is to synthesize oligonucleotides bearing the cisand trans isomeric crosslinks to investigate the relationship between the ICL structure and the cellular response. Our hypothesis is that the trans and cis‐ICLs trigger different cellular responses. There is evidence that the ICL produced by DMC triggers a p53 independent cell death and research uncovering mechanisms through which p53 independent cell death occurs is crucial to identify molecular targets to treat tumors with a mutant p53. These tumors represent at least 50% of all cancers. We present here the different synthetic routes to access cis and trans isomeric interstrand crosslinks formed by mitomycins in their reaction with DNA and preliminary label free proteomic studies to identify the stereoisomeric ICLs molecular targets. Proteomic studies were accomplished from K562 leukemia cells transfected with the purified ICLs for 24 hours and showed several interesting proteins differently regulated by the trans‐ and cis‐ICLs: (1) Histone‐lysine N‐methyltransferase EHMT2 which down‐regulates a cell cycle inhibitory gene expression network (decreased 46% upon treatment with the trans‐ICL, but no change was observed with the cis‐ICL); (2) Target of EGR1 protein 1 which inhibits cell growth rate and cell cycle (increased 69% upon treatment with the trans‐ICL, but decreased 30% with the cis‐ICL); (3) Poly(ADP‐ribose) glycohydrolase DNA repair, involved in DNA repair (decreased 19% upon treatment with the trans‐ICL, but increased 63% with the cis‐ICL); (4) Telomerases Cajal body protein 1, involved in cell cycle regulation (no change observed upon tratment with the trans‐ICL but decreased 75% with the cis‐ICL. In addition, Ingenuity Pathway Analysis was conducted to identify the major molecular pathways impacted by each ICL treatment. In conclusion, we have identified both ICL molecular targets and cellular responses involved in DNA repair and cell cycle regulation in K‐562cell lines. Further validations are needed to confirm the results.

Identification of Potential Cellular Responses Triggered by Stereoisomeric DNA Interstrand Crosslinks Produced by Mitomycins in MCF‐7 Cells

Mitomycin C (MC) is a DNA alkylating agent broadly used in chemotherapy. MC and Decarbamoylmitomycin C (DMC), a synthetic MC derivative which lacks the carbamoyl at the C10 position, can form interstrand crosslinks (ICLs) between the exocyclic amines of opposing deoxyguanosine moieties. These ICLs are responsible for the cytotoxicity of mitomycins towards cancer cells since they prevent replication of highly proliferating cells. MC and DMC can crosslink DNA in both 5’‐CpG and 5’‐GpC sequence contexts, and the crosslinking is diastereospecific: At 5’‐CpG, mitomycins produce trans‐ICLs whereas cis‐ICLs are found at 5’‐GpC steps. The major ICL formed by Mitomycin C in culture cells is the trans‐ICL whereas decarbamoylmitomycin C yields the stereoisomeric cis‐ICL preferentially. Our overarching goal is to synthesize oligonucleotides bearing the cisand trans isomeric crosslinks to investigate the relationship between ICL structure and cellular response. Our hypothesis is that the trans and cis‐ICLs trigger different cellular responses. There is evidence that the ICL produced by DMC triggers a p53 independent cell death and research uncovering mechanisms through which p53 independent cell death occurs is crucial to identify molecular targets to treat tumors with a mutant p53. These tumors represent at least 50% of all cancers. We present here the different synthetic routes to access cis and trans isomeric interstrand crosslinks formed by mitomycins in their reaction with DNA and preliminary label free proteomic studies to identify the stereoisomeric ICLs molecular targets. Proteomic studies were accomplished from MCF‐7 cells transfected with the purified ICLs for 24 hours. They showed several interesting proteins were differently regulated by the trans‐ and cis‐ICLs: (1) TBC1 domain family member 9 which regulates cell proliferation (increased 41% upon treatment with the trans‐ICL treatment, but decreased 50% with the cis‐ICL); (2) Core histone macro‐H2A.2, involved in DNA repair (decreased 75% upon treatment with the trans‐ICL, but increased 89% with the cis‐ICL); (3) Aurora kinase B which inhibits p21 expression and regulates cell cycle (increased 50% upon treatment with the trans‐ICL, but decreased 73% by the cis‐ICL); and (4) Membrane‐associated tyrosine‐ and threonine‐specific cdc2‐inhibitory kinase which acts a negative regulator of G2 to M transition (decreased 36% upon treatment with the trans‐ICL, but increased 5% with the cis‐ICL). In addition, Ingenuity Pathway Analysis was conducted to identify the major molecular pathways impacted by each ICL treatment. In conclusion, we now have identified both ICL molecular targets and cellular responses involved in DNA repair and cell cycle regulation in MCF7 cell lines. Further validations are needed to confirm the results.

The rate of formation and stability of abasic site interstrand crosslinks in the DNA duplex

DNA interstrand crosslinks (ICLs) strands pose an impenetrable barrier for DNA replication. Different ICLs are known to recruit distinct DNA repair pathways. NEIL3 glycosylase has been known to remove an abasic (Ap) site derived DNA crosslink (Ap-ICL). An Ap-ICL forms spontaneously from the Ap site with an adjacent adenine in the opposite strand. Lack of genetic models and a poor understanding of the fate of these lesions leads to many questions about the occurrence and the toxicity of Ap-ICL in cells.Here, we investigate the circumstances of Ap-ICL formation. With an array of different oligos, we have investigated the rates of formation, the yields, and the stability of Ap-ICL. Our findings point out how different bases in the vicinity of the Ap site change crosslink formation in vitro. We reveal that AT-rich rather than GC-rich regions in the surrounding Ap site lead to higher rates of Ap-ICL formation. Overall, our data reveal that Ap-ICL can be formed in virtually any DNA sequence context surrounding a hot spot of a 5′-Ap-dT pair, albeit with significantly different rates and yields. Based on Ap-ICL formation in vitro, we attempt to predict the number of Ap-ICLs in the cell.

Interstrand Crosslinking Involving Guanine: A New Major UVC Laser-Induced Biphotonic Oxidatively Generated DNA Damage.

Several classes of oxidatively generated DNA damage including oxidized purine and pyrimidine bases, interstrand base crosslinks and DNA-protein crosslinks have been previously shown to be generated in both isolated DNA and cellular DNA upon exposure to either 266-nm laser irradiation or one-electron oxidants. In this study, we provide evidence that biphotonic ionization of guanine bases by UVC laser irradiation of double-stranded deoxyoligonucleotides in aerated aqueous solutions induces the formation of interstrand crosslinks (ICLs). This is supported by various experiments including sequencing gel analyses of formed photoproducts and effects of UVC laser intensity on their formation. This constitutes a novel example of the diversity of reactions of guanine radical cation that can be generated by various one-electron oxidants including UVC laser biphotonic ionization, direct effect of ionization radiation and type I photosensitizers. However, the exact structure of the interstrand base adducts that is a challenging analytical issue remains to be further established. Examples of relevant biochemical/structural applications of biphotonic induction of ICLs in DNA samples by high-intensity UVC laser pulses are provided.

A review of current treatment strategies based on cisplatin for leishmaniosis.

Neglected tropical diseases as termed by World Health Organization include a group of 20 diverse infectious diseases in the tropical and subtropical regions of the world. Amongst these diseases, high mortality rate is mostly associated with Chagas disease and leishmaniosis due to limited drugs, severe toxicity caused by the available drugs and drug resistance. The above hitches have triggered the researchers to focus on the development of novel alternative therapeutics. Studies reveal that several target-based drugs have emerged which affect the biochemical pathways of the causative parasite. An anti-cancerous molecule and its derivatives might be used as antiprotozoal agents due to biochemical similarities affecting DNA metabolism. Cisplatin is a widely used drug for the treatment of testicular, ovarian, head and neck cancers, melanoma, lymphomas and several others. It exerts anticancer activity via multiple mechanisms but most effective mechanism is binding with DNA, formation of intrastrand and interstrand cross-links and generation of DNA lesions. However, side effects of cisplatin limit its application and effectiveness in the cancer treatment. Moreover, to minimize the side effects of cisplatin, combination therapies are used and have proven to be effective against cancers. Thus, current review is aimed at highlighting potential of cisplatin alone and in combination therapies as an antileishmanial agent.

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.