Long Range 1,4 and 1,6-Interstrand Cross-Links Formed by a Trinuclear Platinum Complex. Minor Groove Preassociation Affects Kinetics and Mechanism of Cross-Link Formation as Well as Adduct Structure

2004 ◽  
Vol 126 (7) ◽  
pp. 2166-2180 ◽  
Author(s):  
Alexander Hegmans ◽  
Susan J. Berners-Price ◽  
Murray S. Davies ◽  
Donald S. Thomas ◽  
Anthony S. Humphreys ◽  
...  
Keyword(s):  
Long Range ◽  
Platinum Complex ◽  
Minor Groove ◽  
Kinetics And Mechanism ◽  
Cross Link ◽  
Interstrand Cross Links ◽  
Cross Links ◽  
Link Formation

Mechanistic and Pharmacodynamic Studies of Adct-301, a Pyrrolobenzodiazepine (PBD) Dimer-Containing Antibody Drug Conjugate (ADC) Targeting CD25-Expressing Hematological Malignancies

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1559-1559 ◽  
Author(s):  
Michael J. Flynn ◽  
Patrick H. van Berkel ◽  
Francesca Zammarchi ◽  
Peter C. Tyrer ◽  
Ayse U. Akarca ◽  
...  
Keyword(s):  
Cell Surface ◽  
Clinical Development ◽  
Cross Linking ◽  
Cross Link ◽  
Employment Equity ◽  
Interstrand Cross Links ◽  
Cross Links ◽  
Equity Ownership ◽  
Dose Dependent ◽  
Link Formation

Abstract ADCT-301, currently in Phase I clinical trial, is an ADC composed of a recombinant human IgG1, HuMax®-TAC against human IL-2R-α (CD25) conjugated through a cleavable linker to a PBD dimer warhead with a drug-antibody ratio of 2.3. In vitro and ex vivo, ADCT-301 binds human CD25 with picomolar affinity. ADCT-301 has highly potent and targeted cytotoxicity against a panel of human lymphoma cell lines. On release, PBD dimers bind in the DNA minor groove and exert their cytotoxic action via the formation of DNA interstrand cross-links. In vivo, ADCT-301 demonstrates dose-dependent antitumor activity against subcutaneous and disseminated lymphoma models. For example, in the Karpas 299 xenograft model, 10/10 tumor-free survivors are observed following a single dose of 0.5 mg/kg, whereas Adcetris® gives only a modest delay in mean tumor growth at 0.5 mg/kg, despite this tumor expressing three-fold higher target antigen levels for this drug. The current study aimed to further define the mechanism of action of ADCT-301 and validate pharmacodynamic assays for clinical development. In Karpas 299 cells, evidence for internalization of ADCT-301 was shown by a reduction of CD25 molecules on the cell surface over the first three hours post-treatment followed by a return to pre-treatment levels by 16 hours. This is consistent with the documented rapid recycling of CD25 to the membrane after exposure to IL-2 (Hemar et al Journal of Cell Biology 1995). Furthermore, ADCT-301 on the cell surface declined by >70% over four hours. Following a two-hour exposure to ADCT-301, DNA interstrand cross-linking, measured using a modification of the single cell gel electrophoresis (comet) assay, reached a peak between 4 and 8 hours after which cross-links persisted up to 36 hours. In contrast, the peak of cross-link formation for an equimolar concentration of warhead was immediately following drug exposure and a non-targeted PBD-containing ADC did not produce crosslinks in these cells. A strong correlation (r = 0.97) between loss of viability and DNA cross-link formation provides support for this DNA damage being the critical initiating mechanism of cytotoxicity of ADCT-301. We have previously shown that PBD-induced DNA interstrand cross-links elicit a robust, but delayed γ-H2AX response (Wu et al Clinical Cancer Research 2013). In Karpas 299 cells phosphorylation of H2AX was observed 24 hours after a two-hour exposure to sub-GI50 concentrations of ADCT-301. In these cells continuous exposure to ADCT-301 resulted in a dose-dependent G2/M arrest, peaking at 48 hours, later than for the naked warhead. The peak of the early apoptosis marker annexin-V on the cell surface of Karpas 299 cells was observed between 60 and 72 hours and maximal loss of viability was at 96 hours. Significant bystander killing of CD25-negative human Burkitt's lymphoma-derived Ramos cells was demonstrated for ADCT-301 both by co-culture experiments with CD25-positive Karpas 299 cells, and by media transfer from Karpas 299 cells treated with ADCT-301. This is important as many lymphomas are heterogeneous in their CD25 expression profile (Strauchen et al American Journal of Pathology 1987). In SCID mice with Karpas 299 subcutaneous tumors a single dose of ADCT-301 was administered at 0.2 or 0.6 mg/kg. 24 hours after treatment, excised tumors showed a dose proportional increase in intensity of membrane and cytoplasmic staining by an anti-PBD payload antibody. Cross-linking was determined as 23% (0.2 mg/kg) vs 49% (0.6 mg/kg) (p ≤ 0.01) reduction in Tail Moment using the comet assay and dose-dependent γ-H2AX formation measured by immunohistochemistry was observed. No cross-linking was observed in matched lymphocyte samples. These data confirm the mechanism of cell killing of ADCT-301 and provide relevant pharmacodynamic assays for use in the clinical development of PBD-based ADCs. Disclosures Flynn: Spirogen/Medimmune: Employment. van Berkel:ADC Therapeutics: Employment, Equity Ownership, Patents & Royalties. Zammarchi:ADC Therapeutics: Employment. Tyrer:Spirogen/Medimmune: Employment. Williams:Spirogen/Medimmune: Employment. Howard:ADCT Spirogen/Medimmune: Employment, Equity Ownership, Patents & Royalties. Hartley:ADCT Spirogen/Medimmune: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Two DD-Carboxypeptidases fromMycobacterium smegmatisAffect Cell Surface Properties through Regulation of Peptidoglycan Cross-Linking and Glycopeptidolipids

2018 ◽  
Vol 200 (14) ◽  
Author(s):  
Satya Deo Pandey ◽  
Shilpa Pal ◽  
Ganesh Kumar N ◽  
Ankita Bansal ◽  
Sathi Mallick ◽  
...  
Keyword(s):  
Cell Surface ◽  
Mycobacterium Smegmatis ◽  
Biofilm Formation ◽  
Cross Linking ◽  
Surface Lipid ◽  
Cross Link ◽  
Content Type ◽  
Murine Macrophages ◽  
Cross Links ◽  
Link Formation

ABSTRACTDuring the peptidoglycan (PG) maturation of mycobacteria, the glycan strands are interlinked by both 3-3 (between twomeso-diaminopimelic acids [meso-DAPs]) and 4-3 cross-links (betweend-Ala andmeso-DAP), though there is a predominance (60 to 80%) of 3-3 cross-links. Thedd-carboxypeptidases (dd-CPases) act on pentapeptides to generate tetrapeptides that are used byld-transpeptidases as substrates to form 3-3 cross-links. Therefore,dd-CPases play a crucial role in mycobacterial PG cross-link formation. However, the physiology ofdd-CPases in mycobacteria is relatively unexplored. In this study, we deleted twodd-CPase genes,msmeg_2433andmsmeg_2432, both individually and in combination, fromMycobacterium smegmatismc2155. Though the singledd-CPase gene deletions had no significant impact on the mycobacterial physiology, many interesting functional alterations were observed in the double-deletion mutant,viz., a predominance in PG cross-link formation was shifted from 3-3 cross-links to 4-3, cell surface glycopeptidolipid (GPL) expression was reduced, and susceptibility to β-lactams and antitubercular agents was enhanced. Moreover, the survival rate of the double mutant within murine macrophages was higher than that of the parent. Interestingly, the complementation with any one of thedd-CPase genes could restore the wild-type phenotype. In a nutshell, we infer that the altered ratio of 4-3 to 3-3 PG cross-links might have influenced the expression of surface GPLs, colony morphology, biofilm formation, drug susceptibility, and subsistence of the cells within macrophages.IMPORTANCEThe glycan strands in mycobacterial peptidoglycan (PG) are interlinked by both 3-3 and 4-3 cross-links. Thedd-CPases generate tetrapeptides by acting on the pentapeptides, andld-transpeptidases use tetrapeptides as substrates to form 3-3 cross-links. In this study, we showed that simultaneous deletions of twodd-CPases alter the nature of PG cross-linking from 3-3 cross-links to 4-3 cross-links. The deletions subsequently decrease the expression of glycopeptidolipids (significant surface lipid present in many nontuberculous mycobacteria, includingMycobacterium smegmatis) and affect other physiological parameters, like cell morphology, growth rate, biofilm formation, antibiotic susceptibility, and survival within murine macrophages. Thus, unraveling the physiology ofdd-CPases might help us design antimycobacterial therapeutics in the future.

Diverse applications of covalent cross-links in duplex DNA: From anti-cancer drugs to the detection of disease-relevant single nucleotide polymorphisms to the synthesis of well-defined substrates for the study of novel DNA repair processes

2018 ◽  
Author(s):  
◽  
Maryam Imani Nejad
Keyword(s):  
Excision Repair ◽  
Alkylating Agents ◽  
Nucleotide Polymorphisms ◽  
Cross Link ◽  
Single Nucleotide ◽  
Synthetic Dna ◽  
Interstrand Cross Links ◽  
Cross Links ◽  
Ap Sites ◽  
Cellular Dna

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Abasic (Ap) sites are a common form of DNA lesion that occur endogenously 50,000-200,000 per cell per day in mammals. The alkylation of the guanine and adenine residues by the alkylating agents such as nitrogen mustards also induces the formation of Ap sites in genomic DNA. Our group recently showed that Ap sites can forge DNA-DNA interstrand cross-links in some sequences via reaction of the Ap aldehyde residue with the exocyclic amino groups of nucleobases, such as adenine and guanine, on the opposing strand of the DNA duplex. The earlier work in the group revealed that formation of these covalent bridges between two DNA strands is highly sequence- dependent. Although interstrand cross-links are one of the most deleterious types of cellular DNA damage, the availability of synthetic DNA duplexes containing chemically well-defined, site-specific interstrand cross-links has been proven to be a valuable tool in biological chemistry and medicine. We prepared and characterized a new Ap-derived interstrand cross-link. In another project, we use these remarkable cross-linking reactions for the covalent capture of disease-relevant single nucleotide polymorphism by using a protein nanopore technology. The complex mechanisms underlying cross-link repair in cells and limited availability of stable and defined cross-link are two major reasons why repair pathways of these lesions are not yet well understood. By preparing a variety of Ap-derived cross-links, we studied the role of a base excision repair DNA glycosylase, NEIL3 in unhooking the lesions.

scholarly journals FANCD2 Binding to H4K20me2 via a Methyl-Binding Domain Is Essential for Efficient DNA Cross-Link Repair

2019 ◽  
Vol 39 (15) ◽  
Author(s):  
Karissa L. Paquin ◽  
Nicholas E. Mamrak ◽  
Jada L. Garzon ◽  
Juan A. Cantres-Velez ◽  
Paul A. Azzinaro ◽  
...  
Keyword(s):  
Genome Instability ◽  
Bone Marrow Failure ◽  
Binding Domain ◽  
Inherited Disease ◽  
Cross Link ◽  
Human Genetic Disease ◽  
Focus Formation ◽  
Interstrand Cross Links ◽  
Cross Links ◽  
Error Prone Repair

ABSTRACT Fanconi anemia (FA) is an inherited disease characterized by bone marrow failure and increased cancer risk. FA is caused by mutation of any 1 of 22 genes, and the FA proteins function cooperatively to repair DNA interstrand cross-links (ICLs). A central step in the activation of the FA pathway is the monoubiquitination of the FANCD2 and FANCI proteins, which occurs within chromatin. How FANCD2 and FANCI are anchored to chromatin remains unknown. In this study, we identify and characterize a FANCD2 histone-binding domain (HBD) and embedded methyl-lysine-binding domain (MBD) and demonstrate binding specificity for H4K20me2. Disruption of the HBD/MBD compromises FANCD2 chromatin binding and nuclear focus formation and its ability to promote error-free DNA interstrand cross-link repair, leading to increased error-prone repair and genome instability. Our study functionally describes the first FA protein chromatin reader domain and establishes an important link between this human genetic disease and chromatin plasticity.

scholarly journals The Structure-Specific Endonuclease Ercc1-Xpf Is Required To Resolve DNA Interstrand Cross-Link-Induced Double-Strand Breaks

2004 ◽  
Vol 24 (13) ◽  
pp. 5776-5787 ◽  
Author(s):  
Laura J. Niedernhofer ◽  
Hanny Odijk ◽  
Magda Budzowska ◽  
Ellen van Drunen ◽  
Alex Maas ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Strand Break ◽  
Sister Chromatid Exchanges ◽  
Wild Type ◽  
Cross Link ◽  
Interstrand Cross Links ◽  
Normal Metabolism ◽  
Cross Links

ABSTRACT Interstrand cross-links (ICLs) are an extremely toxic class of DNA damage incurred during normal metabolism or cancer chemotherapy. ICLs covalently tether both strands of duplex DNA, preventing the strand unwinding that is essential for polymerase access. The mechanism of ICL repair in mammalian cells is poorly understood. However, genetic data implicate the Ercc1-Xpf endonuclease and proteins required for homologous recombination-mediated double-strand break (DSB) repair. To examine the role of Ercc1-Xpf in ICL repair, we monitored the phosphorylation of histone variant H2AX (γ-H2AX). The phosphoprotein accumulates at DSBs, forming foci that can be detected by immunostaining. Treatment of wild-type cells with mitomycin C (MMC) induced γ-H2AX foci and increased the amount of DSBs detected by pulsed-field gel electrophoresis. Surprisingly, γ-H2AX foci were also induced in Ercc1 −/− cells by MMC treatment. Thus, DSBs occur after cross-link damage via an Ercc1-independent mechanism. Instead, ICL-induced DSB formation required cell cycle progression into S phase, suggesting that DSBs are an intermediate of ICL repair that form during DNA replication. In Ercc1 −/− cells, MMC-induced γ-H2AX foci persisted at least 48 h longer than in wild-type cells, demonstrating that Ercc1 is required for the resolution of cross-link-induced DSBs. MMC triggered sister chromatid exchanges in wild-type cells but chromatid fusions in Ercc1 −/− and Xpf mutant cells, indicating that in their absence, repair of DSBs is prevented. Collectively, these data support a role for Ercc1-Xpf in processing ICL-induced DSBs so that these cytotoxic intermediates can be repaired by homologous recombination.

scholarly journals Initiation of DNA interstrand cross-link repair in humans: the nucleotide excision repair system makes dual incisions 5' to the cross-linked base and removes a 22- to 28-nucleotide-long damage-free strand.

1997 ◽  
Vol 17 (12) ◽  
pp. 6822-6830 ◽  
Author(s):  
T Bessho ◽  
D Mu ◽  
A Sancar
Keyword(s):  
Dna Repair ◽  
Mammalian Cells ◽  
Excision Repair ◽  
Repair System ◽  
Cross Link ◽  
Unique Challenge ◽  
Cell Extracts ◽  
Interstrand Cross Links ◽  
Cross Links ◽  
The Cross

Most DNA repair mechanisms rely on the redundant information inherent to the duplex to remove damaged nucleotides and replace them with normal ones, using the complementary strand as a template. Interstrand cross-links pose a unique challenge to the DNA repair machinery because both strands are damaged. To study the repair of interstrand cross-links by mammalian cells, we tested the activities of cell extracts of wild-type or excision repair-defective rodent cell lines and of purified human excision nuclease on a duplex with a site-specific cross-link. We found that in contrast to monoadducts, which are removed by dual incisions bracketing the lesion, the cross-link causes dual incisions, both 5' to the cross-link in one of the two strands. The net result is the generation of a 22- to 28-nucleotide-long gap immediately 5' to the cross-link. This gap may act as a recombinogenic signal to initiate cross-link removal.

scholarly journals The reactivity of an unusual amidase may explain colibactin’s DNA cross-linking activity

2019 ◽  
Author(s):  
Yindi Jiang ◽  
Alessia Stornetta ◽  
Peter W. Villalta ◽  
Matthew R. Wilson ◽  
Paul D. Boudreau ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Amide Bond ◽  
Bond Forming ◽  
Interstrand Cross Links ◽  
Cross Links ◽  
Dna Crosslinking ◽  
Alkylate Dna ◽  
Link Formation

ABSTRACTCertain commensal and pathogenic bacteria produce colibactin, a small molecule genotoxin that causes interstrand cross-links in host cell DNA. Though colibactin has been found to alkylate DNA, the molecular basis for cross-link formation is unclear. Here, we report that the colibactin biosynthetic enzyme ClbL is an amide bond-forming enzyme that links aminoketone and β-keto thioester substrates in vitro and in vivo. The substrate specificity of ClbL strongly supports a role for this enzyme in terminating the colibactin NRPS-PKS assembly line. This transformation would incorporate two electrophilic cyclopropane warheads into the final natural product scaffold. Overall, this work provides a biosynthetic explanation for colibactin’s DNA crosslinking activity and paves the way for further study of its chemical structure.

scholarly journals A synthetic 5,3-cross-link in the cell wall of rod-shaped Gram-positive bacteria

2021 ◽  
Vol 118 (11) ◽  
pp. e2100137118
Author(s):  
David A. Dik ◽  
Nan Zhang ◽  
Emily J. Sturgell ◽  
Brittany B. Sanchez ◽  
Jason S. Chen ◽  
...  
Keyword(s):  
Cell Wall ◽  
Building Blocks ◽  
Cross Link ◽  
Positive Bacterium ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Novel Approach ◽  
Cross Links ◽  
Link Formation ◽  
Cell Wall Architecture

Gram-positive bacteria assemble a multilayered cell wall that provides tensile strength to the cell. The cell wall is composed of glycan strands cross-linked by nonribosomally synthesized peptide stems. Herein, we modify the peptide stems of the Gram-positive bacterium Bacillus subtilis with noncanonical electrophilic d-amino acids, which when in proximity to adjacent stem peptides form novel covalent 5,3-cross-links. Approximately 20% of canonical cell-wall cross-links can be replaced with synthetic cross-links. While a low level of synthetic cross-link formation does not affect B. subtilis growth and phenotype, at higher levels cell growth is perturbed and bacteria elongate. A comparison of the accumulation of synthetic cross-links over time in Gram-negative and Gram-positive bacteria highlights key differences between them. The ability to perturb cell-wall architecture with synthetic building blocks provides a novel approach to studying the adaptability, elasticity, and porosity of bacterial cell walls.

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