RESUMO
Several nucleoside antibiotics are structurally characterized by a 5â³-amino-5â³-deoxyribose (ADR) appended via a glycosidic bond to a high-carbon sugar nucleoside (5'S,6'S)-5'-C-glycyluridine (GlyU). GlyU is further modified with an N-alkylamine linker, the biosynthetic origin of which has yet to be established. By using a combination of feeding experiments with isotopically labeled precursors and characterization of recombinant proteins from multiple pathways, the biosynthetic mechanism for N-alkylamine installation for ADR-GlyU-containing nucleoside antibiotics has been uncovered. The data reveal S-adenosyl-L-methionine (AdoMet) as the direct precursor of the N-alkylamine, but, unlike conventional AdoMet- or decarboxylated AdoMet-dependent alkyltransferases, the reaction is catalyzed by a pyridoxal-5'-phosphate-dependent aminobutyryltransferase (ABTase) using a stepwise γ-replacement mechanism that couples γ-elimination of AdoMet with aza-γ-addition onto the disaccharide alkyl acceptor. In addition to using a conceptually different strategy for AdoMet-dependent alkylation, the newly discovered ABTases require a phosphorylated disaccharide alkyl acceptor, revealing a cryptic intermediate in the biosynthetic pathway.
Assuntos
Antibacterianos/química , Fosfato de Piridoxal/química , Alquilação/fisiologia , Antibacterianos/farmacologia , Fenômenos Bioquímicos , Metionina/metabolismo , Nucleosídeos/química , Fosfatos , Fosforilação , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/químicaRESUMO
Alkylation of guanine bases in DNA is detrimental to cells due to its high mutagenic and cytotoxic potential and is repaired by the alkyltransferase AGT. Additionally, alkyltransferase-like proteins (ATLs), which are structurally similar to AGTs, have been identified in many organisms. While ATLs are per se catalytically inactive, strong evidence has suggested that ATLs target alkyl lesions to the nucleotide excision repair system (NER). Using a combination of single-molecule and ensemble approaches, we show here recruitment of UvrA, the initiating enzyme of prokaryotic NER, to an alkyl lesion by ATL. We further characterize lesion recognition by ATL and directly visualize DNA lesion search by highly motile ATL and ATL-UvrA complexes on DNA at the molecular level. Based on the high similarity of ATLs and the DNA-interacting domain of AGTs, our results provide important insight in the lesion search mechanism, not only by ATL but also by AGT, thus opening opportunities for controlling the action of AGT for therapeutic benefit during chemotherapy.
Assuntos
Adenosina Trifosfatases/metabolismo , Alquil e Aril Transferases/metabolismo , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/fisiologia , Alquilação/fisiologia , DNA/metabolismo , Dano ao DNA , Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiologia , Guanina/metabolismo , Microscopia de Força Atômica/métodos , Mutagênese , O(6)-Metilguanina-DNA Metiltransferase/genética , Pinças ÓpticasRESUMO
Duocarmycins [including cyclopropyl pyrroloindole (CPI) or cyclopropyl benzoindole (CBI)] are a class of DNA minor-groove alkylators and seco-CPI/CBIs are synthetic pro-forms that can spirocyclize to CPI/CBI. Bis-CPI/CBIs are potential drug candidates because of their enhanced cytotoxicity from DNA crosslinking, but it is difficult to analyze them for structure-activity correlation because of their DNA reactivity. To study their DNA alkylation, neutral thermal hydrolysis has been frequently applied to process depurination. However, unwanted side reactions under this condition have been reported, which could lead to poor correlation of DNA alkylation data with efficacy results, especially for bis-CPI/CBIs. In this study, an acidic depurination method was developed and applied for analysis of DNA alkylation and shown to be an easier and milder method than the traditional neutral thermal hydrolysis. DNA alkylation and stability of three bis-seco-CBIs were characterized in comparison with two mono-seco-CPIs. The results suggested that: 1) The acidic depurination method was capable of capturing a more representative population, sometimes a different population, of DNA adducts as they existed on DNA compared with the heat depurination method. 2) Di-adenine adducts were captured as expected for the CBI dimers, although the major type of adduct was still mono-adenine adducts. 3) The rate of DNA alkylation, DNA adduct profile, and relative amounts of di-adduct versus mono-adduct were significantly affected by the size, and possibly lipophilicity, of the nonalkylating part of the molecules. 4) Spirocyclization and amide hydrolysis represented two major pathways of degradation. Overall, by applying acidic depurination analyses, this study has illustrated DNA adduct characteristics of novel bis-seco-CBIs with dominating mono-alkylation and provides an alternative method for evaluating DNA minor-groove alkylators. These findings provide an effective analytical tool to evaluate DNA alkylators and to study the DNA alkylation that is a disposition mechanism of these compounds.
Assuntos
Alquilação/fisiologia , Antineoplásicos Alquilantes/metabolismo , DNA/metabolismo , Duocarmicinas/metabolismo , Adenina/metabolismo , Alquilantes/metabolismo , Adutos de DNA/metabolismoRESUMO
BACKGROUND: Alkylated DNA-protein alkyltransferases (AGTs) are conserved proteins that repair alkylation damage in DNA by using a single-step mechanism leading to irreversible alkylation of the catalytic cysteine in the active site. Trans-alkylation induces inactivation and destabilization of the protein, both in vitro and in vivo, likely triggering conformational changes. A complete picture of structural rearrangements occurring during the reaction cycle is missing, despite considerable interest raised by the peculiarity of AGT reaction, and the contribution of a functional AGT in limiting the efficacy of chemotherapy with alkylating drugs. METHODS: As a model for AGTs we have used a thermostable ortholog from the archaeon Sulfolobus solfataricus (SsOGT), performing biochemical, structural, molecular dynamics and in silico analysis of ligand-free, DNA-bound and mutated versions of the protein. RESULTS: Conformational changes occurring during lesion recognition and after the reaction, allowed us to identify a novel interaction network contributing to SsOGT stability, which is perturbed when a bulky adduct between the catalytic cysteine and the alkyl group is formed, a mandatory step toward the permanent protein alkylation. CONCLUSIONS: Our data highlighted conformational changes and perturbation of intramolecular interaction occurring during lesion recognition and catalysis, confirming our previous hypothesis that coordination between the N- and C-terminal domains of SsOGT is important for protein activity and stability. GENERAL SIGNIFICANCE: A general model of structural rearrangements occurring during the reaction cycle of AGTs is proposed. If confirmed, this model might be a starting point to design strategies to modulate AGT activity in therapeutic settings.
Assuntos
Alquil e Aril Transferases/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Alquilantes/metabolismo , Alquilação/fisiologia , Catálise , Reparo do DNA/fisiologia , Domínios Proteicos , Estabilidade Proteica , Sulfolobus solfataricus/metabolismoRESUMO
Pyrrolobenzodiazepine (PBD)-dimer is a DNA minor groove alkylator, and its CD22 THIOMAB antibody drug conjugate (ADC) demonstrated, through a disulfide linker, an efficacy in tumor reduction for more than 7 weeks with minimal body weight loss in xenograft mice after a single 0.5-1 mg/kg i.v. dose. The DNA alkylation was investigated here in tumors and healthy organs of mice to understand the sustained efficacy and tolerability. The experimental procedures included the collection of tumors and organ tissues of xenograft mice treated with the ADC followed by DNA isolation/hydrolysis/quantitation and payload recovery from reversible DNA alkylation. PBD-dimer formed a considerable amount of adducts with tissue DNA, representing approximately 98% (at 24 hours), and 99% (at 96 hours) of the total PBD-dimer in tumors, and 78-89% in liver and lung tissues, suggesting highly efficient covalent binding of the released PBD-dimer to tissue DNA. The amount of PBD-DNA adducts in tumor tissues was approximately 24-fold (at 24 hours) and 70-fold (at 96 hours) greater than the corresponding amount of adducts in liver and lung tissues. In addition, the DNA alkylation levels increased 3-fold to 4-fold from 24 to 96 hours in tumors [41/106 base pairs (bp) at 96 hours] but remained at the same level (1/106 bp) in livers and lungs. These results support the typical target-mediated cumulative uptake of ADC into tumors and payload release that offers an explanation for its sustained antitumor efficacy. In addition, the low level of DNA alkylation in normal tissues is consistent with the tolerability observed in mice.
Assuntos
Alquilação/fisiologia , Anticorpos/metabolismo , Benzodiazepinas/metabolismo , DNA/metabolismo , Pirróis/metabolismo , Animais , Adutos de DNA/metabolismo , Xenoenxertos/metabolismo , Imunoconjugados/metabolismo , Fígado/metabolismo , Pulmão/metabolismo , Camundongos , Neoplasias/metabolismoRESUMO
PURPOSE: Melflufen (melphalan flufenamide, previously designated J1) is an optimized and targeted derivative of melphalan, hydrolyzed by aminopeptidases overexpressed in tumor cells resulting in selective release and trapping of melphalan, and enhanced activity in preclinical models. METHODS: This was a prospective, single-armed, open-label, first-in-human, dose-finding phase I/IIa study in 45 adult patients with advanced and progressive solid tumors without standard treatment options. Most common tumor types were ovarian carcinoma (n = 20) and non-small-cell lung cancer (NSCLC, n = 11). RESULTS: In the dose-escalating phase I part of the study, seven patients were treated with increasing fixed doses of melflufen (25-130 mg) Q3W. In the subsequent phase IIa part, 38 patients received in total 115 cycles of therapy at doses of 30-75 mg. No dose-limiting toxicities (DLTs) were observed at 25 and 50 mg; at higher doses DLTs were reversible neutropenias and thrombocytopenias, particularly evident in heavily pretreated patients, and the recommended phase II dose (RPTD) was set to 50 mg. Response Evaluation Criteria In Solid Tumors (RECIST) evaluation after 3 cycles of therapy (27 patients) showed partial response in one (ovarian cancer), and stable disease in 18 patients. One NSCLC patient received nine cycles of melflufen and progressed after 7 months of therapy. CONCLUSIONS: In conclusion, melflufen can safely be given to cancer patients, and the toxicity profile was as expected for alkylating agents; RPTD is 50 mg Q3W. Reversible and manageable bone marrow suppression was identified as a DLT. Clinical activity is suggested in ovarian cancer, but modest activity in treatment of refractory NSCLC.
Assuntos
Antineoplásicos Alquilantes/administração & dosagem , Melfalan/análogos & derivados , Neoplasias/tratamento farmacológico , Fenilalanina/análogos & derivados , Adulto , Idoso , Idoso de 80 Anos ou mais , Alquilação/efeitos dos fármacos , Alquilação/fisiologia , Antineoplásicos Alquilantes/efeitos adversos , Progressão da Doença , Relação Dose-Resposta a Droga , Esquema de Medicação , Feminino , Doenças Hematológicas/induzido quimicamente , Humanos , Masculino , Melfalan/administração & dosagem , Melfalan/efeitos adversos , Pessoa de Meia-Idade , Neoplasias/diagnóstico , Peptídeo Hidrolases/metabolismo , Fenilalanina/administração & dosagem , Fenilalanina/efeitos adversos , Estudos ProspectivosRESUMO
Repair of DNA alkylation damage is critical for genomic stability and involves multiple conserved enzymatic pathways. Alkylation damage resistance, which is critical in cancer chemotherapy, depends on the overexpression of alkylation repair proteins. However, the mechanisms responsible for this upregulation are unknown. Here, we show that an OTU domain deubiquitinase, OTUD4, is a positive regulator of ALKBH2 and ALKBH3, two DNA demethylases critical for alkylation repair. Remarkably, we find that OTUD4 catalytic activity is completely dispensable for this function. Rather, OTUD4 is a scaffold for USP7 and USP9X, two deubiquitinases that act directly on the AlkB proteins. Moreover, we show that loss of OTUD4, USP7, or USP9X in tumor cells makes them significantly more sensitive to alkylating agents. Taken together, this work reveals a novel, noncanonical mechanism by which an OTU family deubiquitinase regulates its substrates, and provides multiple new targets for alkylation chemotherapy sensitization of tumors.
Assuntos
Alquilação/fisiologia , Dano ao DNA/fisiologia , Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA/fisiologia , Dioxigenases/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteases Específicas de Ubiquitina/metabolismo , Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato , Alquilação/genética , Western Blotting , Dano ao DNA/genética , Reparo do DNA/genética , Células HEK293 , Humanos , Imunoprecipitação , Microscopia de Fluorescência , Modelos Biológicos , Espectrometria de Massas em TandemRESUMO
In the ER (endoplasmic reticulum) of human cells, disulfide bonds are predominantly generated by the two isoforms of Ero1 (ER oxidoreductin-1): Ero1α and Ero1ß. The activity of Ero1α is tightly regulated through the formation of intramolecular disulfide bonds to help ensure balanced ER redox conditions. Ero1ß is less tightly regulated, but the molecular details underlying control of activity are not as well characterized as for Ero1α. Ero1ß contains an additional cysteine residue (Cys262), which has been suggested to engage in an isoform-specific regulatory disulfide bond with Cys100 However, we show that the two regulatory disulfide bonds in Ero1α are likely conserved in Ero1ß (Cys90-Cys130 and Cys95-Cys100). Molecular modelling of the Ero1ß structure predicted that the side chain of Cys262 is completely buried. Indeed, we found this cysteine to be reduced and partially protected from alkylation in the ER of living cells. Furthermore, mutation of Cys100-but not of Cys262-rendered Ero1ß hyperactive in cells, as did mutation of Cys130 Ero1ß hyperactivity induced the UPR (unfolded protein response) and resulted in oxidative perturbation of the ER redox state. We propose that features other than a distinct pattern of regulatory disulfide bonds determine the loose redox regulation of Ero1ß relative to Ero1α.
Assuntos
Retículo Endoplasmático/enzimologia , Glicoproteínas de Membrana/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , Alquilação/fisiologia , Linhagem Celular , Cisteína/genética , Cisteína/metabolismo , Retículo Endoplasmático/genética , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Glicoproteínas de Membrana/genética , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genéticaRESUMO
Tyrosyl-DNA phosphodiesterase 1 (Tdp1) repairs topoisomerase I cleavage complexes (Top1cc) by hydrolyzing their 3'-phosphotyrosyl DNA bonds and repairs bleomycin-induced DNA damage by hydrolyzing 3'-phosphoglycolates. Yeast Tdp1 has also been implicated in the repair of topoisomerase II-DNA cleavage complexes (Top2cc). To determine whether vertebrate Tdp1 is involved in the repair of various DNA end-blocking lesions, we generated Tdp1 knock-out cells in chicken DT40 cells (Tdp1-/-) and Tdp1-complemented DT40 cells with human TDP1. We found that Tdp1-/- cells were not only hypersensitive to camptothecin and bleomycin but also to etoposide, methyl methanesulfonate (MMS), H(2)O(2), and ionizing radiation. We also show they were deficient in mitochondrial Tdp1 activity. In biochemical assays, recombinant human TDP1 was found to process 5'-phosphotyrosyl DNA ends when they mimic the 5'-overhangs of Top2cc. Tdp1 also processes 3'-deoxyribose phosphates generated from hydrolysis of abasic sites, which is consistent with the hypersensitivity of Tdp1-/- cells to MMS and H(2)O(2). Because recent studies established that CtIP together with BRCA1 also repairs topoisomerase-mediated DNA damage, we generated dual Tdp1-CtIP-deficient DT40 cells. Our results show that Tdp1 and CtIP act in parallel pathways for the repair of Top1cc and MMS-induced lesions but are epistatic for Top2cc. Together, our findings reveal a broad involvement of Tdp1 in DNA repair and clarify the role of human TDP1 in the repair of Top2-induced DNA damage.
Assuntos
Antígenos de Neoplasias/metabolismo , Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , DNA Topoisomerases Tipo II/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neoplasias/enzimologia , Diester Fosfórico Hidrolases/metabolismo , Alquilação/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Transporte/metabolismo , Células Cultivadas , Galinhas , DNA de Cadeia Simples/metabolismo , Endodesoxirribonucleases , Humanos , Dados de Sequência Molecular , Neoplasias/genética , Proteínas Nucleares/metabolismo , Diester Fosfórico Hidrolases/genética , Proteínas de Ligação a Poli-ADP-Ribose , Especificidade por Substrato/fisiologia , VertebradosRESUMO
Agents that form methylation adducts in DNA are highly mutagenic and carcinogenic, and organisms have evolved specialized cellular pathways devoted to their repair, including DNA alkyltransferases. These are proteins conserved in eucarya, bacteria and archaea, acting by a unique reaction mechanism, which leads to direct repair of DNA alkylation damage and irreversible protein alkylation. The alkylated form of DNA alkyltransferases is inactive, and in eukaryotes, it is rapidly directed to degradation. We report here in vitro and in vivo studies on the DNA alkyltransferase from the thermophilic archaeon Sulfolobus solfataricus (SsOGT). The development of a novel, simple, and sensitive fluorescence-based assay allowed a careful characterization of the SsOGT biochemical and DNA binding activities. In addition, transcriptional and post-translational regulation of SsOGT by DNA damage was studied. We show that although the gene transcription is induced by alkylating agent treatment, the protein is degraded in vivo by an alkylation-dependent mechanism. These experiments suggest a striking conservation, from archaea to humans, of this important pathway safeguarding genome stability.
Assuntos
Alquil e Aril Transferases/metabolismo , Proteínas Arqueais/metabolismo , Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , DNA Arqueal/metabolismo , Sulfolobus solfataricus/enzimologia , Alquil e Aril Transferases/genética , Alquilação/fisiologia , Proteínas Arqueais/genética , DNA Arqueal/genética , Sulfolobus solfataricus/genéticaRESUMO
O(6)-Alkylguanine-DNA alkyltransferase (AGT) plays a major role in repair of the cytotoxic and mutagenic lesion O(6)-methylguanine (m(6)G) in DNA. Unlike the Escherichia coli alkyltransferase Ogt that also repairs O(4)-methylthymine (m(4)T) efficiently, the human AGT (hAGT) acts poorly on m(4)T. Here we made several hAGT mutants in which residues near the cysteine acceptor site were replaced by corresponding residues from Ogt to investigate the basis for the inefficiency of hAGT in repair of m(4)T. Construct hAGT-03 (where hAGT sequence -V(149)CSSGAVGN(157)- was replaced with the corresponding Ogt -I(143)GRNGTMTG(151)-) exhibited enhanced m(4)T repair activity in vitro compared with hAGT. Three AGT proteins (hAGT, hAGT-03, and Ogt) exhibited similar protection from killing by N-methyl-N'-nitro-N-nitrosoguanidine and caused a reduction in m(6)G-induced G:C to A:T mutations in both nucleotide excision repair (NER)-proficient and -deficient Escherichia coli strains that lack endogenous AGTs. hAGT-03 resembled Ogt in totally reducing the m(4)T-induced T:A to C:G mutations in NER-proficient and -deficient strains. Surprisingly, wild type hAGT expression caused a significant but incomplete decrease in NER-deficient strains but a slight increase in T:A to C:G mutation frequency in NER-proficient strains. The T:A to C:G mutations due to O(4)-alkylthymine formed by ethylating and propylating agents were also efficiently reduced by either hAGT-03 or Ogt, whereas hAGT had little effect irrespective of NER status. These results show that specific alterations in the hAGT active site facilitate efficient recognition and repair of O(4)-alkylthymines and reveal damage-dependent interactions of base and nucleotide excision repair.
Assuntos
Adutos de DNA/metabolismo , Reparo do DNA/fisiologia , O(6)-Metilguanina-DNA Metiltransferase/genética , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Timina/metabolismo , Alquilação/fisiologia , Domínio Catalítico , Dano ao DNA/fisiologia , Escherichia coli/genética , Etano/metabolismo , Humanos , Metano/metabolismo , Mutagênese/fisiologia , O(6)-Metilguanina-DNA Metiltransferase/química , Propano/metabolismo , Estrutura Terciária de ProteínaRESUMO
O(6)-methylguanine DNA methyltransferase (MGMT) suppresses mutations and cell death that result from alkylation damage. MGMT expression is lost by epigenetic silencing in a variety of human cancers including nearly half of sporadic colorectal cancers, suggesting that this loss maybe causal. Using mice with a targeted disruption of the Mgmt gene, we tested whether Mgmt protects against azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF), against AOM and dextran sulfate sodium (DSS)-induced colorectal adenomas and against spontaneous intestinal adenomas in Apc(Min) mice. We also examined the genetic interaction of the Mgmt null gene with a DNA mismatch repair null gene, namely Msh6. Both Mgmt and Msh6 independently suppress AOM-induced ACF, and combination of the two mutant alleles had a multiplicative effect. This synergism can be explained entirely by the suppression of alkylation-induced apoptosis when Msh6 is absent. In addition, following AOM+DSS treatment Mgmt protected against adenoma formation to the same degree as it protected against AOM-induced ACF formation. Finally, Mgmt deficiency did not affect spontaneous intestinal adenoma development in Apc(Min/+) mice, suggesting that Mgmt suppresses intestinal cancer associated with exogenous alkylating agents, and that endogenous alkylation does not contribute to the rapid tumor development seen in Apc(Min/+) mice.