RESUMO
Although MYC is a significant oncogenic transcription factor driver of cancer, directly targeting MYC has remained challenging due to its intrinsic disorder and poorly defined structure, deeming it "undruggable." Whether transient pockets formed within unstructured regions of proteins can be selectively targeted with small molecules remains an outstanding challenge. Here, we developed a stereochemically paired spirocyclic oxindole aziridine covalent library and screened this library for degradation of MYC. We identified a hit covalent ligand, KL2-236, bearing a unique sulfinyl aziridine warhead, that engaged MYC as a pure MYC/MAX protein complex, and in cancer cells to destabilize MYC, inhibit MYC transcriptional activity and degrade MYC in a proteasome-dependent manner through targeting intrinsically disordered C203 and D205 residues. Notably, this reactivity was most pronounced for specific stereoisomers of KL2-236 with a diastereomer, KL4-019, that was largely inactive. Mutagenesis of both C203 and D205 completely attenuated KL2-236-mediated MYC degradation. We also optimized our KL2-236 hit compound to generate a more potent, selective, and durable MYC degrader, KL4-219A. Our results reveal a novel ligandable site within MYC and indicate that certain intrinsically disordered regions within transcription factors, such as MYC, can be interrogated by isomerically unique chiral small molecules, leading to destabilization and degradation.
Assuntos
Aziridinas , Proteínas Proto-Oncogênicas c-myc , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/genética , Humanos , Aziridinas/química , Aziridinas/farmacologia , Estereoisomerismo , Linhagem Celular Tumoral , Estrutura MolecularRESUMO
The non-proteinogenic tris-amino acid labionin was discovered in 2010 and since has been described in many natural products, defining an entire family of lanthipeptides. The unusual amino acid is biosynthetically produced by the sequential condensation of a cysteine onto two dehydroalanines. An attempt in 2011 to synthesize the amino acid monomeric unit was unsuccessful yet served as a valuable foundation and guide to the present work. A recently disclosed methodology for the selective opening of aziridines by mercaptan nucleophiles was applied to another methodology for contrasteric aziridination, thus enabling a short synthesis of challenging and elusive labionin.
Assuntos
Aminoácidos , Aziridinas , Alanina/química , Alanina/análogos & derivados , Alanina/síntese química , Alanina/análogos & derivados , Aminoácidos/química , Aminoácidos/síntese química , Aziridinas/química , Aziridinas/síntese química , Cisteína/química , Estrutura Molecular , PeptídeosRESUMO
Eudesmane-type sesquiterpene lactone isoalantolactone 1 is of great interest due to its availability, biological activity and synthetic application. Respective series of original spirocyclic (11S,5') (1,2,3-triazoline-eudesma-4,15-enolides) and (11S)-aziridine-eudesma-4,15-enolides were efficiently synthesized via a chemoselective 1,3-dipolar cycloaddition reaction of organic azides to the exocyclic double bond of the lactone ring of isoalantolactone or 13E-(aryl)isoalantolactones by heating in DMF or toluene. The thermal reactions of isoalantolactone with benzyl azide, 2-azidoethanol, or n-butyl azide in 2-methoxyethanol afforded 13-(alkyamino)isoalantolactones formed as a mixture of (Z) and (E)-isomers. The results of in vitro biological assays showed that novel spirocyclic isoalantolactone derivatives exhibited cytotoxicity against human breast cancer and glioblastoma cells at low micromolar concentrations. The most cytotoxic and selective (11S,5')-spiro-1,2,3-triazoline from 13E-(fluorophenyl)isoalantolactone 20 (IC50(MCF-7) = 8 ± 0.1 µM, SI(MCF-7) > 12.5) was found to induce ROS-dependent death of MCF-7 human breast cancer cells via mitochondrial apoptosis. The corresponding (11S)-spiroaziridine derivatives 21 at non-toxic concentrations (10 and 20 µM) effectively suppressed motility, clonogenicity and adhesion of glioblastoma cells and exhibited synergistic cytotoxicity in combination with temozolomide. In silico analysis revealed the potential ability of the 13-aryl (11S)-spiroaziridine derivative 21 to bypass the blood-brain barrier and exhibit anti-glioblastoma activity probably based on the direct interaction with Hsp90α.
Assuntos
Antineoplásicos , Aziridinas , Proliferação de Células , Ensaios de Seleção de Medicamentos Antitumorais , Sesquiterpenos , Compostos de Espiro , Triazóis , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Relação Estrutura-Atividade , Aziridinas/química , Aziridinas/farmacologia , Aziridinas/síntese química , Compostos de Espiro/química , Compostos de Espiro/farmacologia , Compostos de Espiro/síntese química , Estrutura Molecular , Proliferação de Células/efeitos dos fármacos , Sesquiterpenos/farmacologia , Sesquiterpenos/química , Sesquiterpenos/síntese química , Triazóis/química , Triazóis/farmacologia , Triazóis/síntese química , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Apoptose/efeitos dos fármacos , Sesquiterpenos de Eudesmano/farmacologia , Sesquiterpenos de Eudesmano/química , Sesquiterpenos de Eudesmano/síntese química , Produtos Biológicos/farmacologia , Produtos Biológicos/química , Produtos Biológicos/síntese químicaRESUMO
Cyclopropane and azacyclopropane, also known as aziridine, moieties are found in natural products. These moieties serve as pivotal components that lead to a broad spectrum of biological activities. While diverse strategies involving various classes of enzymes are utilized to catalyze formation of these strained three-membered rings, how non-heme iron and 2-oxoglutarate (Fe/2OG) dependent enzymes enable regio- and stereo-selective C-C and C-N ring closure has only been reported very recently. Herein, we present detailed experimental protocols for mechanistically studying Fe/2OG enzymes that catalyze cyclopropanation and aziridination reactions. These protocols include protein purification, in vitro assays, biophysical spectroscopies, and isotope-tracer experiments. We also report how to use in silico approaches to look for Fe/2OG aziridinases. Furthermore, our current mechanistic understanding of three-membered ring formation is discussed. These results not only shed light on the reaction mechanisms of Fe/2OG enzymes-catalyzed cyclopropanation and aziridination, but also open avenues for expanding the reaction repertoire of the Fe/2OG enzyme superfamily.
Assuntos
Aziridinas , Ciclopropanos , Ácidos Cetoglutáricos , Ciclopropanos/química , Ciclopropanos/metabolismo , Aziridinas/química , Aziridinas/metabolismo , Ácidos Cetoglutáricos/metabolismo , Ácidos Cetoglutáricos/química , Ferro/química , Ferro/metabolismo , Ferroproteínas não Heme/química , Ferroproteínas não Heme/metabolismo , Biocatálise , Ensaios Enzimáticos/métodos , CatáliseRESUMO
The total synthesis of two new marine natural products, (±)-marinoaziridine B 7 and (±)-N-methyl marinoaziridine A 8, was accomplished. The (±)-marinoaziridine 7 was prepared in a six-step linear sequence with a 2% overall yield. The key steps in our strategy were the preparation of the chiral epoxide (±)-5 using the Johnson Corey Chaykovsky reaction, followed by the ring-opening reaction and the Staudinger reaction. The N,N-dimethylation of compound (±)-7 gives (±)-N-methyl marinoaziridine A 8. The NMR spectra of synthetized (±)-marinoaziridine B 7 and isolated natural product did not match. The compounds are biologically characterized using relevant in silico, in vitro and in vivo methods. In silico ADMET and bioactivity profiling predicted toxic and neuromodulatory effects. In vitro screening by MTT assay on three cell lines (MCF-7, H-460, HEK293T) showed that both compounds exhibited moderate to strong antiproliferative and cytotoxic effects. Antimicrobial tests on bacterial cultures of Escherichia coli and Staphylococcus aureus demonstrated the dose-dependent inhibition of the growth of both bacteria. In vivo toxicological tests were performed on zebrafish Danio rerio and showed a significant reduction of zebrafish mortality due to N-methylation in (±)-8.
Assuntos
Aziridinas , Staphylococcus aureus , Humanos , Aziridinas/farmacologia , Aziridinas/química , Aziridinas/síntese química , Animais , Staphylococcus aureus/efeitos dos fármacos , Células HEK293 , Antibacterianos/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Escherichia coli/efeitos dos fármacos , Peixe-Zebra , Células MCF-7 , Testes de Sensibilidade Microbiana , Produtos Biológicos/farmacologia , Produtos Biológicos/química , Produtos Biológicos/síntese química , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacosRESUMO
Bacterial nitroreductase enzymes capable of activating imaging probes and prodrugs are valuable tools for gene-directed enzyme prodrug therapies and targeted cell ablation models. We recently engineered a nitroreductase (E. coli NfsB F70A/F108Y) for the substantially enhanced reduction of the 5-nitroimidazole PET-capable probe, SN33623, which permits the theranostic imaging of vectors labeled with oxygen-insensitive bacterial nitroreductases. This mutant enzyme also shows improved activation of the DNA-alkylation prodrugs CB1954 and metronidazole. To elucidate the mechanism behind these enhancements, we resolved the crystal structure of the mutant enzyme to 1.98 Å and compared it to the wild-type enzyme. Structural analysis revealed an expanded substrate access channel and new hydrogen bonding interactions. Additionally, computational modeling of SN33623, CB1954, and metronidazole binding in the active sites of both the mutant and wild-type enzymes revealed key differences in substrate orientations and interactions, with improvements in activity being mirrored by reduced distances between the N5-H of isoalloxazine and the substrate nitro group oxygen in the mutant models. These findings deepen our understanding of nitroreductase substrate specificity and catalytic mechanisms and have potential implications for developing more effective theranostic imaging strategies in cancer treatment.
Assuntos
Metronidazol , Nitroimidazóis , Nitrorredutases , Nitrorredutases/metabolismo , Nitrorredutases/química , Nitrorredutases/genética , Nitroimidazóis/química , Nitroimidazóis/metabolismo , Metronidazol/química , Metronidazol/metabolismo , Metronidazol/farmacologia , Pró-Fármacos/metabolismo , Pró-Fármacos/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Tomografia por Emissão de Pósitrons/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Domínio Catalítico , Engenharia de Proteínas , Modelos Moleculares , Aziridinas/química , Aziridinas/metabolismoRESUMO
Breast cancer is associated with high mortality and morbidity rates. As about 20-30% of patients exhibiting ER-positive phenotype are resistant to hormonal treatment with the standard drug tamoxifen, finding new therapies is a necessity. Postbiotics, metabolites, and macromolecules isolated from probiotic bacteria cultures have been proven to have sufficient bioactivity to exert prohealth and anticancer effects, making them viable adjunctive agents for the treatment of various neoplasms, including breast cancer. In the current study, postbiotics derived from L. plantarum and L. rhamnosus cultures were assessed on an in vitro breast cancer model as potential adjunctive agents to therapy utilizing tamoxifen and a candidate aziridine-hydrazide hydrazone derivative drug. Cell viability and cell death processes, including apoptosis, were analyzed for neoplastic MCF-7 cells treated with postbiotics and synthetic compounds. Cell cycle progression and proliferation were analyzed by PI-based flow cytometry and Ki-67 immunostaining. Postbiotics decreased viability and triggered apoptosis in MCF-7, modestly affecting the cell cycle and showing a lack of negative impact on normal cell viability. Moreover, they enhanced the cytotoxic effect of tamoxifen and the new candidate drug toward MCF-7, accelerating apoptosis and the inhibition of proliferation. This illustrates postbiotics' potential as natural adjunctive agents supporting anticancer therapy based on synthetic drugs.
Assuntos
Apoptose , Aziridinas , Neoplasias da Mama , Proliferação de Células , Tamoxifeno , Humanos , Tamoxifeno/farmacologia , Tamoxifeno/química , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Células MCF-7 , Feminino , Aziridinas/farmacologia , Aziridinas/química , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Hidrazonas/farmacologia , Hidrazonas/química , Probióticos/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/química , Ciclo Celular/efeitos dos fármacosRESUMO
A series of optically pure aziridine phosphines and their corresponding phosphine oxides were synthesized through established chemical methodologies. The compounds were systematically investigated for their biological properties. Notably, all synthesized compounds demonstrated moderate antibacterial activity only against the reference strain of Staphylococcus aureus. However, compounds 5 and 7 exhibited noteworthy cell viability inhibition of human cervical epithelioid carcinoma HeLa cells and endometrial adenocarcinoma Ishikawa cells. Further studies of these compounds revealed additional biological effects, including disruption of the cell membrane in high concentrations, cell cycle arrest in the S phase, and the induction of reactive oxygen species (ROS). Comparative analysis of the two classes of chiral organophosphorus derivatives of aziridines indicated that chiral phosphine oxides displayed significantly higher biological activity. Consequently, these findings suggest that chiral phosphine oxides may be potential candidates for the development of anticancer drugs. In light of the significant interest in preparations whose structure is based on a three-membered aziridine ring in terms of potential anticancer therapy, this research fits into the current research trend and should constitute a valuable addition to the current state of knowledge and the existing library of aziridine derivatives with anticancer properties.
Assuntos
Aziridinas , Fosfinas , Humanos , Células HeLa , Aziridinas/farmacologia , ÓxidosRESUMO
AST-001 is a chemically synthesized inactive nitrogen mustard prodrug that is selectively cleaved to a cytotoxic aziridine (AST-2660) via aldo-keto reductase family 1 member C3 (AKR1C3). The purpose of this study was to investigate the pharmacokinetics and tissue distribution of the prodrug, AST-001, and its active metabolite, AST-2660, in mice, rats, and monkeys. After single and once daily intravenous bolus doses of 1.5, 4.5, and 13.5 mg/kg AST-001 to Sprague-Dawley rats and once daily 1 h intravenous infusions of 0.5, 1.5, and 4.5 mg/kg AST-001 to cynomolgus monkeys, AST-001 exhibited dose-dependent pharmacokinetics and reached peak plasma levels at the end of the infusion. No significant accumulation and gender differences were observed after 7 days of repeated dosing. In rats, the half-life of AST-001 was dose independent and ranged from 4.89 to 5.75 h. In cynomolgus monkeys, the half-life of AST-001 was from 1.66 to 5.56 h and increased with dose. In tissue distribution studies conducted in Sprague-Dawley rats and in liver cancer PDX models in female athymic nude mice implanted with LI6643 or LI6280 HepG2-GFP tumor fragments, AST-001 was extensively distributed to selected tissues. Following a single intravenous dose, AST-001 was not excreted primarily as the prodrug, AST-001 or the metabolite AST-2660 in the urine, feces, and bile. A comprehensive analysis of the preclinical data and inter-species allometric scaling were used to estimate the pharmacokinetic parameters of AST-001 in humans and led to the recommendation of a starting dose of 5 mg/m2 in the first-in-human dose escalation study.
Assuntos
Compostos de Mostarda Nitrogenada , Pró-Fármacos , Animais , Feminino , Camundongos , Ratos , Membro C3 da Família 1 de alfa-Ceto Redutase/efeitos dos fármacos , Macaca fascicularis , Camundongos Nus , Ratos Sprague-Dawley , Compostos de Mostarda Nitrogenada/farmacocinética , Aziridinas/farmacocinética , Relação Dose-Resposta a DrogaRESUMO
Epoxides, aziridines, and cyclopropanes are found in various medicinal natural products, including polyketides, terpenes, peptides, and alkaloids. Many classes of biosynthetic enzymes are involved in constructing these ring structures during their biosynthesis. This review summarizes our current knowledge regarding how α-ketoglutarate-dependent nonheme iron enzymes catalyze the formation of epoxides, aziridines, and cyclopropanes in nature, with a focus on enzyme mechanisms.
Assuntos
Aziridinas , Ferro , Ferro/química , Ácidos Cetoglutáricos/química , Catálise , Ciclopropanos , Compostos de EpóxiRESUMO
In previous works, we demonstrated that tertiary 3-chloropiperidines are potent chemotherapeutics, alkylating the DNA through the formation of bicyclic aziridinium ions. Herein, we report the synthesis of novel secondary 3-chloropiperidine analogues. The synthesis incorporates a new procedure to monochlorinate unsaturated primary amines utilizing N-chlorosuccinimide, while carefully monitoring the temperature to prevent dichlorination. Furthermore, we successfully isolated highly strained bicyclic aziridines by treating the secondary 3-chloropiperidines with a sufficient amount of base. We conclude this work with a DNA cleavage assay as a proof of principle, comparing our previously known substrates to the novel compounds. In this, the secondary 3-chloropiperidine as well as the isolated bicyclic aziridine, proved to be more effective than their tertiary counterpart.
Assuntos
Piperidinas , Piperidinas/química , Piperidinas/síntese química , Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/síntese química , Alquilantes/química , Clivagem do DNA/efeitos dos fármacos , Humanos , Aziridinas/química , DNA/química , SuccinimidasRESUMO
Azaheterocycles are three-membered rings, known as aziridines, that occur naturally and have pharmaceutical applications.These compounds are present as several secondary metabolites produced by plants and microorganisms.Recent studies have demonstrated the effectiveness of aziridine derivatives (N-H/N-Me) as anticancer agents.We synthesized 18 compounds containing an N-Me enone aziridine group, the chemistry of which has been previously published. However, these compounds have drug-likeness properties; therefore, we aimed to demonstrate their drug-like properties using in silico and in vitro investigations.The molecular structures of the compounds were optimized using density functional theory (DFT). The ADMET parameters of the derivatives were calculated using SwissADME and PreADMET. Additionally, these derivatives were evaluated for their ability to bind to caspase-3 and caspase-9 and then subjected to molecular docking. The lead chemical AY128 maintained stable complexes with target proteins during molecular dynamics simulations, as evidenced by the root mean square deviation (RMSD) and root mean square fluctuation (RMSF) parameters. In vitro cytotoxicity and ELISA tests showed that the novel aziridine derivatives, especially AY128, had strong anticancer activity against HepG2 hepatocellular carcinoma cells.Our study suggests that AY128 may be a potential drug candidate for hepatocellular carcinoma through the caspase-3 and caspase-9-dependent apoptotic pathways.Communicated by Ramaswamy H. Sarma.
Assuntos
Antineoplásicos , Aziridinas , Carcinoma Hepatocelular , Descoberta de Drogas , Neoplasias Hepáticas , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Aziridinas/química , Aziridinas/farmacologia , Humanos , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Antineoplásicos/farmacologia , Antineoplásicos/química , Células Hep G2 , Caspase 9/metabolismo , Caspase 3/metabolismo , Relação Estrutura-Atividade , Caspases/metabolismo , Caspases/química , Estrutura Molecular , Transdução de Sinais/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Ligação ProteicaRESUMO
GH127 and GH146 microorganismal retaining ß-l-arabinofuranosidases, expressed by human gut microbiomes, feature an atypical catalytic domain and an unusual mechanism of action. We recently reported that both Bacteroides thetaiotaomicron BtGH146 and Bifidobacterium longum HypBA1 are inhibited by ß-l-arabinofuranosyl cyclophellitol epoxide, supporting the action of a zinc-coordinated cysteine as a catalytic nucleophile, where in most retaining GH families, an aspartate or glutamate is employed. This work presents a panel of ß-l-arabinofuranosyl cyclophellitol epoxides and aziridines as mechanism-based BtGH146/HypBA1 inhibitors and activity-based probes. The ß-l-arabinofuranosyl cyclophellitol aziridines both inhibit and label ß-l-arabinofuranosidase efficiently (however with different activities), whereas the epoxide-derived probes favor BtGH146 over HypBA1. These findings are accompanied by X-ray structural analysis of the unmodified ß-l-arabinofuranosyl cyclophellitol aziridine in complex with both isozymes, which were shown to react by nucleophilic opening of the aziridine, at the pseudoanomeric carbon, by the active site cysteine nucleophile to form a stable thioether bond. Altogether, our activity-based probes may serve as chemical tools for the detection and identification of low-abundance ß-l-arabinofuranosidases in complex biological samples.
Assuntos
Aziridinas , Cisteína , Humanos , Glicosídeo Hidrolases/química , Aziridinas/química , Compostos de EpóxiRESUMO
Tumor therapy escape due to undesired side effects induced by treatment, such as prosurvival autophagy or cellular senescence, is one of the key mechanisms of resistance that eventually leads to tumor dormancy and recurrence. Glioblastoma is the most frequent and practically incurable neoplasm of the central nervous system; thus, new treatment modalities have been investigated to find a solution more effective than the currently applied standards based on temozolomide. The present study examined the newly synthesized compounds of aziridine-hydrazide hydrazone derivatives to determine their antineoplastic potential against glioblastoma cells in vitro. Although the output of our investigation clearly demonstrates their proapoptotic activity, the cytotoxic effect appeared to be blocked by treatment-induced autophagy, the phenomenon also detected in the case of temozolomide action. The addition of an autophagy inhibitor, chloroquine, resulted in a significant increase in apoptosis triggered by the tested compounds, as well as temozolomide. The new aziridine-hydrazide hydrazone derivatives, which present cytotoxic potential against glioblastoma cells comparable to or even higher than that of temozolomide, show promising results and, thus, should be further investigated as antineoplastic agents. Moreover, our findings suggest that the combination of an apoptosis inducer with an autophagy inhibitor could optimize chemotherapeutic efficiency, and the addition of an autophagy inhibitor should be considered as an optional adjunctive therapy minimizing the risk of tumor escape from treatment.
Assuntos
Antineoplásicos , Aziridinas , Glioblastoma , Humanos , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Temozolomida/farmacologia , Temozolomida/uso terapêutico , Cloroquina/farmacologia , Hidrazonas/farmacologia , Hidrazinas/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Autofagia , Aziridinas/farmacologia , Aziridinas/uso terapêuticoRESUMO
In this study, we report a series of newly synthesised sulphonamides of aziridine-2-carboxylic acid (Az-COOH) ester and amide analogues as potent protein disulphide isomerase (PDI, EC 5.3.4.1) inhibitors. The inhibitory activity on PDI was determined against recombinant human PDIA1 and PDIA3 proteins using an insulin reduction assay. These compounds in low micromolar to low nanomolar concentrations showed the effective in vitro inhibitory properties of PDIA1 with weaker effects on PDIA3. Complexes of 15N- and 15N,13C- uniformly labelled recombinant human PDIA1a with two PDIA1 inhibitors were produced and investigated by a protein nuclear magnetic resonance (NMR) spectroscopy. It was found that both C53 and C56 of the PDIA1 enzyme were involved in covalent binding. Finally, in a range of pharmacological studies, we demonstrated that investigated compounds displayed anti-cancer and anti-thrombotic activity. These findings demonstrate that sulphonamides of Az-COOH derivatives are promising candidates for the development of novel anti-cancer and anti-thrombotic agents.
Assuntos
Aziridinas , Isomerases de Dissulfetos de Proteínas , Sulfonamidas , Humanos , Aziridinas/farmacologia , Isomerases de Dissulfetos de Proteínas/antagonistas & inibidores , Isomerases de Dissulfetos de Proteínas/química , Sulfonamidas/farmacologiaRESUMO
Aziridines are compounds with a nitrogen-containing three-membered ring. When it is incorporated into natural products, the reactivity of the strained ring often drives the biological activities of aziridines. Despite its importance, the enzymes and biosynthetic strategies deployed to install this reactive moiety remain understudied. Herein, we report the use of in silico methods to identify enzymes with potential aziridine-installing (aziridinase) functionality. To validate candidates, we reconstitute enzymatic activity in vitro and demonstrate that an iron(IV)-oxo species initiates aziridine ring closure by the C-H bond cleavage. Furthermore, we divert the reaction pathway from aziridination to hydroxylation using mechanistic probes. This observation, isotope tracing experiments using H218O and 18O2, and quantitative product analysis, provide evidence for the polar capture of a carbocation species by the amine in the pathway to aziridine installation.
Assuntos
Aziridinas , Ferro , Ferro/química , Hidroxilação , CatáliseRESUMO
The ubiquitin-proteasome pathway (UPP) is the major proteolytic system in the cytosol and nucleus of all eukaryotic cells. The role of proteasome inhibitors (PIs) as critical agents for regulating cancer cell death has been established. Aziridine derivatives are well-known alkylating agents employed against cancer. However, to the best of our knowledge, aziridine derivatives showing inhibitory activity towards proteasome have never been described before. Herein we report a new class of selective and nonPIs bearing an aziridine ring as a core structure. In vitro cell-based assays (two leukemia cell lines) also displayed anti-proliferative activity for some compounds. In silico studies indicated non-covalent binding mode and drug-likeness for these derivatives. Taken together, these results are promising for developing more potent PIs.
Assuntos
Antineoplásicos , Aziridinas , Neoplasias , Humanos , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/uso terapêutico , Complexo de Endopeptidases do Proteassoma/metabolismo , Antineoplásicos/uso terapêutico , Aziridinas/farmacologia , Aziridinas/química , Neoplasias/metabolismo , Alquilantes , UbiquitinasRESUMO
Natural products containing an aziridine ring, such as mitomycin C and azinomycin B, exhibit antitumor activities by alkylating DNA via their aziridine rings; however, the biosynthetic mechanisms underlying the formation of these rings have not yet been elucidated. We herein investigated the biosynthesis of vazabitide A, the structure of which is similar to that of azinomycin B, and demonstrated that Vzb10/11, with no similarities to known enzymes, catalyzed the formation of the aziridine ring via sulfate elimination. To elucidate the detailed reaction mechanism, crystallization of Vzb10/11 and the homologous enzyme, AziU3/U2, in the biosynthesis of azinomycin B was attempted, and the structure of AziU3/U2, which had a new protein fold overall, was successfully determined. The structural analysis revealed that these enzymes adjusted the dihedral angle between the amino group and the adjacent sulfate group of the substrate to almost 180° and enhanced the nucleophilicity of the C6-amino group temporarily, facilitating the SN2-like reaction to form the aziridine ring. The present study reports for the first time the molecular basis for aziridine ring formation.
Assuntos
Aziridinas , Sulfatos , Aziridinas/química , DNA/química , MitomicinaRESUMO
Naturally occurring oligoamines, such as spermine, spermidine, and putrescine, are well-known regulators of gene expression. These oligoamines frequently have short alkyl spacers with varying lengths between the amines. Linear polyethylenimine (PEI) is a polyamine that has been widely applied as a gene vector, with various formulations currently in clinical trials. In order to emulate natural oligoamine gene regulators, linear random copolymers containing both PEI and polypropylenimine (PPI) repeat units were designed as novel gene delivery agents. In general, statistical copolymerization of 2-oxazolines and 2-oxazines leads to the formation of gradient copolymers. In this study, however, we describe for the first time the synthesis of near-ideal random 2-oxazoline/2-oxazine copolymers through careful tuning of the monomer structures and reactivity as well as polymerization conditions. These copolymers were then transformed into near-random PEI-PPI copolymers by controlled side-chain hydrolysis. The prepared PEI-PPI copolymers formed stable polyplexes with GFP-encoding plasmid DNA, as validated by dynamic light scattering. Furthermore, the cytotoxicity and transfection efficiency of polyplexes were evaluated in C2C12 mouse myoblasts. While the polymer chain length did not significantly increase the toxicity, a higher PPI content was associated with increased toxicity and also lowered the amount of polymers needed to achieve efficient transfection. The transfection efficiency was significantly influenced by the degree of polymerization of PEI-PPI, whereby longer polymers resulted in more transfected cells. Copolymers with 60% or lower PPI content exhibited a good balance between high plasmid-DNA transfection efficiency and low toxicity. Interestingly, these novel PEI-PPI copolymers revealed exceptional serum tolerance, whereby transfection efficiencies of up to 53% of transfected cells were achieved even under 50% serum conditions. These copolymers, especially PEI-PPI with DP500 and a 1:1 PEI/PPI ratio, were identified as promising transfection agents for plasmid DNA.
Assuntos
DNA , Polímeros , Animais , Aziridinas , DNA/química , Técnicas de Transferência de Genes , Camundongos , Plasmídeos/genética , Polietilenoimina/química , Polímeros/química , TransfecçãoRESUMO
Advances in the field of cancer immunotherapy have stimulated renewed interest in adenoviruses as oncolytic agents. Clinical experience has shown that oncolytic adenoviruses are safe and well tolerated but possess modest single-agent activity. One approach to improve the potency of oncolytic viruses is to utilise their tumour selectivity to deliver genes encoding prodrug-activating enzymes. These enzymes can convert prodrugs into cytotoxic species within the tumour; however, these cytotoxins can interfere with viral replication and limit utility. In this work, we evaluated the activity of a nitroreductase (NTR)-armed oncolytic adenovirus ONYX-411NTR in combination with the clinically tested bioreductive prodrug PR-104. Both NTR-expressing cells in vitro and xenografts containing a minor population of NTR-expressing cells were highly sensitive to PR-104. Pharmacologically relevant prodrug exposures did not interfere with ONYX-411NTR replication in vitro. In vivo, prodrug administration increased virus titre and improved virus distribution within tumour xenografts. Colonisation of tumours with high ONYX-411NTR titre resulted in NTR expression and prodrug activation. The combination of ONYX-411NTR with PR-104 was efficacious against HCT116 xenografts, whilst neither prodrug nor virus were active as single agents. This work highlights the potential for future clinical development of NTR-armed oncolytic viruses in combination with bioreductive prodrugs.