RESUMEN
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.
Asunto(s)
Reparación del ADN , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , O(6)-Metilguanina-ADN Metiltransferasa , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , O(6)-Metilguanina-ADN Metiltransferasa/química , O(6)-Metilguanina-ADN Metiltransferasa/antagonistas & inhibidores , Humanos , Teoría Cuántica , Resistencia a Antineoplásicos/efectos de los fármacos , Compuestos de Nitrosourea/química , Compuestos de Nitrosourea/farmacología , Compuestos de Nitrosourea/metabolismoRESUMEN
Methyl damage to DNA bases is common in the cell nucleus. O6-alkylguanine-DNA alkyl transferase (AGT) may be a promising candidate for direct damage reversal in methylated DNA (mDNA) at the O6 point of the guanine. Indeed, atomic-level investigations in the contact region of AGT-DNA complex can provide an in-depth understanding of their binding mechanism, allowing to evaluate the silico-drug nature of AGT and its utility in removing methyl damage in DNA. In this study, molecular dynamics (MD) simulation was utilized to examine the flipping of methylated nucleotide, the binding mechanism between mDNA and AGT, and the comparison of binding strength prior and post methyl transfer to AGT. The study reveals that methylation at the O6 atom of guanine weakens the hydrogen bond (H-bond) between guanine and cytosine, permitting for the flipping of such nucleotide. The formation of a H-bond between the base pair of methylated nucleotide (i.e., cytosine) and the intercalated arginine of AGT also forces the nucleotide to rotate. Following that, electrostatics and van der Waals contacts as well as hydrogen bonding contribute to form the complex of DNA and protein. The stronger binding of AGT with DNA before methyl transfer creates the suitable condition to transfer methyl adduct from DNA to AGT.
Asunto(s)
Reparación del ADN , O(6)-Metilguanina-ADN Metiltransferasa , O(6)-Metilguanina-ADN Metiltransferasa/química , O(6)-Metilguanina-ADN Metiltransferasa/genética , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , Nucleótidos/química , ADN/química , Guanina/química , Guanina/metabolismo , CitosinaRESUMEN
The conjugation of proteins with synthetic molecules can be conducted in many different ways. In this Perspective, we focus on tag-based techniques and specifically on the SNAP-tag technology. The SNAP-tag technology makes use of a fusion protein between a protein of interest and an enzyme tag that enables the actual conjugation reaction. The SNAP-tag is based on the O6-alkylguanine-DNA alkyltransferase (AGT) enzyme and is optimized to react selectively with O6-benzylguanine (BG) substrates. BG-containing dye derivatives have frequently been used to introduce a fluorescent tag to a specific protein. We believe that the site-specific conjugation of polymers to proteins can significantly benefit from the SNAP-tag technology. Especially, polymers synthesized via reversible deactivation radical polymerization allow for the facile introduction of a BG end group to enable SNAP-tag conjugation.
Asunto(s)
O(6)-Metilguanina-ADN Metiltransferasa , Proteínas , O(6)-Metilguanina-ADN Metiltransferasa/genética , O(6)-Metilguanina-ADN Metiltransferasa/química , O(6)-Metilguanina-ADN Metiltransferasa/metabolismoRESUMEN
SignificanceWe directly visualize DNA translocation and lesion recognition by the O6-alkylguanine DNA alkyltransferase (AGT). Our data show bidirectional movement of AGT monomers and clusters on undamaged DNA that depended on Zn2+ occupancy of AGT. A role of cooperative AGT clusters in enhancing lesion search efficiencies by AGT has previously been proposed. Surprisingly, our data show no enhancement of DNA translocation speed by AGT cluster formation, suggesting that AGT clusters may serve a different role in AGT function. Our data support preferential cluster formation by AGT at alkyl lesions, suggesting a role of these clusters in stabilizing lesion-bound complexes. From our data, we derive a new model for the lesion search and repair mechanism of AGT.
Asunto(s)
Transferasas Alquil y Aril/química , Transferasas Alquil y Aril/metabolismo , Reparación del ADN , ADN/química , ADN/genética , Imagen Individual de Molécula , ADN/metabolismo , ADN de Cadena Simple , Humanos , Iones , Modelos Moleculares , O(6)-Metilguanina-ADN Metiltransferasa/química , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , Multimerización de Proteína , Imagen Individual de Molécula/métodos , Relación Estructura-Actividad , Zinc/químicaRESUMEN
The control of endogenous protein activity with light inside live cells is helpful for the high spatiotemporal probing of their dynamic roles. Herein, we report the first small-molecule-ligand-directed caging approach to control the endogenous human O6 -alkylguanine-DNA alkyltransferase (AGT) activity with light, and the caged AGT is constructed from the native intracellular AGT. The photo-responsive O6 -benzylguanine derivative O6 -NBG3 is developed to site-specifically cage the AGT's catalytic cysteine residue, and the light irradiation on-demand restores AGT's activity in vitro, in bacteria, and in mammalian cells. With O6 -NBG3, the alkylated AGT is dealkylated for the first time to recover the DNA repair activity in breast cancer MCF-7 cells by the dose-dependent light irradiation. This decaging strategy enables the localized modulation of endogenous AGT activity with high temporal precision without genetic engineering, which holds great potential for therapeutic applications.
Asunto(s)
Transferasas Alquil y Aril , Guanina , Animales , Guanina/química , Humanos , Ligandos , Mamíferos/metabolismo , O(6)-Metilguanina-ADN Metiltransferasa/químicaRESUMEN
O6-Methylguanine-DNA methyltransferase (MGMT) removes cytotoxic O6-alkyl adducts on the guanine base and protects the cell from genomic damage induced by alkylating agents. Although there are reports of computational studies on the activity of the enzyme with mutations at tyrosine residues, no studies concerning the crystal structure of its mutants have been found. In this study, the function of Tyr91 was investigated in detail by comparing the crystal structures of mutants and their complexes with substrate analogs. In this study, tyrosine, a conserved amino acid near the active-site loop in the C-terminal domain of Sulfurisphaera tokodaii MGMT (StoMGMT), was mutated to phenylalanine to produce a Y91F mutant, and the cysteine which is responsible for receiving the methyl group in the active site was mutated to a serine to produce a C120S mutant. A Y91F/C120S double-mutant StoMGMT was also created. The function of tyrosine is discussed based on the crystal structure of Y91F mutant StoMGMT. The crystal structures of StoMGMT were determined at resolutions of 1.13-2.60â Å. They showed no structural changes except in the mutated part. No electron density for deoxyguanosine or methyl groups was observed in the structure of Y91F mutant crystals immersed in O6-methyl-2'-deoxyguanosine, nor was the group oxidized in wild-type StoMGMT. Therefore, the hydroxy group of Tyr91 may prevent the oxidant from entering the active site. This suggests that tyrosine, which is highly conserved at the N-terminus of the helix-turn-helix motif across species, protects the active site of MGMTs, which are deactivated after repairing only one alkyl adduct. Overall, the results may provide a basis for understanding the molecular mechanisms by which high levels of conserved amino acids play a role in ensuring the integrity of suicide enzymes, in addition to promoting their activity.
Asunto(s)
O(6)-Metilguanina-ADN Metiltransferasa , Sulfolobaceae , Cristalografía por Rayos X , ADN , Reparación del ADN , Humanos , Metiltransferasas/química , O(6)-Metilguanina-ADN Metiltransferasa/química , O(6)-Metilguanina-ADN Metiltransferasa/genética , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , Sulfolobaceae/genética , Sulfolobaceae/metabolismo , TirosinaRESUMEN
The self-labeling protein tags (SLPs) HaloTag7, SNAP-tag, and CLIP-tag allow the covalent labeling of fusion proteins with synthetic molecules for applications in bioimaging and biotechnology. To guide the selection of an SLP-substrate pair and provide guidelines for the design of substrates, we report a systematic and comparative study of the labeling kinetics and substrate specificities of HaloTag7, SNAP-tag, and CLIP-tag. HaloTag7 reaches almost diffusion-limited labeling rate constants with certain rhodamine substrates, which are more than 2 orders of magnitude higher than those of SNAP-tag for the corresponding substrates. SNAP-tag labeling rate constants, however, are less affected by the structure of the label than those of HaloTag7, which vary over 6 orders of magnitude for commonly employed substrates. Determining the crystal structures of HaloTag7 and SNAP-tag labeled with fluorescent substrates allowed us to rationalize their substrate preferences. We also demonstrate how these insights can be exploited to design substrates with improved labeling kinetics.
Asunto(s)
Colorantes Fluorescentes/química , O(6)-Metilguanina-ADN Metiltransferasa/química , Proteínas Recombinantes de Fusión/química , Cinética , Modelos Moleculares , O(6)-Metilguanina-ADN Metiltransferasa/genética , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Rodaminas/química , Coloración y Etiquetado , Especificidad por SustratoRESUMEN
We present a new class of DNA-based nanoswitches that, upon enzymatic repair, could undergo a conformational change mechanism leading to a change in fluorescent signal. Such folding-upon-repair DNA nanoswitches are synthetic DNA sequences containing O6 -methyl-guanine (O6 -MeG) nucleobases and labelled with a fluorophore/quencher optical pair. The nanoswitches are rationally designed so that only upon enzymatic demethylation of the O6 -MeG nucleobases they can form stable intramolecular Hoogsteen interactions and fold into an optically active triplex DNA structure. We have first characterized the folding mechanism induced by the enzymatic repair activity through fluorescent experiments and Molecular Dynamics simulations. We then demonstrated that the folding-upon-repair DNA nanoswitches are suitable and specific substrates for different methyltransferase enzymes including the human homologue (hMGMT) and they allow the screening of novel potential methyltransferase inhibitors.
Asunto(s)
ADN/metabolismo , Nanotecnología , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , ADN/química , Reparación del ADN , Humanos , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , O(6)-Metilguanina-ADN Metiltransferasa/químicaRESUMEN
Synthetic chemical fluorescent dyes promise to be useful for many applications in biology. Covalent, targeted labeling, such as with a SNAP-tag, uses synthetic dyes to label specific proteins in vivo for studying processes such as endocytosis or for imaging via super-resolution microscopy. Despite its potential, such chemical tagging has not been used effectively in plants. A major drawback has been the limited knowledge regarding cell wall and membrane permeability of the available synthetic dyes. Of 31 synthetic dyes tested here, 23 were taken up into BY-2 cells, while eight were not. This creates sets of dyes that can serve to measure endocytosis. Three of the dyes that were able to enter the cells, SNAP-tag ligands of diethylaminocoumarin, tetramethylrhodamine, and silicon-rhodamine 647, were used to SNAP-tag α-tubulin. Successful tagging was verified by live cell imaging and visualization of microtubule arrays in interphase and during mitosis in Arabidopsis (Arabidopsis thaliana) seedlings. Fluorescence activation-coupled protein labeling with DRBG-488 was used to observe PIN-FORMED2 (PIN2) endocytosis and delivery to the vacuole as well as preferential delivery of newly synthesized PIN2 to the actively forming cell plate during mitosis. Together, the data demonstrate that specific self-labeling of proteins can be used effectively in plants to study a wide variety of cellular and biological processes.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Colorantes Fluorescentes/farmacocinética , Células Vegetales/química , Arabidopsis/citología , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Endocitosis , Colorantes Fluorescentes/química , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , O(6)-Metilguanina-ADN Metiltransferasa/química , Células Vegetales/efectos de los fármacos , Células Vegetales/metabolismo , Plantas Modificadas Genéticamente , Rodaminas/química , Rodaminas/farmacocinética , Plantones , Imagen de Lapso de Tiempo , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismoRESUMEN
Promutagenic O6-alkylguanine adducts in DNA are repaired in humans by O6-methylguanine-DNA-methyltransferase (MGMT) in an irreversible reaction. Here we describe the synthesis of a phosphoramidite that allows the preparation of oligodeoxyribonucleotides (ODNs) containing a novel tricyclic thio analogue of O6-methylguanine in which the third ring bridges the 6-thio group and C7 of a 7-deazapurine. These ODNs are very poor substrates for MGMT and poorly recognised by the alkyltransferase-like protein, Atl1. Examination of the active sites of both MGMT and Atl1 suggest large steric clashes hindering binding of the analogue. Such analogues, if mutagenic, are likely to be highly toxic.
Asunto(s)
Transferasas Alquil y Aril/química , Guanina/análogos & derivados , O(6)-Metilguanina-ADN Metiltransferasa/química , Oligodesoxirribonucleótidos/química , Compuestos de Sulfhidrilo/química , Transferasas Alquil y Aril/metabolismo , Guanina/química , Guanina/metabolismo , Humanos , Modelos Moleculares , Estructura Molecular , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , Oligodesoxirribonucleótidos/síntesis química , Oligodesoxirribonucleótidos/metabolismo , Compuestos de Sulfhidrilo/metabolismoRESUMEN
1,1,2,2-cis-diamminedichloroplatinum (II) (cisplatin) is a chemotherapeutic agent widely used in the clinic to treat various cancers. The antitumor activity of cisplatin is generally attributed to its ability to form intrastrand and interstrand DNA-DNA cross-links via sequential platination of two nucleophilic sites within the DNA duplex. However, cisplatin also induces DNA- protein lesions (DPCs) that may contribute to its biological effects due to their ability to block DNA replication and transcription. We previously reported that over 250 nuclear proteins including high mobility group proteins, histone proteins, and elongation factors formed DPCs in human HT1080 cells treated with cisplatin (Ming et al. Chem. Res. Toxicol. 2017, 30, 980-995). Interestingly, cisplatin-induced DNA-protein conjugates were reversed upon heating, by an unknown mechanism. In the present work, DNA repair protein O6-alkylguanine DNA alkyltransferase (AGT) was used as a model to investigate the molecular details of cisplatin-mediated DNA-protein cross-linking and to establish the mechanism of their reversal. We found that AGT is readily cross-linked to DNA in the presence of cisplatin. HPLC-ESI+-MS/MS sequencing of tryptic peptides originating from dG-Pt-AGT complexes revealed that the cross-linking occurred at six sites within this protein including Glu110, Lys125, Cys145, His146, Arg147, and Cys150. Cisplatin-induced Lys-Gua cross-links (1,1-cis-diammine-2-(5-amino-5-carboxypentyl)amino-2-(2'-deoxyguanosine-7-yl)-platinum(II) (dG-Pt-Lys) were detected by HPLC-ESI+-MS/MS of total digests of modified protein in comparison with the corresponding authentic standard. Upon heating, dG-Pt-AGT complexes were subject to platination migration from protein to DNA, forming cis-[Pt(NH3)2{d(GpG)}] cross-links which were detected by HPLC-ESI+-MS/MS. Our results provide a new insight into the mechanism of cisplatin-mediated DNA-protein cross-linking and their dynamic equilibrium with the corresponding DNA-DNA lesions.
Asunto(s)
Cisplatino/farmacología , Aductos de ADN , ADN/química , Modelos Moleculares , O(6)-Metilguanina-ADN Metiltransferasa/química , Antineoplásicos/farmacología , Cromatografía Líquida de Alta Presión , Reactivos de Enlaces Cruzados/farmacología , Humanos , Conformación Proteica , Proteínas Recombinantes , Análisis de Secuencia de Proteína , Espectrometría de Masas en TándemRESUMEN
The genome of living cells is continuously exposed to endogenous and exogenous attacks, and this is particularly amplified at high temperatures. Alkylating agents cause DNA damage, leading to mutations and cell death; for this reason, they also play a central role in chemotherapy treatments. A class of enzymes known as AGTs (alkylguanine-DNA-alkyltransferases) protects the DNA from mutations caused by alkylating agents, in particular in the recognition and repair of alkylated guanines in O6-position. The peculiar irreversible self-alkylation reaction of these enzymes triggered numerous studies, especially on the human homologue, in order to identify effective inhibitors in the fight against cancer. In modern biotechnology, engineered variants of AGTs are developed to be used as protein tags for the attachment of chemical ligands. In the last decade, research on AGTs from (hyper)thermophilic sources proved useful as a model system to clarify numerous phenomena, also common for mesophilic enzymes. This review traces recent progress in this class of thermozymes, emphasizing their usefulness in basic research and their consequent advantages for in vivo and in vitro biotechnological applications.
Asunto(s)
Reparación del ADN , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , Alquilación , Biotecnología , Daño del ADN , Replicación del ADN , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Neoplasias/etiología , Neoplasias/metabolismo , Neoplasias/patología , O(6)-Metilguanina-ADN Metiltransferasa/química , O(6)-Metilguanina-ADN Metiltransferasa/genética , Relación Estructura-Actividad , Termodinámica , Thermoproteus/genética , Thermoproteus/metabolismoRESUMEN
Most cell behaviors are the outcome of processing information from multiple signals generated upon cell stimulation. Thus, a systematic understanding of cellular systems requires methods that allow the activation of more than one specific signaling molecule or pathway within a cell. However, the construction of tools suitable for such multiplexed signal control remains challenging. In this work, we aimed to develop a platform for chemically manipulating multiple signaling molecules/pathways in living mammalian cells based on self-localizing ligand-induced protein translocation (SLIPT). SLIPT is an emerging chemogenetic tool that controls protein localization and cell signaling using synthetic self-localizing ligands (SLs). Focusing on the inner leaflet of the plasma membrane (PM), where there is a hub of intracellular signaling networks, here we present the design and engineering of two new PM-specific SLIPT systems based on an orthogonal eDHFR and SNAP-tag pair. These systems rapidly induce translocation of eDHFR- and SNAP-tag-fusion proteins from the cytoplasm to the PM specifically in a time scale of minutes upon addition of the corresponding SL. We then show that the combined use of the two systems enables chemically inducible, individual translocation of two distinct proteins in the same cell. Finally, by integrating the orthogonal SLIPT systems with fluorescent reporters, we demonstrate simultaneous multiplexed activation and fluorescence imaging of endogenous ERK and Akt activities in a single cell. Collectively, orthogonal PM-specific SLIPT systems provide a powerful new platform for multiplexed chemical signal control in living single cells, offering new opportunities for dissecting cell signaling networks and synthetic cell manipulation.
Asunto(s)
Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas de la Membrana/metabolismo , O(6)-Metilguanina-ADN Metiltransferasa/farmacología , Transporte de Proteínas/efectos de los fármacos , Pirimidinas/farmacología , Tetrahidrofolato Deshidrogenasa/farmacología , Membrana Celular/metabolismo , Escherichia coli/enzimología , Células HeLa , Humanos , Proteínas de la Membrana/genética , O(6)-Metilguanina-ADN Metiltransferasa/química , O(6)-Metilguanina-ADN Metiltransferasa/genética , Ingeniería de Proteínas , Pirimidinas/química , Tetrahidrofolato Deshidrogenasa/química , Tetrahidrofolato Deshidrogenasa/genéticaRESUMEN
O6-alkylguanine-DNA alkyltransferase (AGT) is the main cause of tumor cell resistance to DNA-alkylating agents, so it is valuable to design tumor-targeted AGT inhibitors with hypoxia activation. Based on the existing benchmark inhibitor O6-benzylguanine (O6-BG), four derivatives with hypoxia-reduced potential and their corresponding reduction products were synthesized. A reductase system consisting of glucose/glucose oxidase, xanthine/xanthine oxidase, and catalase were constructed, and the reduction products of the hypoxia-activated prodrugs under normoxic and hypoxic conditions were determined by high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). The results showed that the reduction products produced under hypoxic conditions were significantly higher than that under normoxic condition. The amount of the reduction product yielded from ANBP (2-nitro-6-(3-amino) benzyloxypurine) under hypoxic conditions was the highest, followed by AMNBP (2-nitro-6-(3-aminomethyl)benzyloxypurine), 2-NBP (2-nitro-6-benzyloxypurine), and 3-NBG (O6-(3-nitro)benzylguanine). It should be noted that although the levels of the reduction products of 2-NBP and 3-NBG were lower than those of ANBP and AMNBP, their maximal hypoxic/normoxic ratios were higher than those of the other two prodrugs. Meanwhile, we also investigated the single electron reduction mechanism of the hypoxia-activated prodrugs using density functional theory (DFT) calculations. As a result, the reduction of the nitro group to the nitroso was proven to be a rate-limiting step. Moreover, the 2-nitro group of purine ring was more ready to be reduced than the 3-nitro group of benzyl. The energy barriers of the rate-limiting steps were 34-37 kcal/mol. The interactions between these prodrugs and nitroreductase were explored via molecular docking study, and ANBP was observed to have the highest affinity to nitroreductase, followed by AMNBP, 2-NBP, and 3-NBG. Interestingly, the theoretical results were generally in a good agreement with the experimental results. Finally, molecular docking and molecular dynamics simulations were performed to predict the AGT-inhibitory activity of the four prodrugs and their reduction products. In summary, simultaneous consideration of reduction potential and hypoxic selectivity is necessary to ensure that such prodrugs have good hypoxic tumor targeting. This study provides insights into the hypoxia-activated mechanism of nitro-substituted prodrugs as AGT inhibitors, which may contribute to reasonable design and development of novel tumor-targeted AGT inhibitors.
Asunto(s)
Sistemas de Liberación de Medicamentos , Inhibidores Enzimáticos/química , Simulación del Acoplamiento Molecular , O(6)-Metilguanina-ADN Metiltransferasa , Profármacos/química , Cromatografía Líquida de Alta Presión , Humanos , Hipoxia , O(6)-Metilguanina-ADN Metiltransferasa/antagonistas & inhibidores , O(6)-Metilguanina-ADN Metiltransferasa/química , Espectrometría de Masas en TándemRESUMEN
Antibody-based diagnostic and therapeutic agents play a substantial role in medicine, especially in cancer management. A variety of chemical, genetic and enzymatic site-specific conjugation methods have been developed for equipping antibodies with effector molecules to generate homogeneous antibody conjugates with tailored properties. However, most of these methods are relatively complicated and expensive and require several reaction steps. Self-labeling proteins such as the SNAP-tag are an innovative solution for addressing these challenges. The SNAP-tag is a modified version of the human DNA repair enzyme alkylguanine-DNA alkyltransferase (AGT), which reacts specifically with O(6)-benzylguanine (BG)-modified molecules via irreversible transfer of an alkyl group to a cysteine residue. It provides a simple, controlled and robust site-specific method for labeling antibodies with different synthetic small effector molecules. Fusing a SNAP-tag to recombinant antibodies allows efficient conjugation of BG-containing substrates by autocatalytic, irreversible transfer of the alkyl group to a cysteine residue in the enzyme's active site under physiological conditions and with a 1:1 stoichiometry. This protocol describes how to generate site-specific SNAP-tag single-chain antibody fragment (scFv) conjugates with different types of BG-modified effector molecules. A specific example is included for the design and production of an scFv-photosensitizer conjugate and its characterization as an immuno-theranostic agent. This protocol includes DNA sequences encoding scFV-SNAP-tag fusion proteins and outlines strategies for expression, purification and testing of the resulting scFv-SNAP-tag-based immuno-conjugates. All experiments can be performed by a graduate-level researcher with basic molecular biology skills within an 8-week time frame.
Asunto(s)
Inmunoconjugados/química , Anticuerpos de Cadena Única/química , Línea Celular , Colorantes Fluorescentes/química , Humanos , O(6)-Metilguanina-ADN Metiltransferasa/química , Proteínas Recombinantes/química , Coloración y EtiquetadoRESUMEN
OBJECTIVE: To screen ideal lead compounds from a drug library (ZINC15 database) with potential inhibition effect against O6-methylguanine-DNA methyltransferase (MGMT) to contribute to medication design and refinement. METHODS: A series of computer-aided virtual screening techniques were used to identify potential inhibitors of MGMT. Structure-based virtual screening by LibDock was carried out to calculate LibDock scores, followed by absorption, distribution, metabolism, and excretion and toxicity predictions. Molecule docking was employed to demonstrate binding affinity and mechanism between the selected ligands and MGMT protein. Molecular dynamics simulation was performed to evaluate stability of the ligand-MGMT complex under natural circumstances. RESULTS: Two novel natural compounds, ZINC000008220033 and ZINC000001529323, from the ZINC15 database were found to bind with MGMT with a higher binding affinity together with more favorable interaction energy. Also, they were predicted to have less rodent carcinogenicity, Ames mutagenicity, and developmental toxicity potential as well as noninhibition with cytochrome P-450 2D6. Molecular dynamics simulation analysis demonstrated that the 2 complexes ZINC000008220033-MGMT and ZINC000001529323-MGMT had more favorable potential energy compared with reference ligand O6-benzylguanine, and they could exist stably in the natural environment. CONCLUSIONS: This study elucidated that ZINC000008220033 and ZINC000001529323 were ideal lead compounds with potential inhibition targeting to MGMT protein. These compounds were selected as safe drug candidates and may contribute a solid basis for MGMT target medication design and improvement.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Descubrimiento de Drogas/métodos , Inhibidores Enzimáticos/farmacología , Simulación del Acoplamiento Molecular/métodos , O(6)-Metilguanina-ADN Metiltransferasa/antagonistas & inhibidores , Bases de Datos Factuales , Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Humanos , O(6)-Metilguanina-ADN Metiltransferasa/química , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
DNA repair mechanism is a process through which the cell repairs its damaged DNA. Although there are several mechanisms involved in the DNA repair mechanisms, the direct reversal method is the simplest and does not require a reference template, in which the guanine bases are often methylated, and the methyl guanine methyl transferase protein (MGMT) reverses them. The mutations occurring in the MGMT protein might result in dysfunction of such DNA repair mechanism. In this study, we attempted to evaluate the impact of six missense mutations (Y114E, Y114A, R128G, R128A, R128K, and C145A) at three active-site positions (Y114, C145, and R128) as this might hinder the DNA binding to the protein. These six mutations were subjected to pathogenicity, stability, and conservation analysis using online servers such as PredictSNP, iStable, and ConSurf, respectively. From the predictions, all the six mutations were almost predicted to be significant. Considering true positives, true negatives, false positives, and false negatives, three mutations (Y114E, R128G, and C145A) showed "loss of DNA repair activity," and were analyzed further using molecular dynamics simulations (MDS) using GROMACS for 50ns. MDS run showed that the C145A mutant demonstrated higher structural deviation, decreased compactness, and the binding patterns. The Y114E mutant showed almost a null effect from the structural analysis. Finally, the R128G mutant showed structural variations in between the C145A and Y114E mutations of MGMT protein. We believe that the observed findings in this computational approach might further pave a way of providing better treatment measures by understanding the DNA repair mechanisms.
Asunto(s)
Modelos Genéticos , Simulación de Dinámica Molecular , Mutación Missense , O(6)-Metilguanina-ADN Metiltransferasa/química , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , Humanos , O(6)-Metilguanina-ADN Metiltransferasa/genética , Conformación ProteicaRESUMEN
O6 -Alkylguanine DNA-alkyltransferase (AGT), a DNA repair protein, can form crosslinks with DNA. The AGT-DNA crosslinks are known to be mutagenic when AGT is heterologously expressed in Escherichia coli, as well as in mammalian cells. To understand the biological consequences, reliable access to AGT-oligonucleotide crosslinks is needed. This article describes the synthesis and characterization of site-specific AGT-oligonucleotide crosslinks at the N2-position of deoxyguanosine and N6-position of deoxyadenosine. We developed a post-oligomerization strategy for the synthesis of propargyl-modified oligonucleotides. Copper-catalyzed azide-alkyne cycloaddition was used as a key step to obtain the iodoacetamide-linked oligonucleotides, which serve as good electrophiles for the crosslinking reaction with cysteine-145 of the active site of AGT. Trypsinization of AGT and hydrolysis of oligonucleotides, combined with analysis by liquid chromatography-tandem mass spectrometry, was utilized to confirm the nucleobase-adducted peptides. This method provides a useful strategy for the synthesis and characterization of site-specific DNA-protein crosslinks, which can be further used to understand proteolytic degradation-coupled DNA repair mechanisms. © 2019 by John Wiley & Sons, Inc.
Asunto(s)
O(6)-Metilguanina-ADN Metiltransferasa/síntesis química , Oligonucleótidos/química , Catálisis , Dominio Catalítico , Cromatografía Liquida/métodos , Cobre/química , Reactivos de Enlaces Cruzados/química , Escherichia coli/genética , O(6)-Metilguanina-ADN Metiltransferasa/química , O(6)-Metilguanina-ADN Metiltransferasa/genética , Polimerizacion , Espectrometría de Masas en Tándem/métodos , Moldes Genéticos , Tripsina/químicaRESUMEN
O 6-Alkylguanine DNA alkyltransferases (AGTs) are proteins found in most organisms whose role is to remove alkylation damage from the O6- and O4-positions of 2'-deoxyguanosine (dG) and thymidine (dT), respectively. Variations in active site residues between AGTs from different organisms leads to differences in repair proficiency: The human variant (hAGT) has a proclivity for removal of alkyl groups at the O6-position of guanine and the E. coli OGT protein has activity towards the O4-position of thymine. A chimeric protein (hOGT) that our laboratory has engineered with twenty of the active site residues mutated in hAGT to those found in OGT, exhibited activity towards a broader range of substrates relative to native OGT. Among the substrates that the hOGT protein was found to act upon was interstrand cross-linked DNA connected by an alkylene linkage at the O6-position of dG to the complementary strand. In the present study the activity of hOGT towards DNA containing alkylene intrastrand cross-links (IaCL) at the O6- and O4-positions respectively of dG and dT, which lack a phosphodiester linkage between the connected residues, was evaluated. The hOGT protein exhibited proficiency at removal of an alkylene linkage at the O6-atom of dG but the O4-position of dT was refractory to protein activity. The activity of the chimeric hOGT protein towards these IaCLs to prepare well defined DNA-protein cross-linked conjugates will enable mechanistic and high resolution structural studies to address the differences observed in the repair adeptness of O4-alkylated dT by the OGT protein relative to other AGT variants.
Asunto(s)
ADN/metabolismo , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Metiltransferasas/genética , O(6)-Metilguanina-ADN Metiltransferasa/genética , Ingeniería de Proteínas/métodos , Dominio Catalítico , Reparación del ADN , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Humanos , Metiltransferasas/química , Metiltransferasas/metabolismo , Modelos Moleculares , Mutación , O(6)-Metilguanina-ADN Metiltransferasa/química , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , Especificidad por SustratoRESUMEN
Environmental exposure and cellular metabolism can give rise to DNA alkylation, which can occur on the nitrogen and oxygen atoms of nucleobases, as well as on the phosphate backbone. Although O6-alkyl-2'-deoxyguanosine (O6-alkyl-dG) lesions are known to be associated with cancer, not much is known about how the alkyl group structures in these lesions affect their repair and replicative bypass in vivo or how translesion synthesis DNA polymerases influence the latter process. To answer these questions, here we synthesized oligodeoxyribonucleotides harboring seven O6-alkyl-dG lesions, with the alkyl group being Me, Et, nPr, iPr, nBu, iBu, or sBu, and examined the impact of these lesions on DNA replication in Escherichia coli cells. We found that replication past all the O6-alkyl-dG lesions was highly efficient and that SOS-induced DNA polymerases play redundant roles in bypassing these lesions. Moreover, these lesions directed exclusively the G â A mutation, the frequency of which increased with the size of the alkyl group on the DNA. This could be attributed to the varied repair efficiencies of these lesions by O6-alkylguanine DNA alkyltransferase (MGMT) in cells, which involve the MGMT Ogt and, to a lesser extent, Ada. In conclusion, our study provides important new knowledge about the repair of the O6-alkyl-dG lesions and their recognition by the E. coli DNA replication machinery. Our results suggest that the lesions' carcinogenic potentials may be attributed, at least in part, to their strong mutagenic potential and their efficient bypass by the DNA replication machinery.