RESUMO
Apurinic/apyrimidinic endonuclease-1 (APE1) is a base excision repair (BER) enzyme that is also engaged in transcriptional regulation. Previous work demonstrated that the enzymatic stalling of APE1 on a promoter G-quadruplex (G4) recruits transcription factors during oxidative stress for gene regulation. Also, during oxidative stress, cysteine (Cys) oxidation is a post-translational modification (PTM) that can change a protein's function. The current study provides a quantitative survey of cysteine oxidation to sulfenic acid in APE1 and how this PTM at specific cysteine residues affects the function of APE1 toward the NEIL3 gene promoter G4 bearing an abasic site. Of the seven cysteine residues in APE1, five (C65, C93, C208, C296, and C310) were prone to carbonate radical anion oxidation to yield sulfenic acids that were identified and quantified by mass spectrometry. Accordingly, five Cys-to-serine (Ser) mutants of APE1 were prepared and found to have attenuated levels of endonuclease activity, depending on the position, while KD values generally decreased for G4 binding, indicating greater affinity. These data support the concept that cysteine oxidation to sulfenic acid can result in modified APE1 that enhances G4 binding at the expense of endonuclease activity during oxidative stress. Cysteine oxidation to sulfenic acid residues should be considered as one of the factors that may trigger a switch from base excision repair activity to transcriptional modulation by APE1.
Assuntos
Cisteína , Ácidos Sulfênicos , Cisteína/genética , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Endonucleases/metabolismo , Serina/genética , Fatores de Transcrição/metabolismoRESUMO
The commercially available antibody-drug conjugate (ADC) product, Kadcyla® is synthesized using a 2-step reaction, wherein the linker is conjugated to native lysines on the mAb in step 1, followed by drug conjugation to the linker-modified antibody in step 2. In our study, we synthesized a lysine-conjugated ADC (Syn-ADC) on the same trastuzumab scaffold as Kadcyla® using a 1-step reaction. Mass spectrometry of both products revealed a subpopulation of Kadcyla® containing free linkers conjugated to the mAb, but not conjugated to the drug, which were absent in the 1-step reaction ADC product. Differential scanning calorimetry thermograms showed that the drug and linker conjugation significantly reduced the thermal stability and energies of activation for the denaturation of the CH2 domain of the ADCs. The heating induced aggregation events started as early as â¼57°C and â¼45°C for Kadcyla® and Syn-ADC, respectively, compared with 71°C for Herceptin®. The colloidal stability measurements clearly showed that the hydrophobic drug payload on ADCs significantly reduced the repulsive interprotein interactions when compared to the unconjugated antibody under formulation buffer conditions (pH 6.0). Attaching hydrophobic drug and linker moieties onto the antibody lowered the thermal and colloidal stabilities and increased the aggregation propensity of the ADCs.
Assuntos
Antineoplásicos Imunológicos/química , Imunoconjugados/química , Lisina/química , Maitansina/análogos & derivados , Agregados Proteicos , Trastuzumab/química , Ado-Trastuzumab Emtansina , Antineoplásicos Imunológicos/síntese química , Varredura Diferencial de Calorimetria , Cromatografia Líquida , Dicroísmo Circular , Coloides/síntese química , Coloides/química , Calefação , Lisina/síntese química , Espectrometria de Massas , Maitansina/síntese química , Maitansina/química , Estabilidade Proteica , Análise Espectral RamanRESUMO
Antibody drug conjugates are a rapidly growing form of targeted chemotherapeutics. As companies and researchers move to develop new antibody-drug conjugate (ADC) candidates, high-throughput methods will become increasingly common. Here we use advanced characterization techniques to assess two trastuzumab-DM1 (T-DM1) ADCs; one produced using Protein A immobilization and the other produced in solution. Following determination of payload site and distribution with liquid chromatography-mass spectrometry (LC/MS), thermal stability, heat-induced aggregation, tertiary structure, and binding affinity were characterized using differential scanning calorimetry (DSC), dynamic light scattering (DLS), Raman spectroscopy, and isothermal titration calorimetry (ITC), respectively. Small differences in the thermal stability of the CH2 domain of the antibody as well as aggregation onset temperatures were observed from DSC and DLS, respectively. However, no significant differences in secondary and tertiary structure were observed with Raman spectroscopy, or binding affinity as measured by ITC. Lysine-based ADC conjugation produces an innately heterogeneous population that can generate significant variability in the results of sensitive characterization techniques. Characterization of these ADCs indicated nominal differences in thermal stability but not in tertiary structure or binding affinity. Our results lead us to conclude that lysine-based ADCs synthesized following Protein A immobilization, common in small-scale conjugations, are highly similar to equivalent ADCs produced in larger scale, solution-based methods.
RESUMO
Protein self-assembly relies upon the formation of stabilizing noncovalent interactions across subunit interfaces. Identifying the determinants of self-assembly is crucial for understanding structure-function relationships in symmetric protein complexes and for engineering responsive nanoscale architectures for applications in medicine and biotechnology. Lumazine synthases (LS's) comprise a protein family that forms diverse quaternary structures, including pentamers and 60-subunit dodecahedral capsids. To improve our understanding of the basis for this difference in assembly, we attempted to convert the capsid-forming LS from Aquifex aeolicus (AaLS) into pentamers through a small number of rationally designed amino acid substitutions. Our mutations targeted side chains at ionic (R40), hydrogen bonding (H41), and hydrophobic (L121 and I125) interaction sites along the interfaces between pentamers. We found that substitutions at two or three of these positions could reliably generate pentameric variants of AaLS. Biophysical characterization indicates that this quaternary structure change is not accompanied by substantial changes in secondary or tertiary structure. Interestingly, previous homology-based studies of the assembly determinants in LS's had identified only one of these four positions. The ability to control assembly state in protein capsids such as AaLS could aid efforts in the development of new systems for drug delivery, biocatalysis, or materials synthesis.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Mutação Puntual , Dicroísmo Circular , Escherichia coli/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Dobramento de Proteína , Estabilidade ProteicaRESUMO
Continuing from our ongoing effort in modifying aminoglycoside antibiotics with the goal of counteracting drug resistant bacteria, we have further derivatized pyranmycin, a neomycin class aminoglycoside antibiotic, with modifications at O-6 and N-1 positions. The revealed SAR results demonstrated that the antibacterial activity of pyranmycin can be modulated by different acylic substituents at O-6. Among these results, the 6-O-aminoethyl derivative, JT050, showed effective activity against resistant strain Escherichia coli (pTZ19U-3) and E. coli (pSF815), which provides insight into further structural modifications.
Assuntos
Aminoglicosídeos/síntese química , Antibacterianos/síntese química , Antibacterianos/farmacologia , Nitrogênio/química , Oxigênio/química , Aminoglicosídeos/química , Sequência de Carboidratos , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Dados de Sequência Molecular , Relação Estrutura-AtividadeRESUMO
The criteria for controlling the regioselectivity of Staudinger reduction of azides have been investigated. These findings enable a convenient direct N-1 modification of the perazidoneamine and perazidoribostamycin resulting in the synthesis of aminoglycoside antibiotics with activity against drug-resistant bacteria.
Assuntos
Aminoglicosídeos/síntese química , Antibacterianos/síntese química , Azidas/química , Bactérias/efeitos dos fármacos , Aminoglicosídeos/química , Aminoglicosídeos/farmacologia , Antibacterianos/química , Antibacterianos/farmacologia , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/microbiologia , Sequência de Carboidratos , Resistência Microbiana a Medicamentos , Dados de Sequência MolecularAssuntos
Anti-Infecciosos/síntese química , Carboidratos/síntese química , Oxazolidinonas/síntese química , Anti-Infecciosos/farmacologia , Carboidratos/farmacologia , Técnicas de Química Combinatória/métodos , Escherichia coli/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Mycobacterium smegmatis/efeitos dos fármacos , Oxazolidinonas/farmacologia , Rhodotorula/efeitos dos fármacos , Sonicação , Staphylococcus aureus/efeitos dos fármacosRESUMO
A novel broad-spectrum aminoglycoside antibiotic, pyrankacin, has been prepared. In addition to the synthetic innovation in dideoxygenation and regioselective Staudinger reduction, we have obtained prominent antibacterial activity against several clinically important pathogens in the course of this work.
Assuntos
Aminoglicosídeos , Antibacterianos , Bactérias/efeitos dos fármacos , Aminoglicosídeos/síntese química , Aminoglicosídeos/química , Aminoglicosídeos/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Klebsiella pneumoniae/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Staphylococcus aureus/efeitos dos fármacosRESUMO
In an effort to optimize the antibacterial activity of kanamycin class aminoglycoside antibiotics, we have accomplished the synthesis and antibacterial assay of new kanamycin B analogues. A rationale-based glycodiversification strategy was employed. The activity of the lead is comparable to that of commercially available kanamycin. These new members, however, were found to be inactive against aminoglycoside resistant bacteria. Molecular modeling was used to provide the explanation. Thus, a new strategy for structural modifications of kanamycin class aminoglycosides is suggested.
Assuntos
Antibacterianos/síntese química , Canamicina/análogos & derivados , Antibacterianos/química , Antibacterianos/farmacologia , Sequência de Carboidratos , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Canamicina/síntese química , Canamicina/química , Canamicina/farmacologia , Canamicina Quinase/química , Testes de Sensibilidade Microbiana , Modelos Moleculares , Dados de Sequência Molecular , RNA/química , Staphylococcus aureus/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
[reaction: see text] A novel method for achieving the desired regioselective reduction of the N-1 azido group on a tetraazidoneamine has been developed that leads to the synthesis of both kanamycin and neomycin class antibiotics bearing N-1 modification. Both classes of aminoglycosides are active against aminoglycoside-resistant bacteria carrying APH(3')-I and AAC(6')/APH(2'').