RESUMO
PURPOSE: Biotherapeutics can be susceptible to oxidation during manufacturing and storage. Free L-methionine is known to protect methionine residues in proteins from oxidation. Similarly, free tryptophan and other indole derivatives have been shown to protect tryptophan residues from oxidation. N-acetyl-DL-tryptophan was previously identified as a potentially superior antioxidant to tryptophan as it has a lower oxidation potential and produces less peroxide upon light exposure. This study sought to confirm the antioxidant efficacy and safety of N-acetyl-DL-tryptophan and L-methionine as formulation components for biotherapeutic drugs. METHODS: Antibodies were subjected to AAPH and light exposure in the presence of N-acetyl-DL-tryptophan and L-methionine. Oxidation in relevant CDR and Fc residues was quantified by peptide map. In silico, in vitro, and in vivo studies were performed to evaluate the safety of N-acetyl-DL-tryptophan and L-methionine. RESULTS: Peptide mapping demonstrated that N-acetyl-DL-tryptophan was effective at protecting tryptophans from AAPH stress, and that the combination of N-acetyl-DL-tryptophan and L-methionine protected both tryptophan and methionine from AAPH stress. The safety assessment suggested an acceptable safety profile for both excipients. CONCLUSIONS: N-acetyl-tryptophan and L-methionine effectively reduce the oxidation of susceptible tryptophan and methionine residues in antibodies and are safe for use in parenteral biotherapeutic formulations.
Assuntos
Anticorpos Monoclonais/química , Antioxidantes/química , Metionina/química , Triptofano/análogos & derivados , Amidinas/química , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/toxicidade , Antioxidantes/administração & dosagem , Antioxidantes/toxicidade , Produtos Biológicos , Linhagem Celular Tumoral , Simulação por Computador , Composição de Medicamentos , Excipientes/química , Feminino , Humanos , Macaca fascicularis , Masculino , Metionina/administração & dosagem , Metionina/toxicidade , Testes de Mutagenicidade , Oxirredução , Conformação Proteica , Estabilidade Proteica , Coelhos , Triptofano/administração & dosagem , Triptofano/química , Triptofano/toxicidade , Raios UltravioletaRESUMO
Protein oxidation is a major pathway for degradation of biologic drug products. Past literature reports have suggested that 2,2-azobis (2-amidinopropane) dihydrochloride (AAPH), a free radical generator that produces alkoxyl and alkyl peroxyl radicals, is a useful model reagent stress for assessing the oxidative susceptibility of proteins. Here, we expand the applications of the AAPH model by pairing it with a rapid peptide map method to enable site-specific studies of oxidative susceptibility of monoclonal antibodies and their derivatives for comparison between formats, the evaluation of formulation components, and comparisons across the stress models. Comparing the free radical-induced oxidation model by AAPH with a light-induced oxidation model suggests that light-sensitive residues represent a subset of AAPH-sensitive residues and therefore AAPH can be used as a preliminary screen to highlight molecules that need further assessment by light models. In sum, these studies demonstrate that AAPH stress can be used in multiple ways to evaluate labile residues and oxidation sensitivity as it pertains to developability and manufacturability.
Assuntos
Amidinas/química , Anticorpos Monoclonais/química , Oxirredução/efeitos dos fármacos , Radicais Livres/química , Peptídeos/química , Proteínas/químicaRESUMO
Histone deacetylases (HDACs) are key enzymatic regulators of the epigenome and serve as promising targets for anticancer therapeutics. Recently, we developed a photoreactive "clickable" probe, SAHA-BPyne, to report on HDAC activity and complex formation in native biological systems. Here, we investigate the selectivity, sensitivity, and inhibitory properties of SAHA-BPyne and related potential activity-based probes for HDACs. While we identified several probes that are potent HDAC inhibitors and label HDAC complex components in native proteomic preparations, SAHA-BPyne was markedly superior for profiling HDAC activities in live cells. Interestingly, the enhanced performance of SAHA-BPyne as an in situ activity-based probe could not be solely ascribed to potency in HDAC binding, implying that other features of the molecule were key to efficient active site-directed labeling in living systems. Finally, we demonstrate the value of in situ profiling of HDACs by comparing the activity and expression of HDAC1 in cancer cells treated with the cytotoxic agent parthenolide. These results underscore the utility of activity-based protein profiling for studying HDAC function and may provide insight for the future development of click chemistry-based photoreactive probes for the in situ analysis of additional enzyme activities.
Assuntos
Benzofenonas/química , Histona Desacetilases/química , Histona Desacetilases/metabolismo , Ácidos Hidroxâmicos/química , Sondas Moleculares/química , Benzofenonas/síntese química , Benzofenonas/farmacologia , Linhagem Celular Tumoral , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores de Histona Desacetilases , Humanos , Ácidos Hidroxâmicos/síntese química , Ácidos Hidroxâmicos/farmacologia , Sondas Moleculares/síntese química , Sondas Moleculares/farmacologia , Neoplasias/enzimologia , Proteômica/métodos , Relação Estrutura-Atividade , VorinostatRESUMO
Real-time in situ detection of active proteases is crucial for early-stage cancer screening and cell signaling pathway study; however, it is difficult to achieve using fluorescence or radioactive probes at volumes below 1 nL. Here we demonstrated a hybrid optical probe by incorporating nanocrescent particle and peptides with artificial tag molecules. We performed a proof-of-concept study using prostate specific antigen (PSA), one of the most prominent prostate cancer markers, and a serine protease present in patients' seminal fluid and serum. The Raman spectral signal from the tag molecules is enhanced by the nanocrescent and the signal is monitored as the indicator for peptide cleavage in a femtoliter reaction volume, at levels close to a single proteolytically active PSA molecule. The high reaction specificity of the peptides on individual nanoparticles minimizes the false detection of other serine proteases and background Raman signal, which results in a high-fidelity and high-signal-to-noise-ratio cancer nanoprobe that can be easily incorporated into nano/microfluidic devices.
Assuntos
Técnicas de Sonda Molecular , Nanoestruturas/química , Nanotecnologia/métodos , Peptídeo Hidrolases/química , Peptídeos/química , Antígeno Prostático Específico/análise , Análise Espectral Raman/métodos , Cristalização/métodos , Ativação Enzimática , Humanos , Substâncias Macromoleculares/química , Masculino , Teste de Materiais , Conformação Molecular , Nanoestruturas/ultraestrutura , Óptica e Fotônica , Tamanho da Partícula , Sensibilidade e Especificidade , Propriedades de SuperfícieRESUMO
Proteases regulate numerous biological processes with a degree of specificity often dictated by the amino acid sequence of the substrate cleavage site. To map protease/substrate interactions, a 722-member library of fluorogenic protease substrates of the general format Ac-Ala-X-X-(Arg/Lys)-coumarin was synthesized (X=all natural amino acids except cysteine) and microarrayed with fluorescent calibration standards in glycerol nanodroplets on glass slides. Specificities of 13 serine proteases (activated protein C, plasma kallikrein, factor VIIa, factor IXabeta, factor XIa and factor alpha XIIa, activated complement C1s, C1r, and D, tryptase, trypsin, subtilisin Carlsberg, and cathepsin G) and 11 papain-like cysteine proteases (cathepsin B, H, K, L, S, and V, rhodesain, papain, chymopapain, ficin, and stem bromelain) were obtained from 103,968 separate microarray fluorogenic reactions (722 substrates x 24 different proteases x 6 replicates). This is the first comprehensive study to report the substrate specificity of rhodesain, a papain-like cysteine protease expressed by Trypanasoma brucei rhodesiense, a parasitic protozoa responsible for causing sleeping sickness. Rhodesain displayed a strong P2 preference for Leu, Val, Phe, and Tyr in both the P1=Lys and Arg libraries. Solution-phase microarrays facilitate protease/substrate specificity profiling in a rapid manner with minimal peptide library or enzyme usage.