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1.
Biochem Pharmacol ; 182: 114230, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32979352

RESUMO

L-asparaginase (ASNase) from Escherichia coli (EcAII) is used in the treatment of acute lymphoblastic leukaemia (ALL). EcAII activity in vivo has been described to be influenced by the human lysosomal proteases asparaginyl endopeptidase (AEP) and cathepsin B (CTSB); these hydrolases cleave and could expose epitopes associated with the immune response against EcAII. In this work, we show that ASNase resistance to CTSB and/or AEP influences the formation of anti-ASNase antibodies, one of the main causes of hypersensitivity reactions in patients. Error-prone polymerase chain reaction was used to produce variants of EcAII more resistant to proteolytic cleavage by AEP and CTSB. The variants with enzymatic activity and cytotoxicity levels equivalent to or better than EcAII WT were submitted to in vivo assays. Only one of the mutants presented increased serum half-life, so resistance to these proteases is not the only feature involved in EcAII stability in vivo. Our results showed alteration of the phenotypic profile of B cells isolated after animal treatment with different protease-resistant proteoforms. Furthermore, mice that were exposed to the protease-resistant proteoforms presented lower anti-asparaginase antibodies production in vivo. Our data suggest that modulating resistance to lysosomal proteases can result in less immunogenic protein drugs.


Assuntos
Antineoplásicos/farmacologia , Asparaginase/farmacologia , Produtos Biológicos/farmacologia , Fenômenos Imunogenéticos/efeitos dos fármacos , Lisossomos/imunologia , Peptídeo Hidrolases/farmacologia , Sequência de Aminoácidos , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Asparaginase/química , Asparaginase/uso terapêutico , Produtos Biológicos/química , Produtos Biológicos/uso terapêutico , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Galinhas , Relação Dose-Resposta a Droga , Escherichia coli , Feminino , Cavalos , Humanos , Fenômenos Imunogenéticos/fisiologia , Células Jurkat , Lisossomos/química , Camundongos , Camundongos Endogâmicos BALB C , Peptídeo Hidrolases/química , Peptídeo Hidrolases/uso terapêutico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Estrutura Secundária de Proteína
2.
Mol Inform ; 34(2-3): 84-96, 2015 02.
Artigo em Inglês | MEDLINE | ID: mdl-27490031

RESUMO

Alkylphosphocholines (APCs) and alkyl-lysophosphocholines (ALPs) are antineoplastic agents that interfere with cellular membranes and signaling proteins. Protein kinase Cα (PKCα) is a signaling protein composed by catalytic (C3, C4) and regulatory domains (C1, C2). The C2 needs calcium (Ca(2+) ) and phosphatidylserine (PS) for activation. Miltefosine inhibits PKCα competitively with regard to PS and non-competitively with regard to Ca(2+) , however, the mechanism of action is unknown. We employed molecular docking, molecular dynamics and chemometric methods to verify how 7 APCs and ALPs derivatives and PS interact with the C2 domain. All ligands except PS were grouped in 2 clusters according to their interactions inside the enzyme. The findings showed that PS's phosphoryl oxygens interact with Ca(2+) , the serine moiety interacts with Asn189, and the carbonyl oxygen of the alkylic chain interacts with Arg249 and Thr251. On the other hand, ligands' phosphoryl oxygens interact with Asn189, Arg249, Thr250, and one water molecule instead of Ca(2+) . Because of the different binding mode, we hypothesize that the ligands cause conformational changes in the calcium binding region. Moreover, the packing mismatch between bilayer-forming lipids and ALP/APC chain impedes the C2 domain from docking to the internal leaflet of cellular membranes, interrupting PKCα activation.


Assuntos
Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Fosforilcolina/química , Proteína Quinase C-alfa/química , Humanos , Domínios Proteicos
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