RESUMO
Refractory T cell acute leukaemias that no longer respond to treatment would benefit from new modalities that target T cell-specific surface proteins. T cell associated surface proteins (the surfaceome) offer possible therapy targets to reduce tumour burden but also target the leukaemia-initiating cells from which tumours recur. Recent studies of the T cell leukaemia surfaceome confirmed that CD7 is highly expressed in overt disease. We have used an anti-CD7 antibody drug conjugate (ADC) to show that the binding of antibody to surface CD7 protein results in rapid internalization of the antigen together with the ADC. As a consequence, cell killing was observed via induction of apoptosis and was dependent on cell surface CD7. The in vitro cytotoxic activity (EC50) of the anti-CD7 ADC on T cell acute leukaemia (T-ALL) cells Jurkat and KOPT-K1 was found to be in the range of 5-8 ng/mL. In a pre-clinical xenograft model of human tumour growth expressing CD7 antigen, growth was curtailed by a single dose of ADC. The data indicate that CD7 targeting ADCs may be developed into an important second stage therapy for T cell acute leukaemia, for refractory CD7-positive leukaemias and for subsets of acute myeloid leukaemia (AML) expressing CD7.
Assuntos
Anticorpos Monoclonais/química , Antígenos CD7/imunologia , Apoptose , Liberação Controlada de Fármacos , Imunoconjugados/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Animais , Antígenos CD7/metabolismo , Proliferação de Células , Humanos , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Nus , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Using site directed mutagenesis we altered an active site residue (Phe107) of the enzyme encoded by fabF3 (SCO3248) in the Streptomyces coelicolor gene cluster required for biosynthesis of the calcium dependent antibiotics (CDAs), successfully generating two novel CDA derivatives comprising truncated (C4) lipid side chains and confirming that fabF3 encodes a KAS-II homologue that is involved in determining CDA fatty acid chain length.
Assuntos
3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Ácidos Graxos/biossíntese , Lipopeptídeos/biossíntese , Peptídeos/metabolismo , Streptomyces coelicolor/enzimologia , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/química , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/genética , Domínio Catalítico , Ácidos Graxos/química , Leucina/química , Leucina/metabolismo , Lipopeptídeos/química , Lipopeptídeos/metabolismo , Mutagênese , Peptídeos/química , Fenilalanina/química , Fenilalanina/metabolismo , Streptomyces coelicolor/genéticaRESUMO
Feeding 5-hydroxy and 5-fluorotryptophan to a Streptomyces coelicolor Trp-auxotrophic strain WH101 results in the production of a number of new calcium-dependent antibiotics (CDAs) possessing modified Trp residues. It is anticipated that this method could be used to modulate the biological properties of Trp-containing nonribosomal peptide natural products, or to generate analogues with useful fluorescent properties for studying biological mechanisms of action.
Assuntos
5-Hidroxitriptofano/metabolismo , Lipoproteínas/biossíntese , Biossíntese de Peptídeos Independentes de Ácido Nucleico/fisiologia , Peptídeos/metabolismo , Streptomyces coelicolor/metabolismo , Triptofano/análogos & derivados , 5-Hidroxitriptofano/farmacologia , Antibacterianos/biossíntese , Cálcio/metabolismo , Estrutura Molecular , Biossíntese de Peptídeos Independentes de Ácido Nucleico/efeitos dos fármacos , Peptídeos/química , Engenharia de Proteínas/métodos , Streptomyces coelicolor/efeitos dos fármacos , Triptofano/metabolismo , Triptofano/farmacologiaRESUMO
The acidic lipopeptides, including the calcium-dependent antibiotics (CDA), daptomycin, and A54145, are important macrocyclic peptide natural products produced by Streptomyces species. All three compounds contain a 3-methyl glutamate (3-MeGlu) as the penultimate C-terminal residue, which is important for bioactivity. Here, biochemical in vitro reconstitution of the 3-MeGlu biosynthetic pathway is presented, using exclusively enzymes from the CDA producer Streptomyces coelicolor. It is shown that the predicted 3-MeGlu methyltransferase GlmT and its homologues DptI from the daptomycin producer Streptomyces roseosporus and LptI from the A54145 producer Streptomyces fradiae do not methylate free glutamic acid, PCP-bound glutamate, or Glu-containing CDA in vitro. Instead, GlmT, DptI, and LptI are S-adenosyl methionine (SAM)-dependent alpha-ketoglutarate methyltransferases that catalyze the stereospecific methylation of alpha-ketoglutarate (alphaKG) leading to (3R)-3-methyl-2-oxoglutarate. Subsequent enzyme screening identified the branched chain amino acid transaminase IlvE (SCO5523) as an efficient catalyst for the transformation of (3R)-3-methyl-2-oxoglutarate into (2S,3R)-3-MeGlu. Comparison of reversed-phase HPLC retention time of dabsylated 3-MeGlu generated by the coupled enzymatic reaction with dabsylated synthetic standards confirmed complete stereocontrol during enzymatic catalysis. This stereospecific two-step conversion of alphaKG to (2S,3R)-3-MeGlu completes our understanding of the biosynthesis and incorporation of beta-methylated amino acids into the nonribosomal lipopeptides. Finally, understanding this pathway may provide new possibilities for the production of modified peptides in engineered microbes.
Assuntos
Ácido Glutâmico/análogos & derivados , Ácidos Cetoglutáricos/metabolismo , Lipoproteínas/biossíntese , Metiltransferases/metabolismo , Streptomyces coelicolor/metabolismo , Aminação , Escherichia coli/enzimologia , Escherichia coli/genética , Ácido Glutâmico/química , Ácido Glutâmico/metabolismo , Ácidos Cetoglutáricos/química , Cinética , Lipoproteínas/síntese química , Metilação , Metiltransferases/química , Metiltransferases/genética , Metiltransferases/isolamento & purificação , Estereoisomerismo , Streptomyces coelicolor/enzimologia , Streptomyces coelicolor/genética , Especificidade por SubstratoRESUMO
[reaction: see text] Hydrogen atoms are abstracted from the C2' and C3'-pro-S positions of an (S)-tryptophanyl precursor, with overall syn stereochemistry, during the biosynthesis of the C-terminal Z-2',3'-dehydrotryptophan residue of the calcium-dependent lipopeptide antibiotics (CDAs) in Streptomyces coelicolor. The absence of beta-hydroxytryptophanyl, or other possible intermediates, further suggests a direct dehydrogenation mechanism similar to that proposed for the l-tryptophan 2',3'-oxidase from Chromobacterium violaceum.