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1.
Bioorg Med Chem ; 22(13): 3309-15, 2014 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-24842619

RESUMEN

Conventional chemotherapy has undesirable toxic side-effects to healthy tissues due to low cell selectivity of cytotoxic drugs. One approach to increase the specificity of a cytotoxic drug is to make a less toxic prodrug which becomes activated at the tumour site. The cysteine protease legumain have remarkable restricted substrate specificity and is the only known mammalian asparaginyl (Asn) endopeptidase. Over-expression of legumain is reported in cancers and unstable atherosclerotic plaques, and utilizing legumain is a promising approach to activate prodrugs. In this study we have synthesized the legumain-cleavable peptide sequence N-Boc-Ala-Ala-Asn-Val-OH. The peptide was subsequently conjugated to deacetyl colchicine during three steps to produce Suc-Ala-Ala-Asn-Val-colchicine (prodrug) with >90% chemical purity. Several cell lines with different expressions and activities of legumain were used to evaluate the general toxicity, specificity and efficacy of the microtubule inhibitor colchicine, valyl colchicine and the legumain-cleavable colchicine prodrug. The prodrug was more toxic to the colorectal cancer HCT116 cells (expressing both the 36kDa active and 56kDa proform of legumain) than SW620 cells (only expressing the 56kDa prolegumain) indicating a relationship between toxicity of the prodrug and activity of legumain in the cells. Also, in monoclonal legumain over-expressing HEK293 cells the prodrug toxicity was higher compared to native HEK293 cells. Furthermore, co-administration of the prodrug either with the potent legumain inhibitor cystatin E/M or the endocytosis inhibitor Dyngo-4a inhibited cell death, indicating that the prodrug toxicity was dependent on both asparaginyl endopeptidase activity and endocytosis. This colchicine prodrug adds to a legumain-activated prodrug strategy approach and could possibly be of use both in targeted anticancer and anti-inflammatory therapy.


Asunto(s)
Colchicina/farmacología , Cisteína Endopeptidasas/química , Profármacos/farmacología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Colchicina/síntesis química , Colchicina/química , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Células HCT116 , Células HEK293 , Humanos , Estructura Molecular , Profármacos/síntesis química , Profármacos/química , Relación Estructura-Actividad , Células Tumorales Cultivadas
2.
Acta Radiol ; 51(9): 1014-20, 2010 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-20849319

RESUMEN

BACKGROUND: Contrast-induced nephrotoxicity is a significant risk when using radiographic contrast media clinically, especially in high risk patients. Consequently, there is a need for a new contrast agent with improved clinical safety with regards to nephrotoxicity. PURPOSE: To evaluate the physicochemical properties as well as the preclinical safety and biodistribution parameters of the newly developed radiographic contrast medium GE-145. MATERIAL AND METHODS: Standard methods for radiographic contrast media were used for evaluation of physicochemical properties. The acute toxicity in rats was studied at 8, 10, and 12.5 gI/kg, the clinical chemistry parameters were determined, and histology of the kidneys was performed. Biodistribution was studied in rats using ¹²³I-labeled GE-145. RESULTS: GE-145 is more hydrophilic than iodixanol and has a considerably lower osmolality. The viscosity is similar to that of iodixanol and the protein binding is low. The acute toxicity is similar to that of iodixanol and the biodistribution is similar to that of other radiographic contrast media, showing mainly renal excretion. Kidney histology showed a moderate reversible vacuolization, similar to that of iodixanol. CONCLUSION: GE-145 exhibits similar preclinical properties to other dimeric radiographic contrast media. In addition, the low osmolality enables an iso-osmolar formulation containing a significantly higher concentration of electrolytes than Visipaque.


Asunto(s)
Medios de Contraste/toxicidad , Formamidas/toxicidad , Riñón/efectos de los fármacos , Ácidos Triyodobenzoicos/toxicidad , Análisis de Varianza , Animales , Medios de Contraste/química , Formamidas/administración & dosificación , Concentración Osmolar , Unión Proteica , Ratas , Ratas Wistar , Distribución Tisular , Ácidos Triyodobenzoicos/administración & dosificación , Ácidos Triyodobenzoicos/química , Viscosidad
3.
Rapid Commun Mass Spectrom ; 19(24): 3633-42, 2005.
Artículo en Inglés | MEDLINE | ID: mdl-16285022

RESUMEN

We have recently described the discovery of the Maltacines--a new family of cyclic peptide antibiotics from Bacillus subtilis. In this paper the mass spectrometric characterisation of two of the members is reported. A chemoselective ring opening with base to give the linear peptides was necessary before mass spectrometric characterisation could be performed. MS(n) of the singly and doubly charged protonated molecules gave uninterrupted series of Bn and Y''n ions that allowed determination of the amino acid sequence. By using a combination of derivatisation with phenylisothiocyanate (PITC), high-resolution mass spectrometry and H/D exchange, the identities of three unknown residues were determined. The nature and position of the cyclic structure were disclosed by a chemoselective ring opening with Na 18OH and it was found to be a lactone formed between a hydroxyl of residue number 4 and the C-terminal amino acid number 12. Peptides with different combinations of P/Q and P/K at the N-terminus were synthesised to verify the sequence of the N-terminal B2 ion. The structure of the two peptides is proposed to be: E1a: cyclo-4,12(P-Q-Y-Adip-V-E-T-Y-Orn-S-Y-I-OH) and E1b: cyclo-4,12(P-Q-Y-Adip-V-E-T-Y-K-S-Y-I-OH). Adip = (2-amino-4,5-dihydroxypentanoic acid).


Asunto(s)
Antibacterianos/química , Péptidos Cíclicos/química , Secuencia de Aminoácidos , Antibacterianos/farmacología , Bacillus subtilis/química , Espectrometría de Masas , Péptidos Cíclicos/farmacología
4.
J Mass Spectrom ; 40(10): 1276-86, 2005 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-16177955

RESUMEN

A new complex of cyclic peptide lactone antibiotics from Bacillus subtilis, which we named Maltacines, has recently been described. The structure elucidation of four of them is reported in this paper. The amino acid sequences and structures of the peptides were found by MS(n) of the ring-opened linear peptides, which gave uninterrupted sequences of Bn and Y''n ions. The identities of three unknown residues in the sequences were solved by a combination of derivatisation with phenylisothiocyanate (PITC), high-resolution mass spectrometry and H/D exchange. The nature and position of the cyclic structure was disclosed by a chemo-selective ring opening with Na18OH and was found to be a lactone formed between a hydroxyl of residue number 4 and the C-terminal amino acid number 12. For verification of the structure of the B2+ ion, peptides with different combinations of P/Q and P/K at the N-terminus were synthesised. The structure of the four peptides were found to be: C1a and C2a: cyclo-4,12(P-Q-Y-Adip-V-E-T-Y-Orn-103-Y-I-OH) and C1b/C2b: cyclo-4,12(P-Q-Y-Adip-V-E-T-Y-K-103-Y-I-OH). Adip = aminodihydroxy pentanoic acid.


Asunto(s)
Bacillus subtilis/química , Lactonas/química , Péptidos Cíclicos/química , Secuencia de Aminoácidos , Bacillus subtilis/metabolismo , Microquímica/métodos , Datos de Secuencia Molecular , Estructura Molecular , Nanotecnología , Espectrometría de Masa por Ionización de Electrospray/métodos
5.
J Mass Spectrom ; 40(10): 1287-99, 2005 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-16178056

RESUMEN

A new complex of cyclic peptide lactone antibiotics from Bacillus subtilis, which we named Maltacines has recently been described. The structure elucidation of three of them is reported in this paper. The amino acid sequences and structures of the peptides were found by MS(n) of the ring-opened linear peptides that gave uninterrupted sequences of Bn and Y''n ions. The identities of four unknown residues in the sequences were solved by a combination of derivatisation with phenylisothiocyanate (PITC), high-resolution mass spectrometry and H/D exchange. The nature and position of the cyclic structure was disclosed by a chemo-selective ring opening with Na18OH and was found to be a lactone formed between a hydroxyl of residue number 4 and the C-terminal amino acid number 12. For verification of the structure of the B2 + ion, peptides with different combinations of P/Q and P/K at the N-terminus were synthesized. The structures of the four peptides is tentatively suggested to be: D1a: cyclo(4,12)-P-Q-Y-Adip-A-E-T-Y-Orn-HGly-Y-I-OH, D1b: cyclo(4,12)-P-Q-Y-Adip-A-E-T-Y-Orn-S-Y-I-OH and D1c: cyclo(4,12)-P-Q-Y-Adip-A-E-T-Y-K-S-Y-I-OH. Adip = aminodihydroxy pentanoic acid and HGly = hydroxyglycine.


Asunto(s)
Bacillus subtilis/química , Lactonas/química , Péptidos Cíclicos/química , Secuencia de Aminoácidos , Bacillus subtilis/metabolismo , Microquímica/métodos , Datos de Secuencia Molecular , Estructura Molecular , Nanotecnología , Espectrometría de Masa por Ionización de Electrospray/métodos
6.
J Mass Spectrom ; 40(4): 527-38, 2005 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-15724270

RESUMEN

A new complex of cyclic peptide lactone antibiotics from Bacillus subtilis, which we named maltacines, has recently been described. The structure elucidation of four of them is reported in this paper. The amino acid sequences and structures of the peptides were found by MSn of the ring-opened linear peptides that gave uninterrupted sequences of Bn and Y''n ions. The identities of three unknown residues in the sequences were solved by a combination of derivatization with phenyl isothiocyanate (PITC), high-resolution mass spectrometry and H/D exchange. The nature and position of the cyclic structure were revealed by a chemoselective ring opening with Na18OH and was found to be a lactone formed between a hydroxyl of residue number 4 and the C-terminal amino acid number 12. For verification of the structure of the B2+ ion, peptides with different combinations of P/Q and P/K at the N-terminus were synthesized. The structures of the four peptides were found to be as follows: B1a/B2a, cyclo-4,12(P-Q-Y-HNLeu-A-E-T-Y-Orn-103-Y-I-OH); and B1b/B2b, cyclo-4,12(P-Q-Y-HNLeu-A-E-T-Y-K-103-Y-I-OH).


Asunto(s)
Bacillus subtilis/química , Lactonas/química , Péptidos Cíclicos/química , Secuencia de Aminoácidos , Medición de Intercambio de Deuterio , Espectrometría de Masas , Datos de Secuencia Molecular , Estructura Molecular
7.
Artículo en Inglés | MEDLINE | ID: mdl-15522727

RESUMEN

Purification of secondary metabolites from fermentation broths can be a challenging task both due to the complexity of the medium, inherently unstable molecular structures or by the action of enzymes present in the fermentation broth leading to poor isolation yield and loss of antibiotic activity. A combination of different purification techniques has usually been used to arrive at acceptable purities for characterisation of the target molecules. Due to rapid decay of antimicrobial activity a rapid preparative high-performance liquid chromatography (HPLC) method was developed that provided separation and resolution of a family of 18 closely related cyclic peptides within 110 min with minimal loss of activity. Characterisation of the peptides with LC-MS, UV/IR spectroscopy and amino acid analysis disclosed 20 different peptides with cyclic structures (lactones) with molecular weights between 1447.7 and 1519.8 Da. No peptide antibiotics with identical molecular weights have previously been reported in the literature, which lead us to conclude that this peptide complex has not been discovered before. We have named them Maltacines.


Asunto(s)
Antibacterianos/análisis , Bacillus subtilis/química , Cromatografía Líquida de Alta Presión/métodos , Péptidos , Espectrometría de Masas , Peso Molecular , Espectrofotometría Infrarroja , Espectrofotometría Ultravioleta
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