RESUMEN
XIAP, a member of the inhibitor of apoptosis family of proteins, is a critical regulator of apoptosis. Inhibition of the BIR domain-caspase interaction is a promising approach towards treating cancer. Previous work has been directed towards inhibiting the BIR3-caspase-9 interaction, which blocks the intrinsic apoptotic pathway; selectively inhibiting the BIR2-caspase-3 interaction would also block the extrinsic pathway. The BIR2 domain of XIAP has successfully been crystallized; peptides and small-molecule inhibitors can be soaked into these crystals, which diffract to high resolution. Here, the BIR2 apo crystal structure and the structures of five BIR2-tetrapeptide complexes are described. The structural flexibility observed on comparing these structures, along with a comparison with XIAP BIR3, affords an understanding of the structural elements that drive selectivity between BIR2 and BIR3 and which can be used to design BIR2-selective inhibitors.
Asunto(s)
Caspasa 3/química , Caspasa 3/metabolismo , Inhibidores de Caspasas/química , Proteínas Inhibidoras de la Apoptosis/química , Nucleopoliedrovirus/química , Proteínas Virales/química , Proteína Inhibidora de la Apoptosis Ligada a X/química , Secuencia de Aminoácidos , Apoproteínas/química , Apoproteínas/genética , Apoptosis/genética , Cristalografía por Rayos X , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Datos de Secuencia Molecular , Familia de Multigenes/genética , Nucleopoliedrovirus/genética , Oligopéptidos/química , Oligopéptidos/genética , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína/genética , Proteínas Virales/genética , Proteína Inhibidora de la Apoptosis Ligada a X/genéticaRESUMEN
Recombinant human serum albumin (HSA) conjugates of a 15-amino-acid truncated peptide YY (PYY) analogue were prepared using three heterobifunctional linkers [succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC), 6-maleimidohexanoic acid N-hydroxysuccinimide ester (MHS), and N-[γ-maleimidobutyryloxy]sulfosuccinimide ester (GMBS)] in 2 synthetic steps involving (1) reaction of succinimidyl ester on linker with ε-amine of Lys2 on the peptide and (2) reaction of maleimide on peptide linker with free thiol of Cysteine 34 (Cys34) on albumin. In-process controls using ESI LC-MS were used to follow reactions and identify reaction products. Proteolytic digests of the conjugate revealed that peptide conjugation occurs at Cys34 on HSA. Conjugates were assayed in cell-based assays to determine potency at the human Y2-receptor, and selectivity at the human Y1-, Y4-, and Y5-receptors using a calcium flux assay. All three conjugates assayed were selective agonists of the Y2-receptor, and displayed nanomolar potencies. MCC and MH conjugates were selected for acute PK/PD studies in DIO mice. Significant reduction in food intake was observed with the MH conjugate, which lasted for 24 h at the 10 mg (or 4 µmol)/kg dose. While the MCC conjugate exhibited greater potency in vitro, it was slightly less effective than the MH conjugate in vivo with respect to reduction in food intake. Both conjugates were significantly less active than the peptide coupled to a 30 kDa PEG. The observed T1/2 (8-9 h) for both conjugates was significantly lower than that observed for the PEGylated peptide (â¼25 h). These results suggest that, as compared with the unmodified and PEGylated peptide, the extended circulation half-life of albumin conjugates is mediated through uptake and recirculation by FcRn, and allometric scaling methods are necessary to account for interspecies variation in pharmacokinetic properties.
Asunto(s)
Péptido YY/metabolismo , Albúmina Sérica/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Ingestión de Alimentos/efectos de los fármacos , Semivida , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Datos de Secuencia Molecular , Peso Molecular , Péptido YY/química , Unión Proteica , Conformación Proteica , Albúmina Sérica/química , Albúmina Sérica/farmacología , Especificidad por SustratoRESUMEN
The EphA4 receptor tyrosine kinase plays a role in neurodegenerative diseases, inhibition of nerve regeneration, cancer progression and other diseases. Therefore, EphA4 inhibition has potential therapeutic value. Selective EphA4 kinase inhibitors are not available, but we identified peptide antagonists that inhibit ephrin ligand binding to EphA4 with high specificity. One of these peptides is the cyclic APY-d3 (ßAPYCVYRßASWSC-NH2), which inhibits ephrin-A5 ligand binding to EphA4 with low nanomolar binding affinity and is highly protease resistant. Here we describe modifications of APY-d3 that yield two different key derivatives with greatly increased half-lives in the mouse circulation, the lipidated APY-d3-laur8 and the PEGylated APY-d3-PEG4. These two derivatives inhibit ligand induced EphA4 activation in cells with sub-micromolar potency. Since they retain high potency and specificity for EphA4, lipidated and PEGylated APY-d3 derivatives represent new tools for discriminating EphA4 activities in vivo and for preclinical testing of EphA4 inhibition in animal disease models.
Asunto(s)
Efrina-A5 , Receptor EphA4 , Ratones , Animales , Receptor EphA4/metabolismo , Ligandos , Semivida , Efrina-A5/metabolismo , PolietilenglicolesRESUMEN
Nuclear magnetic resonance (NMR) methods were employed to study the interaction of the cytokine Interleukin-2 (IL-2) with the alpha-subunit of its receptor (IL-2Ralpha), and to help understand the behavior of small molecule inhibitors of this interaction. Heteronuclear (1)H-(15)N HSQC experiments were used to identify the interaction surface of (15)N-enriched Interleukin-2 ((15)N-IL-2) in complex with human IL-2Ralpha. In these experiments, chemical shift and line width changes in the heteronuclear single-quantum coherence (HSQC) spectra upon binding of (15)N-IL-2 enabled classification of NH atoms as either near to, or far from, the IL-2Ralpha interaction surface. These data were complemented by hydrogen/deuterium (H/D) exchange measurements, which illustrated enhanced protection of slowly-exchanging IL-2 NH protons near the site of interaction with IL-2Ralpha. The interaction surface defined by NMR compared well with the IL-2Ralpha binding site identified previously using mutagenesis of human and murine IL-2. Two low molecular weight inhibitors of the IL-2/IL-2Ralpha interaction were studied: one (a cyclic peptide derivative) was found to mimic a part of the cytokine and bind to IL-2Ralpha; the other (an acylphenylalanine derivative) was found to bind to IL-2. For the interaction between IL-2 and the acylphenylalanine, chemical shift perturbations of (15)N and (15)NH backbone resonances were tracked as a function of ligand concentration. The perturbation pattern observed for this complex revealed that the acylphenylalanine is a competitive inhibitor-it binds to the same site on IL-2 that interacts with IL-2Ralpha.
Asunto(s)
Interleucina-2/metabolismo , Receptores de Interleucina/metabolismo , Subunidad alfa del Receptor de Interleucina-2 , Ligandos , Espectroscopía de Resonancia Magnética , Pichia/metabolismoRESUMEN
The phosphatase of regenerating liver (PRL) family, a unique class of oncogenic phosphatases, consists of three members: PRL-1, PRL-2, and PRL-3. Aberrant overexpression of PRL-3 has been found in multiple solid tumor types. Ectopic expression of PRLs in cells induces transformation, increases mobility and invasiveness, and forms experimental metastases in mice. We have now shown that small interfering RNA-mediated depletion of PRL expression in cancer cells results in the down-regulation of p130Cas phosphorylation and expression and prevents tumor cell anchorage-independent growth in soft agar. We have also identified a small molecule, 7-amino-2-phenyl-5H-thieno[3,2-c]pyridin-4-one (thienopyridone), which potently and selectively inhibits all three PRLs but not other phosphatases in vitro. The thienopyridone showed significant inhibition of tumor cell anchorage-independent growth in soft agar, induction of the p130Cas cleavage, and anoikis, a type of apoptosis that can be induced by anticancer agents via disruption of cell-matrix interaction. Unlike etoposide, thienopyridone-induced p130Cas cleavage and apoptosis were not associated with increased levels of p53 and phospho-p53 (Ser(15)), a hallmark of genotoxic drug-induced p53 pathway activation. This is the first report of a potent selective PRL inhibitor that suppresses tumor cell three-dimensional growth by a novel mechanism involving p130Cas cleavage. This study reveals a new insight into the role of PRL-3 in priming tumor progression and shows that PRL may represent an attractive target for therapeutic intervention in cancer.
Asunto(s)
Proteína Sustrato Asociada a CrK/metabolismo , Inhibidores Enzimáticos/farmacología , Proteínas de Neoplasias/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Anoicis/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Células Endoteliales/efectos de los fármacos , Células HT29 , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas de Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Proteínas Tirosina Fosfatasas/genética , Piridinas/farmacología , ARN Interferente Pequeño/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Two libraries of hMC4R agonists, X-Y-DPhe(7)-Arg(8)-2-Nal(9)-Z-NH(2) and X-Y-DPhe(7)-Arg(8)-Trp(9)-Z-NH(2), totaling 185 peptides were prepared using Irori radiofrequency tagging technology and Argonaut Quest 210 Synthesizer, where X stands for N-caps, Y for His(6) surrogates and Z for Gly(10) surrogates. As a result of this study, His-modified pentapeptides with Trp were found to be more hMC4R potent than the corresponding 2-Nal analogs, novel N-caps and Gly surrogates were identified and 19 new peptides which are potent hMC4R agonists (EC(50) 1-15nM) and selective against hMC1R were discovered.
Asunto(s)
Oligopéptidos/síntesis química , Biblioteca de Péptidos , Receptor de Melanocortina Tipo 4/agonistas , Secuencia de Aminoácidos , Glicina , Humanos , Oligopéptidos/química , Oligopéptidos/farmacología , Relación Estructura-ActividadRESUMEN
Linear pentapeptides (Penta-cis-Apc-DPhe-Arg-Trp-Gly-NH2) containing 1-amino-4-phenylcyclohexane-1-carboxylic acid (cis-Apc) and substituted Apc are potent hMC4R agonists and they are inactive or weakly active in hMC1R, hMC3R, and hMC5R agonist assays. This study, together with our earlier report on 5-BrAtc, demonstrated the importance of replacing His6 with phenyl-containing rigid templates in achieving good hMC4R agonist potency and selectivity against hMC1R in linear pentapeptides.
Asunto(s)
Ácidos Carboxílicos/química , Ácidos Carboxílicos/farmacología , Ciclohexanos/química , Ciclohexanos/farmacología , Receptor de Melanocortina Tipo 1/agonistas , Receptor de Melanocortina Tipo 4/agonistas , Secuencia de Aminoácidos , Humanos , Concentración 50 Inhibidora , Estructura Molecular , Sensibilidad y Especificidad , Especificidad por SustratoRESUMEN
A series of pentapeptides, based on Bu-His(6)-DPhe(7)-Arg(8)-Trp(9)-Gly(10)-NH(2) and modified at the Arg(8) position, was prepared and pharmacologically characterized. Peptides containing either cyanoguanidine or acylguanidine, two substantially less basic arginine surrogates, were found to retain the agonist activity of the parent peptide at both hMC1R and hMC4R. This study unequivocally shows that the positive charge of Arg(8) is not essential for efficient interactions of our pentapeptide with both hMC1R and hMC4R.
Asunto(s)
Oligopéptidos/síntesis química , Receptores de Corticotropina/agonistas , Sustitución de Aminoácidos , Arginina , Sitios de Unión , Guanidinas , Humanos , Oligopéptidos/farmacología , Unión Proteica , Receptor de Melanocortina Tipo 4 , Receptores de Melanocortina , Relación Estructura-ActividadRESUMEN
Using nuclear magnetic resonance (NMR) spectroscopy, we have determined the solution structures for a series of potent agonists for the human melanocortin-4 receptor (hMC4R), based on the cyclic peptide MT-II [Ac-Nle-cyclo-(Asp-Lys) (Asp-His-(D)Phe-Arg-Trp-Lys)-NH2]. Members of this series were designed to improve selectivity for MC4R versus the other melanocortin receptors, and to reduce the flexibility of the side chains. The most selective and rigid analog [penta-cyclo(D-K)-Asp-Apc-(D)Phe-Arg-(2S,3S)-beta-methylTrp-Lys-NH2] was found to be a full agonist of hMC4R with an EC50 of 11nM against hMC4R, and to exhibit 65-fold selectivity against hMC1R. This compound represents the most constrained hMC4R peptide agonist described to date. A beta-turn structure was conserved among all of the cyclic peptides studied. The rigidity of the analogs allowed an exceptionally well-defined pharmacophore model to be derived. This model was used to perform a virtual screen using a library of 1000 drug-like compounds, to which a small set of known potent ligands had been intentionally added. The utility of the model was validated by its ability to identify the known ligands from among this large library.
Asunto(s)
Modelos Moleculares , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Receptor de Melanocortina Tipo 4/agonistas , Humanos , Espectroscopía de Resonancia Magnética , Conformación Molecular , Estructura Molecular , Péptidos Cíclicos/síntesis química , Unión Proteica , Soluciones , Relación Estructura-ActividadRESUMEN
Systematic substitution of His(6) residue using non-selective hMC4R pentapeptide agonist (Bu-His(6)-DPhe(7)-Arg(8)-Trp(9)-Gly(10)-NH(2)) as the template led to the identification of Bu-Atc(6)(2-aminotetraline-2-carboxylic acid)-DPhe(7)-Arg(8)-Trp(9)-Gly(10)-NH(2) which showed moderate selectivity towards hMC4R over hMC1R. Further SAR studies resulted in the discovery of Penta-5-BrAtc(6)-DPhe(7)-Arg(8)-Trp(9)-Gly(10)-NH(2) and Penta-5-Me(2)NAtc(6)-DPhe(7)-Arg(8)-Trp(9)-Gly(10)-NH(2) which are potent hMC4R agonists and are inactive in hMC1R, hMC3R and hMC5R agonist assays.
Asunto(s)
Oligopéptidos/síntesis química , Receptores de Corticotropina/agonistas , Secuencia de Aminoácidos , Sustitución de Aminoácidos , AMP Cíclico/biosíntesis , Histidina , Humanos , Oligopéptidos/metabolismo , Oligopéptidos/farmacología , Unión Proteica , Receptor de Melanocortina Tipo 4 , Receptores de Corticotropina/metabolismo , Receptores de Melanocortina , Relación Estructura-ActividadRESUMEN
A series of MT-II related cyclic peptides, based on potent but non-selective hMC4R agonist (Penta-c[Asp-His(6)-DPhe(7)-Arg(8)-Trp(9)-Lys]-NH(2)) was prepared in which His(6) residue was systematically substituted. Two of the most interesting peptides identified in this study are Penta-c[Asp-5-ClAtc-DPhe-Arg-Trp-Lys]-NH(2) and Penta-c[Asp-5-ClAtc-DPhe-Cit-Trp-Lys]-NH(2) which are potent hMC4R agonists and are either inactive or weak partial agonists (not tested for their antagonist activities) in hMC1R, hMC3R and hMC5R agonist assays.
Asunto(s)
Histidina/química , Receptores de Corticotropina/agonistas , Sustitución de Aminoácidos , Supervivencia Celular/efectos de los fármacos , Humanos , Espectroscopía de Resonancia Magnética , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/farmacología , Receptor de Melanocortina Tipo 4 , Receptores de Melanocortina , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
A series of pentapeptides, based on hMC4R pentapeptide agonist (Bu-His(6)-DPhe(7)-Arg(8)-Trp(9)-Gly(10)-NH(2)), was prepared in which either DPhe(7) or Trp(9) residue was systematically substituted. A number of interesting DPhe surrogates (D-Thi, D-3-CF(3)Phe, D-2-Nal and D-3,4-diClPhe) as well as Trp surrogates (2-Nal and Bta) were identified in this study.