RESUMEN
Starting from a tripeptide screening hit, a series of dipeptide inhibitors of the proteasome with Thr as the P3 residue has been optimized with the aid of crystal structures in complex with the ß-5/6 active site of y20S. Derivative 25, (ß5 IC(50)=7.4 nM) inhibits only the chymotryptic activity of the proteasome, shows cellular activity against targets in the UPS, and inhibits proliferation.
Asunto(s)
Quimotripsina/antagonistas & inhibidores , Dipéptidos/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Treonina/química , Humanos , Modelos MolecularesRESUMEN
The mammalian 26S proteasome is a 2500 kDa multi-catalytic complex involved in intracellular protein degradation. We describe the synthesis and properties of a novel series of non-covalent di-peptide inhibitors of the proteasome based [corrected] on a capped tri-peptide that was first identified by high-throughput screening of a library of approx. 350000 compounds for inhibitors of the ubiquitin-proteasome system in cells. We show that these compounds are entirely selective for the beta5 (chymotrypsin-like) site over the beta1 (caspase-like) and beta2 (trypsin-like) sites of the 20S core particle of the proteasome, and over a panel of less closely related proteases. Compound optimization, guided by X-ray crystallography of the liganded 20S core particle, confirmed their non-covalent binding mode and provided a structural basis for their enhanced in vitro and cellular potencies. We demonstrate that such compounds show low nanomolar IC50 values for the human 20S beta5 site in vitro, and that pharmacological inhibition of this site in cells is sufficient to potently inhibit the degradation of a tetra-ubiquitin-luciferase reporter, activation of NFkappaB (nuclear factor kappaB) in response to TNF-alpha (tumour necrosis factor-alpha) and the proliferation of cancer cells. Finally, we identified capped di-peptides that show differential selectivity for the beta5 site of the constitutively expressed proteasome and immunoproteasome in vitro and in B-cell lymphomas. Collectively, these studies describe the synthesis, activity and binding mode of a new series of non-covalent proteasome inhibitors with unprecedented potency and selectivity for the beta5 site, and which can discriminate between the constitutive proteasome and immunoproteasome in vitro and in cells.
Asunto(s)
Oligopéptidos/farmacología , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasoma , Secuencia de Aminoácidos , Sitios de Unión , Ácidos Borónicos/farmacología , Bortezomib , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Células HCT116 , Células HT29 , Humanos , Cinética , Luciferasas/genética , Luciferasas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , FN-kappa B/genética , FN-kappa B/metabolismo , Oligopéptidos/química , Oligopéptidos/metabolismo , Inhibidores de Proteasas/química , Inhibidores de Proteasas/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Pirazinas/farmacología , Interferencia de ARN , Homología de Secuencia de Aminoácido , Ubiquitina/genética , Ubiquitina/metabolismoRESUMEN
The design, synthesis, and structure-activity relationship development of naphthalene-derived human CCR8 antagonists is described. In vitro binding assay results of these investigations are reported, critical interactions of the antagonists with CCR8 are defined, and preliminary physicochemical and pharmacokinetic data for the naphthalene scaffold are presented.
Asunto(s)
Naftalenos/síntesis química , Receptores de Quimiocina/antagonistas & inhibidores , Sulfonamidas/síntesis química , Administración Oral , Animales , Disponibilidad Biológica , Transporte Biológico , Calcio/metabolismo , Línea Celular , Cricetinae , Cricetulus , Diseño de Fármacos , Humanos , Naftalenos/farmacocinética , Naftalenos/farmacología , Ratas , Receptores CCR8 , Solubilidad , Estereoisomerismo , Relación Estructura-Actividad , Sulfonamidas/farmacocinética , Sulfonamidas/farmacologíaRESUMEN
N-Substituted cis-4a-(3-hydroxyphenyl)-8a-methyloctahydroisoquinolines (6a-g) were designed and synthesized as conformationally constrained analogues of the trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidine (4) class of opioid receptor pure antagonists. The methyloctahydroisoquinolines 6a-g can exist in conformations where the 3-hydroxyphenyl substituent is either axial or equatorial, similar to the (3-hydroxyphenyl)piperidines 4. The 3-hydroxyphenyl equatorial conformation is responsible for the antagonist activity observed in the (3-hydroxyphenyl)piperidine antagonists. Single-crystal X-ray analysis of 6a shows that the 3-hydroxyphenyl equatorial conformation is favored in the solid state. Molecular modeling studies also suggest that the equatorial conformation has lower potential energy relative to that of the axial conformation. Evaluation of 6a-g in the [(35)S]GTP-gamma-S in vitro functional assay showed that they were opioid receptor pure antagonists. N-[4a-(3-Hydroxyphenyl)-8a-methyl-2-(3-phenylpropyl)octahydroisoquinoline-6-yl]-3-(piperidin-1-yl)propionamide (6d) with a K(e) of 0.27 nM at the kappa opioid receptor with 154- and 46-fold selectivity relative to those of the micro and delta receptors, respectively, possessed the best combination of kappa potency and selectivity.
Asunto(s)
Isoquinolinas/farmacología , Antagonistas de Narcóticos , Animales , Bencenoacetamidas/farmacología , Células CHO , Cricetinae , Encefalina Ala(2)-MeFe(4)-Gli(5)/farmacología , Encefalina D-Penicilamina (2,5)/farmacología , Guanosina 5'-O-(3-Tiotrifosfato)/antagonistas & inhibidores , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Isoquinolinas/síntesis química , Modelos Moleculares , Conformación Molecular , Pirrolidinas/farmacología , Receptores Opioides/metabolismo , Receptores Opioides delta/antagonistas & inhibidores , Receptores Opioides kappa/antagonistas & inhibidores , Receptores Opioides kappa/metabolismo , Receptores Opioides mu/antagonistas & inhibidoresRESUMEN
A library of compounds biased toward opioid receptor antagonist activity was prepared by incorporating N-phenylpropyl-4beta-methyl-5-(3-hydroxyphenyl)morphans as the core scaffold using simultaneous solution phase synthetic methodology. From this library, N-phenylpropyl-4beta-methyl-5-(3-hydroxyphenyl)-7alpha-[3-(1-piperidinyl)propanamido]morphan [(-)-3b] was identified as the first potent and selective kappa opioid receptor antagonist from the 5-phenylmorphan class of opioids.