RESUMEN
Protein tyrosine phosphatase 1B (PTP1B) attenuates insulin signaling by catalyzing dephosphorylation of insulin receptors (IR) and is an attractive target of potential new drugs for treating the insulin resistance that is central to type II diabetes. Several analogues of cholecystokinin(26)(-)(33) (CCK-8) were found to be surprisingly potent inhibitors of PTP1B, and a common N-terminal tripeptide, N-acetyl-Asp-Tyr(SO(3)H)-Nle-, was shown to be necessary and sufficient for inhibition. This tripeptide was modified to reduce size and peptide character, and to replace the metabolically unstable sulfotyrosyl group. This led to the discovery of a novel phosphotyrosine bioisostere, 2-carboxymethoxybenzoic acid, and to analogues that were >100-fold more potent than the CCK-8 analogues and >10-fold selective for PTP1B over two other PTP enzymes (LAR and SHP-2), a dual specificity phosphatase (cdc25b), and a serine/threonine phosphatase (calcineurin). These inhibitors disrupted the binding of PTP1B to activated IR in vitro and prevented the loss of tyrosine kinase (IRTK) activity that accompanied PTP1B-catalyzed dephosphorylation of IR. Introduction of these poorly cell permeant inhibitors into insulin-treated cells by microinjection (oocytes) or by esterification to more lipophilic proinhibitors (3T3-L1 adipocytes and L6 myocytes) resulted in increased potency, but not efficacy, of insulin. In some instances, PTP1B inhibitors were insulin-mimetic, suggesting that in unstimulated cells PTP1B may suppress basal IRTK activity. X-ray crystallography of PTP1B-inhibitor complexes revealed that binding of an inhibitor incorporating phenyl-O-malonic acid as a phosphotyrosine bioisostere occurred with the mobile WPD loop in the open conformation, while a closely related inhibitor with a 2-carboxymethoxybenzoic acid bioisostere bound with the WPD loop closed, perhaps accounting for its superior potency. These CCK-derived peptidomimetic inhibitors of PTP1B represent a novel template for further development of potent, selective inhibitors, and their cell activity further justifies the selection of PTP1B as a therapeutic target.
Asunto(s)
Inhibidores Enzimáticos/química , Insulina/farmacología , Imitación Molecular , Péptidos/química , Fosfotirosina/química , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Células 3T3 , Secuencia de Aminoácidos , Animales , Unión Competitiva , Células CHO , Células CACO-2 , Cricetinae , Cristalografía por Rayos X , Sinergismo Farmacológico , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Isomerismo , Ratones , Datos de Secuencia Molecular , Péptidos/metabolismo , Péptidos/farmacología , Fosfotirosina/metabolismo , Unión Proteica , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , Proteínas Tirosina Fosfatasas/metabolismo , Ratas , Sincalida/análogos & derivados , Sincalida/química , Sincalida/metabolismo , Sincalida/farmacología , Soluciones , Xenopus laevisRESUMEN
One strategy to treat the insulin resistance that is central to type II diabetes mellitus may be to maintain insulin receptors (IR) in the active (tyrosine phosphorylated) form. Because protein tyrosine phosphatase 1B (PTP1B) binds and subsequently dephosphorylates IR, inhibitors of PTP1B-IR binding are potential insulin 'sensitizers.' A Scintillation Proximity Assay (SPA) was developed to characterize and quantitate PTP1B-IR binding. Human IR were solubilized and captured on wheat germ agglutinin (WGA)-coated SPA beads. Subsequent binding of human, catalytically inactive [35S] PTP1B Cys(215)/Ser (PTP1B(C215S)) to the lectin-anchored IR results in scintillation from the SPA beads that can be quantitated. Binding of PTP1B to IR was pH- and divalent cation-sensitive. Ca(2+) and Mn(2+), but not Mg(2+), dramatically attenuated the loss of PTP1B-IR binding observed when pH was raised from 6.2 to 7.8. PTP1B binding to IR from insulin-stimulated cells was much greater than to IR from unstimulated cells and was inhibited by either an antiphosphotyrosine antibody or treatment of IR with alkaline phosphatase, suggesting that tyrosine phosphorylation of IR is required for PTP1B binding. Phosphopeptides modeled after various IR phosphotyrosine domains each only partially inhibited PTP1B-IR binding, indicating that multiple domains of IR are likely involved in binding PTP1B. However, competitive displacement of [35S]PTP1B(C215S) by PTP1B(C215S) fitted best to a single binding site with a K(d) in the range 100-1000 nM, depending upon pH and divalent cations. PNU-200898, a potent and selective inhibitor of PTP1B whose orientation in the active site of PTP1B has been solved, competitively inhibited catalysis and PTP1B-IR binding with equal potency. The results of this novel assay for PTP1B-IR binding suggest that PTP1B binds preferentially to tyrosine phosphorylated IR through its active site and that binding may be susceptible to therapeutic disruption by small molecules.
Asunto(s)
Proteínas Tirosina Fosfatasas/metabolismo , Receptor de Insulina/metabolismo , Secuencia de Aminoácidos , Animales , Unión Competitiva/efectos de los fármacos , Células CHO , Dominio Catalítico/efectos de los fármacos , Cationes Bivalentes/farmacología , Cricetinae , Humanos , Concentración de Iones de Hidrógeno , Cinética , Datos de Secuencia Molecular , Unión Proteica/efectos de los fármacos , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas/química , Receptor de Insulina/química , Vanadatos/farmacologíaRESUMEN
Previous work has shown that the study of host immune responses against Mycobacterium tuberculosis, the causative agent of tuberculosis, requires the availability of multiple mycobacterial antigens. Since purification of protein from M. tuberculosis cells is extremely cumbersome, we developed a protocol for purifying milligram amounts of ten recombinant antigens of M. tuberculosis from E. coli cells. Purified proteins were immunologically active and free of contaminants that confound interpretation of cell-based immunological assays. The method utilizes a three-step purification protocol consisting of immobilized metal-chelate affinity chromatography, size exclusion chromatography and anion-exchange chromatography. The first two chromatographic steps yielded recombinant protein free of protein contaminants, while the third step (anion-exchange chromatography) efficiently removed E. coli lipopolysaccharide, a potent polyclonal activator of lymphoid cells. The recombinant proteins were immunologically indistinguishable from their native (i.e., purified from M. tuberculosis) counterparts. Thus the method provides a way to utilize recombinant proteins for immunological analyses that require highly purified antigens.
Asunto(s)
Antígenos Bacterianos/aislamiento & purificación , Histidina , Mycobacterium tuberculosis/inmunología , Péptidos/química , Proteínas Recombinantes/aislamiento & purificación , Animales , Antígenos Bacterianos/química , Catalasa/metabolismo , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Cobayas , Lipopolisacáridos/química , Proteínas Recombinantes/químicaRESUMEN
The tuberculin skin test currently used to diagnose infection with Mycobacterium tuberculosis has poor diagnostic value, especially in geographic areas where the prevalence of tuberculosis is low or where the environmental burden of saprophytic, nontuberculous mycobacteria is high. Inaccuracy of the tuberculin skin test often reflects a low diagnostic specificity due to the presence in tuberculin of antigens shared by many mycobacterial species. Thus, a skin test specific for tuberculosis requires the development of new tuberculins consisting of antigens specific to M. tuberculosis. We have formulated cocktails of two to eight antigens of M. tuberculosis purified from recombinant Escherichia coli. Multiantigen cocktails were evaluated by skin testing guinea pigs sensitized with M. bovis BCG. Reactivity of multiantigen cocktails was greater than that of any single antigen. Cocktail activity increased with the number of antigens in the cocktail even when the same amount of total protein was used for cocktails and for each single antigen. A cocktail of four purified antigens specific for the M. tuberculosis complex elicited skin test responses only in BCG-immunized guinea pigs, not in control animals immunized with M. avium. These findings open the way to designing a multiantigen formulation for a skin test specific for tuberculosis.
Asunto(s)
Antígenos Bacterianos/inmunología , Mycobacterium tuberculosis/inmunología , Tuberculosis/inmunología , Animales , Femenino , Cobayas , Proteínas Recombinantes de Fusión/inmunología , Pruebas Cutáneas , Tuberculosis/diagnósticoRESUMEN
ANX Sepharose 4 Fast Flow (low sub) and ANX Sepharose 4 Fast Flow (high sub) are two new media developed at Amersham Pharmacia Biotech. They are weak anion exchangers with different amounts of tertiary amine groups attached via a spacer arm to Sepharose 4 Fast Flow. They have been characterised by the separation of some model proteins under different conditions and by determination of breakthrough capacities for proteins of different molecular masses. Functional performance after storage in different solutions at ambient temperature has been monitored. Carbon release after storage at different pH and temperatures has been measured using the total organic carbon technique. The selectivity results show that these new media are interesting complements to already existing Fast Flow anion exchangers. They are very stable and can be especially useful in applications involving the purification of high-molecular-mass proteins.