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1.
Cell ; 179(4): 829-845.e20, 2019 Oct 31.
Artículo en Inglés | MEDLINE | ID: mdl-31675496

RESUMEN

The immune microenvironment of hepatocellular carcinoma (HCC) is poorly characterized. Combining two single-cell RNA sequencing technologies, we produced transcriptomes of CD45+ immune cells for HCC patients from five immune-relevant sites: tumor, adjacent liver, hepatic lymph node (LN), blood, and ascites. A cluster of LAMP3+ dendritic cells (DCs) appeared to be the mature form of conventional DCs and possessed the potential to migrate from tumors to LNs. LAMP3+ DCs also expressed diverse immune-relevant ligands and exhibited potential to regulate multiple subtypes of lymphocytes. Of the macrophages in tumors that exhibited distinct transcriptional states, tumor-associated macrophages (TAMs) were associated with poor prognosis, and we established the inflammatory role of SLC40A1 and GPNMB in these cells. Further, myeloid and lymphoid cells in ascites were predominantly linked to tumor and blood origins, respectively. The dynamic properties of diverse CD45+ cell types revealed by this study add new dimensions to the immune landscape of HCC.


Asunto(s)
Carcinoma Hepatocelular/inmunología , Proteínas de Transporte de Catión/genética , Inflamación/inmunología , Neoplasias Hepáticas/inmunología , Glicoproteínas de Membrana/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Comunicación Celular/genética , Comunicación Celular/inmunología , Linaje de la Célula/genética , Linaje de la Célula/inmunología , Células Dendríticas/inmunología , Células Dendríticas/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Inflamación/genética , Inflamación/patología , Antígenos Comunes de Leucocito/inmunología , Hígado/inmunología , Hígado/patología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Linfocitos/inmunología , Linfocitos/patología , Proteínas de Membrana de los Lisosomas/genética , Macrófagos/inmunología , Macrófagos/patología , Células Mieloides/inmunología , Células Mieloides/patología , Proteínas de Neoplasias/genética , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Transcriptoma/genética , Transcriptoma/inmunología , Microambiente Tumoral/genética , Microambiente Tumoral/inmunología
2.
Cancer Res Commun ; 4(6): 1548-1560, 2024 Jun 21.
Artículo en Inglés | MEDLINE | ID: mdl-38727236

RESUMEN

KRAS inhibitors have demonstrated exciting preclinical and clinical responses, although resistance occurs rapidly. Here, we investigate the effects of KRAS-targeting therapies on the tumor microenvironment using a library of KrasG12D, p53-mutant, murine pancreatic ductal adenocarcinoma-derived cell lines (KPCY) to leverage immune-oncology combination strategies for long-term tumor efficacy. Our findings show that SOS1 and MEK inhibitors (SOS1i+MEKi) suppressed tumor growth in syngeneic models and increased intratumoral CD8+ T cells without durable responses. Single-cell RNA sequencing revealed an increase in inflammatory cancer-associated fibroblasts (iCAF), M2 macrophages, and a decreased dendritic cell (DC) quality that ultimately resulted in a highly immunosuppressive microenvironment driven by IL6+ iCAFs. Agonist CD40 treatment was effective to revert macrophage polarization and overcome the lack of mature antigen-presenting DCs after SOS1i+MEKi therapy. Treatment increased the overall survival of KPCY tumor-bearing mice. The addition of checkpoint blockade to SOS1i+MEKi combination resulted in tumor-free mice with established immune memory. Our data suggest that KRAS inhibition affects myeloid cell maturation and highlights the need for combining KRAS cancer-targeted therapy with myeloid activation to enhance and prolong antitumor effects. SIGNIFICANCE: Combination of SOS1 and MEK inhibitors increase T cell infiltration while blunting pro-immune myeloid cell maturation and highlights the need for combining KRAS cancer-targeted therapy with myeloid activation to enhance and prolong anti-tumor effects.


Asunto(s)
Carcinoma Ductal Pancreático , Inmunoterapia , Neoplasias Pancreáticas , Proteínas Proto-Oncogénicas p21(ras) , Proteína SOS1 , Microambiente Tumoral , Animales , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/inmunología , Carcinoma Ductal Pancreático/patología , Ratones , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunología , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/genética , Proteína SOS1/genética , Proteína SOS1/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Inmunoterapia/métodos , Línea Celular Tumoral , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Ratones Endogámicos C57BL , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , Femenino
3.
Bioorg Med Chem Lett ; 19(19): 5547-51, 2009 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-19716697

RESUMEN

An SAR study that identified a series of thienopyridine-based potent IkappaB Kinase beta (IKKbeta) inhibitors is described. With focuses on the structural optimization at C4 and C6 of structure 1 (Fig. 1), the study reveals that small alkyl and certain aromatic groups are preferred at C4, whereas polar groups with proper orientation at C6 efficiently enhance compound potency. The most potent analogues inhibit IKKbeta with IC50s as low as 40 nM, suppress LPS-induced TNF-alpha production in vitro and in vivo, display good kinase selectivity profiles, and are active in a HeLa cell NF-kappaB reporter gene assay, demonstrating that they directly interfere with the NF-kappaB signaling pathway.


Asunto(s)
Quinasa I-kappa B/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Piridinas/química , Animales , Descubrimiento de Drogas , Células HeLa , Humanos , Quinasa I-kappa B/metabolismo , Lipopolisacáridos/farmacología , Ratones , FN-kappa B/metabolismo , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/síntesis química , Piridinas/farmacología , Transducción de Señal , Relación Estructura-Actividad , Factor de Necrosis Tumoral alfa/metabolismo
4.
J Med Chem ; 46(22): 4669-75, 2003 Oct 23.
Artículo en Inglés | MEDLINE | ID: mdl-14561086

RESUMEN

It has been reported that the diaryl urea class of p38alpha inhibitors binds to p38 map kinase with both high affinity and slow binding kinetics (Pargellis et al. Nat. Struct. Biol. 2002, 9, 268-272). The slow binding kinetics of this class of inhibitors is believed to be the result of binding to an allosteric pocket adjacent to the p38alpha active site. The use of traditional kinetic and equilibrium methods to measure the binding affinity of this class of compounds has created many challenges for determination of structure-activity relationships (SAR). The thermal denaturation method provides a means of measuring high-affinity interactions. In this paper, the method of thermal denaturation will be described as it has been applied to the diaryl urea class of p38 map kinase inhibitors.


Asunto(s)
Inhibidores Enzimáticos/química , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Urea/análogos & derivados , Urea/química , Algoritmos , Sitio Alostérico , Animales , Sitios de Unión , Rastreo Diferencial de Calorimetría , Fluorescencia , Calefacción , Humanos , Ratones , Proteína Quinasa 14 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/química , Unión Proteica , Desnaturalización Proteica , Pliegue de Proteína , Espectrofotometría Ultravioleta , Relación Estructura-Actividad , Termodinámica
5.
J Med Chem ; 45(16): 3394-405, 2002 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-12139450

RESUMEN

An imidazo[4,5-h]isoquinolin-7,9-dione (1) was identified as an adenosine 5'-triphosphate competitive inhibitor of lck by high throughput screening. Initial structure-activity relationship studies identified the dichlorophenyl ring and the imide NH as important pharmacophores. A binding model was constructed to understand how 1 binds to a related kinase, hck. These results suggested that removing the gem-dimethyl group and flattening the ring would enhance activity. This was realized by converting 1 to the imidazo[4,5-h]isoquinolin-9-one (20), resulting in an 18-fold improvement in potency against lck and a 50-fold increase in potency in a cellular assay.


Asunto(s)
Inhibidores Enzimáticos/síntesis química , Imidazoles/síntesis química , Isoquinolinas/síntesis química , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/antagonistas & inhibidores , Animales , Anticuerpos/farmacología , Sitios de Unión , Complejo CD3/inmunología , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Imidazoles/química , Imidazoles/farmacología , Interleucina-2/biosíntesis , Isoquinolinas/química , Isoquinolinas/farmacología , Células Jurkat , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Conformación Molecular , Relación Estructura-Actividad , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo
6.
J Med Chem ; 46(8): 1337-49, 2003 Apr 10.
Artículo en Inglés | MEDLINE | ID: mdl-12672234

RESUMEN

The tyrosine kinase p56lck (lck) is essential for T cell activation; thus, inhibitors of lck have potential utility as autoimmune agents. Our initial disclosure of a new class of lck inhibitors based on the phenylaminoimidazoisoquinolin-9-one showed reasonable cellular activity but did not work in vivo upon oral administration. Our current work highlights the further use of rational drug design and molecular modeling to produce a series of lck inhibitors that demonstrate cellular activity below 100 nM and are as efficacious as cyclosporin A in an in vivo mouse model of anti-CD3-induced IL-2 production.


Asunto(s)
Bencimidazoles/síntesis química , Inhibidores Enzimáticos/síntesis química , Inmunosupresores/síntesis química , Isoquinolinas/síntesis química , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/antagonistas & inhibidores , Administración Oral , Animales , Anticuerpos Monoclonales/farmacología , Bencimidazoles/química , Bencimidazoles/farmacología , Complejo CD3/inmunología , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Inmunosupresores/química , Inmunosupresores/farmacología , Interleucina-2/antagonistas & inhibidores , Interleucina-2/biosíntesis , Interleucina-2/sangre , Isoquinolinas/química , Isoquinolinas/farmacología , Células Jurkat , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Conformación Molecular , Unión Proteica , Relación Estructura-Actividad
7.
Am J Clin Exp Immunol ; 2(2): 172-85, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23885334

RESUMEN

Vascular adhesion protein-1 (VAP-1) has been implicated in the pathogenesis of inflammatory diseases and is suggested to play a role in immune cell trafficking. It is not clear whether this effect is mediated by the oxidase activity or by other features of the protein such as direct adhesion. In order to study the role of VAP-1 oxidase activity in vivo, we have generated mice carrying an oxidase activity-null VAP-1 protein. We demonstrate that the VAP-1 oxidase null mutant mice have a phenotype similar to the VAP-1 null mice in animal models of sterile peritonitis and antibody induced arthritis suggesting that the oxidase activity is responsible for the inflammatory function of VAP-1.

8.
J Biomol Screen ; 16(9): 1106-11, 2011 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-21844327

RESUMEN

Vascular adhesion protein-1 (VAP-1), also known as semicarbazide-sensitive amine oxidase (SSAO) or copper-containing amine oxidase (AOC3, EC 1.4.3.6), catalyzes oxidative deamination of primary amines. One endogenous substrate has recently been described (Siglec 10), and although its mechanism of action in vivo is not completely understood, it is suggested to play a role in immune cell trafficking, making it a target of interest for autoimmune and inflammatory diseases. Much of the enzymology performed around this target has been conducted with absorbance, fluorescent, or radiometric formats that can have some limitations for high-throughput screening and subsequent compound profiling. The authors present the use of a bioluminescent assay, originally developed for monoamine oxidase enzymes, in a high-throughput format. It can be used for related SSAOs such as AOC1 given their substrate similarity with VAP-1. The authors also demonstrate that it is compatible with different sources of VAP-1, both purified recombinant and VAP-1 overexpressed on live cells.


Asunto(s)
Amina Oxidasa (conteniendo Cobre)/metabolismo , Ensayos Analíticos de Alto Rendimiento , Mediciones Luminiscentes , Amina Oxidasa (conteniendo Cobre)/antagonistas & inhibidores , Animales , Células CHO , Línea Celular Transformada , Cricetinae , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Células HEK293 , Humanos , Concentración 50 Inhibidora , Ratones , Ratas
9.
Biochem Pharmacol ; 77(3): 422-32, 2009 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-19027720

RESUMEN

The characterization of the potent p38 inhibitor BIRB796 as a dual inhibitor of p38/Jun N-terminal kinases (JNK) mitogen-activated protein kinases (EC 2.7.11.24) has complicated the interpretation of its reported anti-inflammatory activity. To better understand the contribution of JNK2 inhibition to the anti-inflammatory activities of BIRB796, we explored the relationship between the effects of BIRB796 and analogues on cytokine production and on cellular p38 and JNK signaling. We determined the binding affinity for BIRB796 and structural analogues to p38alpha and JNK2 and characterized compound 2 as a p38 inhibitor that binds to p38alpha with an affinity equivalent to BIRB796 but does not bind to any of the JNK isoforms. High-content imaging enabled us to show that the inhibition of p38 signaling by BIRB796 and analogues correlates with the ability of these compounds to inhibit the lipopolysaccharide (LPS)-induced TNF-alpha production in THP-1 monocytes. This finding was extended to cytokine release by disease-relevant human primary cells: to the production of TNF-alpha by peripheral blood mononuclear cells, and of IL-8 by neutrophils. Furthermore, BIRB796 and compound 2 inhibited the production of TNF-alpha in THP-1 monocytes and the IL-12/IL-18-induced production of interferon-gamma in human T-cells with similar potencies. In contrast, cellular JNK signaling in response to cytokines or stress stimuli was only weakly inhibited by BIRB796 and analogues and not affected by compound 2. In summary, our data suggest that p38 inhibition alone is sufficient to completely suppress cytokine production and that the added inhibition of JNK2 does not significantly contribute to the effects of BIRB796 on cytokine production.


Asunto(s)
Citocinas/biosíntesis , Mediadores de Inflamación/metabolismo , Proteína Quinasa 9 Activada por Mitógenos/antagonistas & inhibidores , Naftalenos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Transducción de Señal/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Células HeLa , Humanos
11.
J Biol Chem ; 277(47): 45129-40, 2002 Nov 22.
Artículo en Inglés | MEDLINE | ID: mdl-12221085

RESUMEN

The IKKbeta and NEMO/IKKgamma subunits of the NF-kappaB-activating signalsome complex are known to be essential for activating NF-kappaB by inflammatory and other stress-like stimuli. However, the IKKalpha subunit is believed to be dispensable for the latter responses and instead functions as an in vivo mediator of other novel NF-kappaB-dependent and -independent functions. In contrast to this generally accepted view of IKKalpha's physiological functions, we demonstrate in mouse embryonic fibroblasts (MEFs) that, akin to IKKbeta and NEMO/IKKgamma, IKKalpha is also a global regulator of tumor necrosis factor alpha- and IL-1-responsive IKK signalsome-dependent target genes including many known NF-kappaB targets such as serum amyloid A3, C3, interleukin (IL)-6, IL-11, IL-1 receptor antagonist, vascular endothelial growth factor, Ptx3, beta(2)-microglobulin, IL-1alpha, Mcp-1 and -3, RANTES (regulated on activation normal T cell expressed and secreted), Fas antigen, Jun-B, c-Fos, macrophage colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor. Only a small number of NF-kappaB-dependent target genes were preferentially dependent on IKKalpha or IKKbeta. Constitutive expression of a trans-dominant IkappaBalpha superrepressor (IkappaBalphaSR) in wild type MEFs confirmed that these signalsome-dependent target genes were also dependent on NF-kappaB. A subset of NF-kappaB target genes were IKK-dependent in the absence of exogenous stimuli, suggesting that the signalsome was also required to regulate basal levels of activated NF-kappaB in established MEFs. Overall, a sizable number of novel NF-kappaB/IKK-dependent genes were identified including Secreted Frizzled, cadherin 13, protocadherin 7, CCAAT/enhancer-binding protein-beta and -delta, osteoprotegerin, FOXC2 and FOXF2, BMP-2, p75 neurotrophin receptor, caspase-11, guanylate-binding proteins 1 and 2, ApoJ/clusterin, interferon (alpha and beta) receptor 2, decorin, osteoglycin, epiregulin, proliferins 2 and 3, stromal cell-derived factor, and cathepsins B, F, and Z. SOCS-3, a negative effector of STAT3 signaling, was found to be an NF-kappaB/IKK-induced gene, suggesting that IKK-mediated NF-kappaB activation can coordinately illicit negative effects on STAT signaling.


Asunto(s)
Regulación de la Expresión Génica/inmunología , Inflamación/genética , FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/fisiología , Animales , Células Cultivadas , Embrión de Mamíferos/anatomía & histología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/fisiología , Regulación de la Expresión Génica/fisiología , Humanos , Quinasa I-kappa B , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Inflamación/metabolismo , Interleucina-1/farmacología , Sustancias Macromoleculares , Ratones , Datos de Secuencia Molecular , Inhibidor NF-kappaB alfa , FN-kappa B/antagonistas & inhibidores , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Serina-Treonina Quinasas/genética , Subunidades de Proteína , Factor de Necrosis Tumoral alfa/farmacología
12.
Bioorg Med Chem Lett ; 13(18): 3101-4, 2003 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-12941343

RESUMEN

BIRB 796, a member of the N-pyrazole-N'-naphthly urea class of p38MAPK inhibitors, binds to the kinase with both slow association and dissociation rates. Prior to binding, the kinase undergoes a reorganization of the activation loop exposing a critical binding domain. We demonstrate that, independent of the loop movement, association rates are governed by low energy conformations of the inhibitor and polar functionality on the tolyl ring. As anticipated, the dissociation rates of the inhibitors from the kinase are slowed by lipophilic and hydrogen bond interactions. The value of structure-kinetic relationships (SKR) in drug design is discussed.


Asunto(s)
Inhibidores Enzimáticos/química , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Naftalenos/química , Pirazoles/química , Enfermedades Autoinmunes/tratamiento farmacológico , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Concentración 50 Inhibidora , Cinética , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/química , Naftalenos/farmacología , Unión Proteica , Estructura Terciaria de Proteína , Pirazoles/farmacología , Relación Estructura-Actividad , Proteínas Quinasas p38 Activadas por Mitógenos
13.
Biochemistry ; 43(37): 11658-71, 2004 Sep 21.
Artículo en Inglés | MEDLINE | ID: mdl-15362850

RESUMEN

A novel inhibitor of p38 mitogen-activated protein kinase (p38), CMPD1, identified by high-throughput screening, is characterized herein. Unlike the p38 inhibitors described previously, this inhibitor is substrate selective and noncompetitive with ATP. In steady-state kinetics experiments, CMPD1 was observed to prevent the p38alpha-dependent phosphorylation (K(i)(app) = 330 nM) of the splice variant of mitogen-activated protein kinase-activated protein kinase 2 (MK2a) that contains a docking domain for p38alpha and p38beta, but it did not prevent the phosphorylation of ATF-2 (K(i)(app) > 20 microM). In addition to kinetic studies, isothermal titration calorimetry and surface plasmon resonance experiments were performed to elucidate the mechanism of inhibition. While isothermal titration calorimetry analysis indicated that CMPD1 binds to p38alpha, CMPD1 was not observed to compete with ATP for p38alpha, nor was it able to interrupt the binding of p38alpha to MK2a observed by surface plasmon resonance. Therefore, deuterium exchange mass spectrometry (DXMS) was employed to study the p38alpha.CMPD1 inhibitory complex, to provide new insight into the mechanism of substrate selective inhibition. The DXMS data obtained for the p38alpha.CMPD1 complex were compared to the data obtained for the p38alpha.MK2a complex and a p38alpha.active site binding inhibitor complex. Alterations in the DXMS behavior of both p38alpha and MK2a were observed upon complex formation, including but not limited to the interaction between the carboxy-terminal docking domain of MK2a and its binding groove on p38alpha. Alterations in the D(2)O exchange of p38alpha produced by CMPD1 suggest that the substrate selective inhibitor binds in the vicinity of the active site of p38alpha, resulting in perturbations to regions containing nucleotide binding pocket residues, docking groove residues (E160 and D161), and a Mg(2+) ion cofactor binding residue (D168). Although the exact mechanism of substrate selective inhibition by this novel inhibitor has not yet been disclosed, the results suggest that CMPD1 binding in the active site region of p38alpha induces perturbations that may result in the suboptimal positioning of substrates and cofactors in the transition state, resulting in selective inhibition of p38alpha activity.


Asunto(s)
Compuestos de Bifenilo/metabolismo , Inhibidores Enzimáticos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Estructura Terciaria de Proteína , Factor de Transcripción Activador 2 , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Compuestos de Bifenilo/química , Calorimetría , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Inhibidores Enzimáticos/química , Péptidos y Proteínas de Señalización Intracelular , Ratones , Proteína Quinasa 14 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Fosforilación , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Especificidad por Sustrato , Resonancia por Plasmón de Superficie , Factores de Transcripción/metabolismo
14.
Biochemistry ; 43(31): 9950-60, 2004 Aug 10.
Artículo en Inglés | MEDLINE | ID: mdl-15287722

RESUMEN

The p38 mitogen-activated protein kinase (p38) pathway is required for the production of proinflammatory cytokines (TNFalpha and IL-1) that mediate the chronic inflammatory phases of several autoimmune diseases. Potent p38 inhibitors, such as the slow tight-binding inhibitor BIRB 796, have recently been reported to block the production of TNFalpha and IL-1beta. Here we analyze downstream signaling complexes and molecular mechanisms, to provide new insight into the function of p38 signaling complexes and the development of novel inhibitors of the p38 pathway. Catalysis, signaling functions, and molecular interactions involving p38alpha and one of its downstream signaling partners, mitogen-activated protein kinase-activated protein kinase 2 (MK2), have been explored by steady-state kinetics, surface plasmon resonance, isothermal calorimetry, and stopped-flow fluorescence. Functional 1/1 signaling complexes (Kd = 1-100 nM) composed of activated and nonactivated forms of p38alpha and a splice variant of MK2 (MK2a) were characterized. Catalysis of MK2a phosphorylation and activation by p38alpha was observed to be efficient under conditions where substrate is saturating (kcat(app) = 0.05-0.3 s(-1)) and nonsaturating (kcat(app)/KM(app) = 1-3 x 10(6) M(-1) s(-1)). Specific interactions between the carboxy-terminal residues of MK2a (370-400) and p38alpha precipitate formation of a high-affinity complex (Kd = 20 nM); the p38alpha-dependent MK2a phosphorylation reaction was inhibited by the 30-amino acid docking domain peptide of MK2a (IC50 = 60 nM). The results indicate that the 30-amino acid docking domain peptide of MK2a is required for the formation of a tight, functional p38alpha.MK2a complex, and that perturbation of the tight-docking interaction between these signaling partners prevents the phosphorylation of MK2a. The thermodynamic and steady-state kinetic characterization of the p38alpha.MK2a signaling complex has led to a clear understanding of complex formation, catalysis, and function on the molecular level.


Asunto(s)
Sistema de Señalización de MAP Quinasas , Proteínas Quinasas Activadas por Mitógenos/química , Proteínas Quinasas Activadas por Mitógenos/fisiología , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/fisiología , Empalme Alternativo , Animales , Calorimetría , Catálisis , Fluoresceína-5-Isotiocianato/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/fisiología , Cinética , Sistema de Señalización de MAP Quinasas/genética , Ratones , Proteína Quinasa 14 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/fisiología , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de Proteína/genética , Espectrometría de Fluorescencia , Relación Estructura-Actividad , Resonancia por Plasmón de Superficie , Termodinámica , Ultracentrifugación
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