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1.
Nat Cancer ; 5(1): 66-84, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-38151625

RESUMEN

Chromosomal instability (CIN) is a hallmark of cancer, caused by persistent errors in chromosome segregation during mitosis. Aggressive cancers like high-grade serous ovarian cancer (HGSOC) and triple-negative breast cancer (TNBC) have a high frequency of CIN and TP53 mutations. Here, we show that inhibitors of the KIF18A motor protein activate the mitotic checkpoint and selectively kill chromosomally unstable cancer cells. Sensitivity to KIF18A inhibition is enriched in TP53-mutant HGSOC and TNBC cell lines with CIN features, including in a subset of CCNE1-amplified, CDK4-CDK6-inhibitor-resistant and BRCA1-altered cell line models. Our KIF18A inhibitors have minimal detrimental effects on human bone marrow cells in culture, distinct from other anti-mitotic agents. In mice, inhibition of KIF18A leads to robust anti-cancer effects with tumor regression observed in human HGSOC and TNBC models at well-tolerated doses. Collectively, our results provide a rational therapeutic strategy for selective targeting of CIN cancers via KIF18A inhibition.


Asunto(s)
Cinesinas , Neoplasias de la Mama Triple Negativas , Humanos , Animales , Ratones , Cinesinas/genética , Cinesinas/metabolismo , Mitosis/genética , Línea Celular , Puntos de Control de la Fase M del Ciclo Celular
2.
Sci Transl Med ; 10(472)2018 12 19.
Artículo en Inglés | MEDLINE | ID: mdl-30567927

RESUMEN

Glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) has been identified in multiple genome-wide association studies (GWAS) as a contributor to obesity, and GIPR knockout mice are protected against diet-induced obesity (DIO). On the basis of this genetic evidence, we developed anti-GIPR antagonistic antibodies as a potential therapeutic strategy for the treatment of obesity and observed that a mouse anti-murine GIPR antibody (muGIPR-Ab) protected against body weight gain, improved multiple metabolic parameters, and was associated with reduced food intake and resting respiratory exchange ratio (RER) in DIO mice. We replicated these results in obese nonhuman primates (NHPs) using an anti-human GIPR antibody (hGIPR-Ab) and found that weight loss was more pronounced than in mice. In addition, we observed enhanced weight loss in DIO mice and NHPs when anti-GIPR antibodies were codosed with glucagon-like peptide-1 receptor (GLP-1R) agonists. Mechanistic and crystallographic studies demonstrated that hGIPR-Ab displaced GIP and bound to GIPR using the same conserved hydrophobic residues as GIP. Further, using a conditional knockout mouse model, we excluded the role of GIPR in pancreatic ß-cells in the regulation of body weight and response to GIPR antagonism. In conclusion, these data provide preclinical validation of a therapeutic approach to treat obesity with anti-GIPR antibodies.


Asunto(s)
Receptor del Péptido 1 Similar al Glucagón/agonistas , Obesidad/tratamiento farmacológico , Receptores de la Hormona Gastrointestinal/antagonistas & inhibidores , Adipocitos/metabolismo , Animales , Anticuerpos/farmacología , Anticuerpos/uso terapéutico , Dieta , Quimioterapia Combinada , Conducta Alimentaria , Polipéptido Inhibidor Gástrico/metabolismo , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Péptidos Similares al Glucagón/análogos & derivados , Péptidos Similares al Glucagón/farmacología , Péptidos Similares al Glucagón/uso terapéutico , Humanos , Fragmentos Fc de Inmunoglobulinas/farmacología , Fragmentos Fc de Inmunoglobulinas/uso terapéutico , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Liraglutida/farmacología , Liraglutida/uso terapéutico , Ratones Obesos , Obesidad/patología , Primates , Receptores de la Hormona Gastrointestinal/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes de Fusión/uso terapéutico , Respiración , Aumento de Peso/efectos de los fármacos , Pérdida de Peso/efectos de los fármacos
3.
PLoS One ; 11(9): e0163366, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27658254

RESUMEN

Panitumumab and cetuximab target the epidermal growth factor receptor for the treatment of metastatic colorectal cancer. These therapies provide a significant survival benefit to patients with metastatic colorectal cancer with wild-type RAS. A single point mutation in the ectodomain of EGFR (S468R) confers acquired or secondary resistance in cetuximab treated patients, which is not observed in panitumumab-treated patients. Structural and biophysical studies presented here show this mutation directly blocks cetuximab binding to EGFR domain III and describes a unique mechanism by which panitumumab uses a central cavity to accommodate this mutation.

4.
Anal Chem ; 88(24): 12427-12436, 2016 12 20.
Artículo en Inglés | MEDLINE | ID: mdl-28193065

RESUMEN

Over the past two decades, orthogonal acceleration time-of-flight has been the de facto analyzer for solution and membrane-soluble protein native mass spectrometry (MS) studies; this however is gradually changing. Three MS instruments are compared, the Q-ToF, Orbitrap, and the FT-ICR, to analyze, under native instrument and buffer conditions, the seven-transmembrane helical protein bacteriorhodopsin-octylglucoside micelle and the empty nanodisc (MSP1D1-Nd) using both MS and tandem-MS modes of operation. Bacteriorhodopsin can be released from the octylglucoside-micelle efficiently on all three instruments (MS-mode), producing a narrow charge state distribution (z = 8+ to 10+) by either increasing the source lens or collision cell (or HCD) voltages. A lower center-of-mass collision energy (0.20-0.41 eV) is required for optimal bacteriorhodopsin liberation on the FT-ICR, in comparison to the Q-ToF and Orbitrap instruments (0.29-2.47 eV). The empty MSP1D1-Nd can be measured with relative ease on all three instruments, resulting in a highly complex spectrum of overlapping, polydisperse charge states. There is a measurable difference in MSP1D1-Nd charge state distribution (z = 15+ to 26+), average molecular weight (141.7 to 169.6 kDa), and phospholipid incorporation number (143 to 184) under low activation conditions. Utilizing tandem-MS, bacteriorhodopsin can be effectively liberated from the octylglucoside-micelle by collisional (Q-ToF and FT-ICR) or continuous IRMPD activation (FT-ICR). MSP1D1-Nd spectral complexity can also be significantly reduced by tandem-MS (Q-ToF and FT-ICR) followed by mild collisional or continuous IRMPD activation, resulting in a spectrum in which the charge state and phospholipid incorporation levels can easily be determined.


Asunto(s)
Bacteriorodopsinas/química , Glucósidos/química , Espectrometría de Masas/métodos , Micelas , Ciclotrones , Análisis de Fourier , Halobacterium salinarum/química , Modelos Moleculares , Nanoestructuras/química , Conformación Proteica , Membrana Púrpura/química
5.
Biochim Biophys Acta ; 1848(10 Pt A): 1974-80, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26074010

RESUMEN

Integral membrane proteins (IMPs) are of therapeutic interest and are targeted by a majority of approved drugs. It's difficult to express, purify, and maintain the functional conformation of IMPs. Nanodisc presents a reliable method to solubilize and stabilize IMPs in detergent-free condition. In this study, we demonstrate the assembly and purification of a chimeric ion channel, KcsA-Kv1.3 Nanodisc. We further detail biophysical analysis of the assembled Nanodisc using analytical ultracentrifugation (AUC), surface plasmon resonance (SPR), and back scattering interferometry (BSI). AUC is employed to determine the molecular composition of the empty and KcsA-Kv1.3 Nanodisc. Combination of SPR and BSI overcomes each other's limitation and provides insight of equilibrium binding properties of peptide and small molecule ligands to KcsA-Kv1.3.


Asunto(s)
Proteínas Bacterianas/química , Canal de Potasio Kv1.3/química , Nanopartículas/química , Nanopartículas/ultraestructura , Bloqueadores de los Canales de Potasio/química , Canales de Potasio/química , Secuencia de Aminoácidos , Proteínas Bacterianas/antagonistas & inhibidores , Sitios de Unión , Canal de Potasio Kv1.3/antagonistas & inhibidores , Datos de Secuencia Molecular , Complejos Multiproteicos/síntesis química , Complejos Multiproteicos/ultraestructura , Unión Proteica
6.
J Biomol Screen ; 19(7): 1014-23, 2014 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-24717911

RESUMEN

In the nuclei of hepatocytes, glucokinase regulatory protein (GKRP) modulates the activity of glucokinase (GK), a key regulator of glucose homeostasis. Currently, direct activators of GK (GKAs) are in development for the treatment of type 2 diabetes. However, this approach is generally associated with a risk of hypoglycemia. To mitigate such risk, we target the GKRP regulation, which indirectly restores GK activity. Here we describe a screening strategy to look specifically for GKRP modulators, in addition to traditional GKAs. Two high-throughput screening campaigns were performed with our compound libraries using a luminescence assay format, one with GK alone and the other with a GK/GKRP complex in the presence of sorbitol-6-phosphate (S6P). By a subtraction method in the hit triage process of these campaigns, we discovered two close analogs that bind GKRP specifically with sub-µM potency to a site distinct from where fructose-1-phosphate binds. These small molecules are first-in-class allosteric modulators of the GK/GKRP interaction and are fully active even in the presence of S6P. Activation of GK by this particular mechanism, without altering the enzymatic profile, represents a novel pharmacologic modality of intervention in the GK/GKRP pathway.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Descubrimiento de Drogas/métodos , Glucoquinasa/química , Adenosina Trifosfato/química , Regulación Alostérica , Animales , Glucemia/análisis , Calorimetría , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Fluorescencia , Fluorometría , Fructosafosfatos/química , Hepatocitos/metabolismo , Hexosafosfatos/química , Homeostasis , Humanos , Hipoglucemia/prevención & control , Concentración 50 Inhibidora , Luminiscencia , Unión Proteica , Conformación Proteica , Mapeo de Interacción de Proteínas , Ratas , Resonancia por Plasmón de Superficie
7.
J Med Chem ; 57(2): 309-24, 2014 Jan 23.
Artículo en Inglés | MEDLINE | ID: mdl-24405172

RESUMEN

Small molecule activators of glucokinase have shown robust efficacy in both preclinical models and humans. However, overactivation of glucokinase (GK) can cause excessive glucose turnover, leading to hypoglycemia. To circumvent this adverse side effect, we chose to modulate GK activity by targeting the endogenous inhibitor of GK, glucokinase regulatory protein (GKRP). Disrupting the GK-GKRP complex results in an increase in the amount of unbound cytosolic GK without altering the inherent kinetics of the enzyme. Herein we report the identification of compounds that efficiently disrupt the GK-GKRP interaction via a previously unknown binding pocket. Using a structure-based approach, the potency of the initial hit was improved to provide 25 (AMG-1694). When dosed in ZDF rats, 25 showed both a robust pharmacodynamic effect as well as a statistically significant reduction in glucose. Additionally, hypoglycemia was not observed in either the hyperglycemic or normal rats.


Asunto(s)
Proteínas Portadoras/metabolismo , Glucoquinasa/metabolismo , Hipoglucemiantes/química , Piperazinas/química , Animales , Sitios de Unión , Proteínas Portadoras/química , Cristalografía por Rayos X , Glucoquinasa/química , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Hipoglucemia/inducido químicamente , Hipoglucemiantes/efectos adversos , Hipoglucemiantes/farmacología , Piperazinas/efectos adversos , Piperazinas/farmacología , Conformación Proteica , Transporte de Proteínas , Ratas , Ratas Zucker , Estereoisomerismo , Relación Estructura-Actividad , Sulfonamidas/efectos adversos , Sulfonamidas/química , Sulfonamidas/farmacología
8.
Nature ; 504(7480): 437-40, 2013 Dec 19.
Artículo en Inglés | MEDLINE | ID: mdl-24226772

RESUMEN

Glucose homeostasis is a vital and complex process, and its disruption can cause hyperglycaemia and type II diabetes mellitus. Glucokinase (GK), a key enzyme that regulates glucose homeostasis, converts glucose to glucose-6-phosphate in pancreatic ß-cells, liver hepatocytes, specific hypothalamic neurons, and gut enterocytes. In hepatocytes, GK regulates glucose uptake and glycogen synthesis, suppresses glucose production, and is subject to the endogenous inhibitor GK regulatory protein (GKRP). During fasting, GKRP binds, inactivates and sequesters GK in the nucleus, which removes GK from the gluconeogenic process and prevents a futile cycle of glucose phosphorylation. Compounds that directly hyperactivate GK (GK activators) lower blood glucose levels and are being evaluated clinically as potential therapeutics for the treatment of type II diabetes mellitus. However, initial reports indicate that an increased risk of hypoglycaemia is associated with some GK activators. To mitigate the risk of hypoglycaemia, we sought to increase GK activity by blocking GKRP. Here we describe the identification of two potent small-molecule GK-GKRP disruptors (AMG-1694 and AMG-3969) that normalized blood glucose levels in several rodent models of diabetes. These compounds potently reversed the inhibitory effect of GKRP on GK activity and promoted GK translocation both in vitro (isolated hepatocytes) and in vivo (liver). A co-crystal structure of full-length human GKRP in complex with AMG-1694 revealed a previously unknown binding pocket in GKRP distinct from that of the phosphofructose-binding site. Furthermore, with AMG-1694 and AMG-3969 (but not GK activators), blood glucose lowering was restricted to diabetic and not normoglycaemic animals. These findings exploit a new cellular mechanism for lowering blood glucose levels with reduced potential for hypoglycaemic risk in patients with type II diabetes mellitus.


Asunto(s)
Proteínas Portadoras/antagonistas & inhibidores , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Hipoglucemiantes/farmacología , Hipoglucemiantes/uso terapéutico , Proteínas Adaptadoras Transductoras de Señales , Animales , Glucemia/metabolismo , Proteínas Portadoras/metabolismo , Núcleo Celular/enzimología , Cristalografía por Rayos X , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/enzimología , Modelos Animales de Enfermedad , Hepatocitos , Humanos , Hiperglucemia/sangre , Hiperglucemia/tratamiento farmacológico , Hiperglucemia/enzimología , Hipoglucemiantes/química , Hígado/citología , Hígado/enzimología , Hígado/metabolismo , Masculino , Modelos Moleculares , Especificidad de Órganos , Fosforilación/efectos de los fármacos , Piperazinas/química , Piperazinas/metabolismo , Piperazinas/farmacología , Piperazinas/uso terapéutico , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Ratas , Ratas Wistar , Sulfonamidas/química , Sulfonamidas/metabolismo , Sulfonamidas/farmacología , Sulfonamidas/uso terapéutico
9.
J Am Chem Soc ; 131(46): 16654-5, 2009 Nov 25.
Artículo en Inglés | MEDLINE | ID: mdl-19886658

RESUMEN

In aqueous solution, azaquinolone inhibitors bind to prolyl 4-hydroxylase in two different orientations, as first detected by (19)F spectroscopy. This contrasts with the crystallographic structure where only one orientation has been determined. Dissection of the metal binding properties of the enzyme allowed structures of both complexes to be obtained in solution from (19)F and (13)C dipolar shifts in a labeled ligand.


Asunto(s)
Inhibidores Enzimáticos/química , Naftiridinas/química , Procolágeno-Prolina Dioxigenasa/antagonistas & inhibidores , Procolágeno-Prolina Dioxigenasa/química , Cristalografía por Rayos X , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Naftiridinas/farmacología , Resonancia Magnética Nuclear Biomolecular
10.
Comb Chem High Throughput Screen ; 12(8): 760-71, 2009 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-19531013

RESUMEN

This manuscript describes the discovery and characterization of inhibitors of the lipid phosphatase SHIP2, an important target for the treatment of Type 2 diabetes, using the Automated Ligand Identification System. ALIS is an affinity selection-mass spectrometry platform for label-free, high throughput screening of mixture-based combinatorial libraries. We detail the mass-encoded synthesis of a library that yielded NGD-61338, a pyrazole-based SHIP2 inhibitor. Quantitative ALIS affinity measurements and inhibition of SHIP2 enzymatic activity indicate that this compound has micromolar binding affinity and inhibitory activity for this target. This inhibitor, which does not contain a phosphatase "warhead," binds the active site of SHIP2 as determined by ALIS-based competition experiments with the enzyme's natural substrate, phosphatidylinositol 3,4,5-triphosphate (PIP3). Structure-activity relationships for NGD-61338 and two other ligand classes discovered by ALIS screening were explored using a combination of combinatorial library synthesis and ALIS-enabled affinity ranking in compound mixtures.


Asunto(s)
Técnicas Químicas Combinatorias , Inhibidores Enzimáticos/análisis , Inhibidores Enzimáticos/química , Ensayos Analíticos de Alto Rendimiento/métodos , Espectrometría de Masas/métodos , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Monoéster Fosfórico Hidrolasas/metabolismo , Dominio Catalítico , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Inositol Polifosfato 5-Fosfatasas , Estructura Molecular , Pirazoles/análisis , Pirazoles/síntesis química , Pirazoles/química , Pirazoles/farmacología , Estereoisomerismo , Relación Estructura-Actividad
11.
Anal Biochem ; 384(2): 213-23, 2009 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-18952043

RESUMEN

The human hypoxia-inducible factor prolyl hydroxylases 1, 2, and 3 (HIF-PHD1, -2, and -3) are thought to act as proximal sensors of cellular hypoxia by virtue of their mechanism-based dependence on molecular oxygen. These 2-oxoglutarate (2-OG) and non-heme iron-dependent oxygenases constitutively hydroxylate HIF, resulting in high-affinity binding to Von Hippel-Lindau protein (pVHL). Some reported affinities for the HIF-PHDs for 2-OG and iron approach the estimated physiological concentrations for these cofactors, suggesting that the system as described is not catalytically optimal. Here we report the enzymatic characterization of full-length recombinant human HIF-PHD2 using a novel and sensitive catalytic assay. We demonstrated submicromolar affinities for 2-OG and ferrous iron and HIF-PHD2 Km values for oxygen that are greater than atmospheric oxygen levels, suggesting that molecular oxygen is indeed the key regulator of this pathway. In addition, we observed enhancement of HIF-PHD2 catalytic activity in the presence of ascorbic acid with only minor modifications of HIF-PHD2 requirements for 2-OG, and a detailed pH study demonstrated optimal HIF-PHD2 catalytic activity at pH 6.0. Lastly, we used this sensitive and facile assay to rapidly perform a large high-throughput screen of a chemical library to successfully identify and characterize novel 2-OG competitive inhibitors of HIF-PHD2.


Asunto(s)
Inhibidores Enzimáticos/farmacología , Transferencia Resonante de Energía de Fluorescencia/métodos , Procolágeno-Prolina Dioxigenasa/antagonistas & inhibidores , Procolágeno-Prolina Dioxigenasa/química , Ácido Ascórbico/metabolismo , Inhibidores Enzimáticos/análisis , Humanos , Concentración de Iones de Hidrógeno , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Ácidos Cetoglutáricos/metabolismo , Cinética , Modelos Biológicos , Oxígeno/metabolismo , Procolágeno-Prolina Dioxigenasa/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo
12.
Assay Drug Dev Technol ; 5(1): 105-15, 2007 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-17355203

RESUMEN

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyzes the interconversion of inert glucocorticoid (cortisone) to the active glucocorticoid (cortisol) and is enriched in liver and fat tissues. Increasing evidence suggests that selective inhibition of 11beta-HSD1 may reduce the excess glucocorticoid levels that underlie the etiology of many common disorders that constitute the metabolic syndrome. Measurement of 11beta-HSD1 activity has historically involved the detection of cortisol by methods unfavorable for large-scale screening, such as high performance liquid chromatography or thin layer chromatography. Here we describe the development and validation of novel homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET) and electrochemiluminescence assays for the measurement of cortisol. These non-radioactive assays were easy to perform and produced robust results with reference compound values comparable to those obtained by conventional methods. The TR-FRET assay was easily automated and was successfully employed for the high-throughput screening of a large compound library for inhibitors of purified human recombinant 11beta-HSD1.


Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/análisis , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/química , Electroquímica/métodos , Transferencia Resonante de Energía de Fluorescencia/métodos , Hidrocortisona/análisis , Mediciones Luminiscentes/métodos , Microquímica/métodos , Humanos , Técnica de Dilución de Radioisótopos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad
13.
Proc Natl Acad Sci U S A ; 103(26): 9814-9, 2006 Jun 27.
Artículo en Inglés | MEDLINE | ID: mdl-16782814

RESUMEN

Cellular and physiological responses to changes in dioxygen levels in metazoans are mediated via the posttranslational oxidation of hypoxia-inducible transcription factor (HIF). Hydroxylation of conserved prolyl residues in the HIF-alpha subunit, catalyzed by HIF prolyl-hydroxylases (PHDs), signals for its proteasomal degradation. The requirement of the PHDs for dioxygen links changes in dioxygen levels with the transcriptional regulation of the gene array that enables the cellular response to chronic hypoxia; the PHDs thus act as an oxygen-sensing component of the HIF system, and their inhibition mimics the hypoxic response. We describe crystal structures of the catalytic domain of human PHD2, an important prolyl-4-hydroxylase in the human hypoxic response in normal cells, in complex with Fe(II) and an inhibitor to 1.7 A resolution. PHD2 crystallizes as a homotrimer and contains a double-stranded beta-helix core fold common to the Fe(II) and 2-oxoglutarate-dependant dioxygenase family, the residues of which are well conserved in the three human PHD enzymes (PHD 1-3). The structure provides insights into the hypoxic response, helps to rationalize a clinically observed mutation leading to familial erythrocytosis, and will aid in the design of PHD selective inhibitors for the treatment of anemia and ischemic disease.


Asunto(s)
Dominio Catalítico , Oxígeno/metabolismo , Procolágeno-Prolina Dioxigenasa/química , Sitios de Unión , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Humanos , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Procolágeno-Prolina Dioxigenasa/antagonistas & inhibidores , Procolágeno-Prolina Dioxigenasa/genética , Conformación Proteica , Enfermedad de von Hippel-Lindau/genética
14.
Assay Drug Dev Technol ; 4(2): 175-83, 2006 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-16712421

RESUMEN

SH2 domain-containing inositol 5-phosphatase 2 (SHIP2) is a potential drug target for the treatment of type 2 diabetes. This enzyme serves as a negative regulator of insulin-mediated signal transduction by catalyzing the dephosphorylation of the second messenger lipid molecule phosphatidylinositol 3,4,5-triphosphate. Traditionally, assays for phosphoinositide phosphatases such as SHIP2 have relied on radiolabeled phosphatidylinositol-containing lipid membranes and chromatographic separation of labeled phospholipid substrate from product by thin-layer chromatography. We have expressed and purified catalytically active phosphatase domain constructs of SHIP2 from Escherichia coli and developed a sensitive and antibody- or binding protein-independent assay for SHIP2 amenable to high-throughput screening of phosphoinositide phosphatases or phosphoinositide kinases. This microfluidic assay, with Z' values approximately 0.8, is based upon the difference in mobility within an electric field between a fluorophore-labeled phosphatidylinositol 3,4,5-triphosphate substrate and the corresponding 3,4-bisphosphate product. High-throughput screening of a 91,060-member compound library in 384-well format resulted in the identification of SHIP2 inhibitors.


Asunto(s)
Técnicas Analíticas Microfluídicas , Monoéster Fosfórico Hidrolasas/análisis , Cromatografía en Capa Delgada , Dimetilsulfóxido/química , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Regulación Enzimológica de la Expresión Génica , Fosfohidrolasa PTEN/análisis , Fosfohidrolasa PTEN/genética , Fosfatidilinositol 3-Quinasas/análisis , Fosfatidilinositol 3-Quinasas/genética , Fosfatos de Fosfatidilinositol/análisis , Fosfatos de Fosfatidilinositol/genética , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Monoéster Fosfórico Hidrolasas/genética , Proteínas Recombinantes/análisis , Transducción de Señal , Transfección
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