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1.
Protein Expr Purif ; 75(1): 104-13, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-20828617

RESUMEN

The sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDHS) isoform is a promising contraceptive target because it is specific to male germ cells, essential for sperm motility and male fertility, and well suited to pharmacological inhibition. However, GAPDHS is difficult to isolate from native sources and recombinant expression frequently results in high production of insoluble enzyme. We chose to use the Bac-to-Bac baculovirus-insect cell system to express a His-tagged form of human GAPDHS (Hu his-GAPDHS) lacking the proline-rich N-terminal sequence. This recombinant Hu his-GAPDHS was successfully produced in Spodoptera frugiperda 9 (Sf9) cells by infection with recombinant virus as a soluble, enzymatically active form in high yield, >35 mg/L culture. Biochemical characterization of the purified enzyme by mass spectrometry and size exclusion chromatography confirmed the presence of the tetrameric form. Further characterization by peptide ion matching mass spectrometry and Edman sequencing showed that unlike the mixed tetramer forms produced in bacterial expression systems, human his-GAPDHS expressed in baculovirus-infected insect cells is homotetrameric. The ability to express and purify active human GAPDHS as homotetramers in high amounts will greatly aid in drug discovery efforts targeting this enzyme for discovery of novel contraceptives and three compounds were identified as inhibitors of Hu his-GAPDHS from a pilot screen of 1120 FDA-approved compounds.


Asunto(s)
Baculoviridae/genética , Clonación Molecular , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Espermatozoides/enzimología , Spodoptera/citología , Secuencia de Aminoácidos , Animales , Línea Celular , Clonación Molecular/métodos , Gliceraldehído-3-Fosfato Deshidrogenasas/química , Gliceraldehído-3-Fosfato Deshidrogenasas/aislamiento & purificación , Humanos , Masculino , Espectrometría de Masas , Datos de Secuencia Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/aislamiento & purificación , Isoformas de Proteínas/metabolismo , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Spodoptera/genética , Spodoptera/virología
2.
J Biomol Screen ; 13(5): 396-405, 2008 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-18480472

RESUMEN

Twenty human proteins encode Phox/Bem1p (PB1) domains, which are involved in forming protein heterodimers. MEKK2, MEKK3, and MEK5 are 3 serine-threonine protein kinases that have PB1 domains. MEKK2, MEKK3, and MEK5 are the MAP3Ks and the MAP2K in the ERK5 mitogen-activated protein kinase (MAPK) signaling module. ERK5 is a critical MAPK for both development of the vasculature and vascular homeostasis in the adult, but no other MAPK has been shown to be critical in vascular maintenance in the adult animal. MEKK2 and MEKK3 are the only MAP3Ks shown to physically interact with and activate the MEK5-ERK5 signaling module. Interaction of MEKK2 or MEKK3 with MEK5 is mediated by heterodimerization of the MEKK2 (or MEKK3) PB1 and MEK5 PB1 domains. The authors have developed a homogeneous, time-resolved fluorescence resonance energy transfer (TR-FRET) assay to monitor PB1-PB1 domain heterodimerization. The assay uses a europium-chelate conjugated GST-MEK5 PB1 domain chimera, biotinylated MEKK2 PB1 domain, and streptavidin-Cy5. Interaction of the MEKK2 and MEK5 PB1 domains gives a robust FRET signal (Z' factor = 0.93), which is completely abrogated by mutation of 2 acidic residues (64D65E-->AA) within the MEK5 PB1 domain that causes loss of stable PB1-PB1 domain interaction. This assay can be used to study the specificity of PB1-PB1 domain interactions and to screen for molecules that can regulate MEKK2/MEKK3-MEK5 interactions. Disruption of PB1 domain interactions represents a novel approach for selectively regulating the ERK5 signaling pathway independent of kinase active site-directed adenosine triphosphate competitive inhibitors.


Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Proteínas/química , Técnicas Químicas Combinatorias , Dimerización , Glutatión Transferasa/química , Humanos , MAP Quinasa Quinasa 5/química , Estructura Terciaria de Proteína
3.
J Biomol Screen ; 12(4): 481-9, 2007 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-17478482

RESUMEN

Inflammatory T cells that are reactive to myelin protein components of the CNS play a critical role in the pathogenesis of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). The authors have previously generated mice that predominantly harbor T cells transgenic for a T-cell receptor (TCR) that is specific to the myelin proteolipid protein (PLP) 139-151 and that spontaneously develop MS-like paralysis. T cells from healthy transgenic mice respond to stimulation with PLP139-151 in a highly specific manner by proliferation and secretion of proinflammatory cytokines such as interleukin (IL)-2 and interferon (INF)-gamma in vitro. To identify druglike compounds that may inhibit inflammatory T-cell responses, the authors have developed a high-throughput screening assay with primary T cells from PLP TCR transgenic mice. They have screened 41,184 small-molecule compounds that follow Lipinski's rules for their inhibitory activity on the proliferation and secretion of proinflammatory cytokines in PLP-reactive T cells. To this end, the screen identified 6 nontoxic compounds with a molecular weight <500 that inhibited inflammatory responses in PLP-reactive T cells in a concentration-dependent fashion. The identified compounds represent valid leads that may be developed into novel therapeutics for MS that could be administered orally.


Asunto(s)
Inmunosupresores/metabolismo , Vaina de Mielina/metabolismo , Biblioteca de Péptidos , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Animales , Femenino , Inmunofenotipificación , Inmunosupresores/aislamiento & purificación , Inmunosupresores/farmacología , Ratones , Ratones Transgénicos , Vaina de Mielina/inmunología , Linfocitos T/inmunología
4.
J Biomol Screen ; 11(7): 729-35, 2006 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-16928982

RESUMEN

Familial amyotrophic lateral sclerosis (ALS) accounts for 10% of all ALS cases; approximately 25% of these cases are due to mutations in the Cu/Zn superoxide dismutase gene (SOD1). To date, 105 different mutations spanning all 5 exons have been identified in the SOD1 gene. Mutant SOD1-associated ALS is caused by a toxic gain of function of the mutated protein. Therefore, regardless of the specific mechanism whereby mutant SOD1 initiates motor neuron death, the authors hypothesize that measures that decrease levels of mutant SOD1 protein should ameliorate the phenotype in transgenic mice and potentially in patients with SOD1-mediated disease. They have designed 2 cell-based screening assays to identify small, brain-permeant molecules that inactivate expression of the SOD1 gene or increase the degradation of the SOD1 protein. Here they describe the development and optimization of these assays and the results of high-throughput screening using a variety of compound libraries, including a total of more than 116,000 compounds. The majority of the hit compounds identified that down-regulated SOD1 were shown to be toxic in a cell-based viability assay or were nonselective transcription inhibitors, but work is continuing on a number of nonspecific inhibitors of SOD1 expression. Ultimately, the authors believe that these 2 cell-based assays will provide powerful strategies to identify novel therapies for the treatment of inherited SOD1-associated forms of ALS.


Asunto(s)
Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Esclerosis Amiotrófica Lateral/enzimología , Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/farmacología , Superóxido Dismutasa/metabolismo , Animales , Línea Celular , Inhibidores Enzimáticos/análisis , Inhibidores Enzimáticos/química , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Ratones , Proteínas Mutantes/metabolismo , Células PC12 , Regiones Promotoras Genéticas/genética , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Superóxido Dismutasa-1
5.
Chem Biol ; 12(7): 811-23, 2005 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-16039528

RESUMEN

Cyclin-dependent kinase 5 (Cdk5) is widely viewed as a possible target for a wide variety of neurological disorders. One pathological role attributed to Cdk5 is the abnormal phosphorylation of tau that may lead to the neuronal inclusions known as neurofibrillary tangles. A high through-put screen for inhibitors of Cdk5-mediated phosphorylation of tau resulted in three compounds with distinct mechanisms of action. One compound is competitive with ATP and has a high affinity for the Cdk5 ATP binding pocket. The second compound also competes with ATP, is noncompetitive with tau, and (uniquely among this class of inhibitors) displaces adjacent amino acid residues to make room for the nitrophenyl group. A third compound did not compete with ATP, but did compete with tau at low concentrations of tau. The SAR and charge optimization derived from cocrystals of the two ATP competitors along with cocrystals of three other ATP competitors map out the importance of filling and properly charging different regions of the ATP binding pocket. Taken together, this analysis shows how the structure of Cdk5 constrains the space of potential inhibitors and reveals a pocket unfilled in all of the structures. These leads could be a starting point for structure-based drug design of more potent and selective inhibitors.


Asunto(s)
Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Neuronas/efectos de los fármacos , Proteínas tau/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/enzimología , Unión Competitiva , Quinasa 5 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/química , Quinasas Ciclina-Dependientes/metabolismo , Activación Enzimática , Ligandos , Modelos Químicos , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Neuronas/enzimología , Fosforilación/efectos de los fármacos , Relación Estructura-Actividad , Proteínas tau/química
6.
J Biomol Screen ; 10(1): 1-12, 2005 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-15695338

RESUMEN

Cerebral accumulation of the amyloid beta-peptide (Abeta) is believed to play a key role in the pathogenesis of Alzheimer's disease (AD). Because Abeta is produced from the proteolysis of amyloid beta-protein precursor (APP) by beta-and gamma-secretases, these enzymes are considered important drug targets for AD. The authors have developed a luciferase-based reporter system that can identify new molecules that inhibit APP processing in a high-throughput manner. Such molecules can help in understanding the biology of APP and APP processing and in developing new drug prototypes for AD. In this system, APP is fused on its C-terminus with Gal4-VP16, a chimeric yeast-viral transcription activator, and luciferase is under control of the yeast Gal4 promoter. Compounds that modulate the luciferase signal may affect the secretases directly, interact with modifiers of these proteases, or interact with APP directly. The authors successfully interfaced this assay with a high-throughput screen, testing approximately 60,000 compounds with diverse chemical structures. In principle, this sensitive, specific, and quantitative assay may be useful for identifying both inhibitors and stimulators of APP processing.


Asunto(s)
Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Secretasas de la Proteína Precursora del Amiloide , Ácido Aspártico Endopeptidasas/metabolismo , Línea Celular , Endopeptidasas , Humanos , Concentración 50 Inhibidora , Transfección
7.
Chem Biol ; 10(9): 837-46, 2003 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-14522054

RESUMEN

Neuronal ubiquitin C-terminal hydrolase (UCH-L1) has been linked to Parkinson's disease (PD), the progression of certain nonneuronal tumors, and neuropathic pain. Certain lung tumor-derived cell lines express UCH-L1 but it is not expressed in normal lung tissue, suggesting that this enzyme plays a role in tumor progression, either as a trigger or as a response. Small-molecule inhibitors of UCH-L1 would be helpful in distinguishing between these scenarios. By utilizing high-throughput screening (HTS) to find inhibitors and traditional medicinal chemistry to optimize their affinity and specificity, we have identified a class of isatin O-acyl oximes that selectively inhibit UCH-L1 as compared to its systemic isoform, UCH-L3. Three representatives of this class (30, 50, 51) have IC(50) values of 0.80-0.94 micro M for UCH-L1 and 17-25 micro M for UCH-L3. The K(i) of 30 toward UCH-L1 is 0.40 micro M and inhibition is reversible, competitive, and active site directed. Two isatin oxime inhibitors increased proliferation of the H1299 lung tumor cell line but had no effect on a lung tumor line that does not express UCH-L1. Inhibition of UCH-L1 expression in the H1299 cell line using RNAi had a similar proproliferative effect, suggesting that the UCH-L1 enzymatic activity is antiproliferative and that UCH-L1 expression may be a response to tumor growth. The molecular mechanism of this response remains to be determined.


Asunto(s)
Antineoplásicos/farmacología , Isatina/farmacología , Neoplasias Pulmonares/patología , Oximas/farmacología , Ubiquitina Tiolesterasa/antagonistas & inhibidores , Antineoplásicos/química , División Celular/efectos de los fármacos , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Concentración 50 Inhibidora , Isatina/química , Isoenzimas/antagonistas & inhibidores , Oximas/química , Relación Estructura-Actividad
8.
Front Biosci ; 9: 2788-95, 2004 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-15353314

RESUMEN

Collagenases are a highly specific class of enzymes. In their native states, collagenases cleave only native triple helical collagen molecules at a single peptide bond between Gly775-Leu776 for Type I collagen and Gly775-Ile776 for Type II collagen. The linear sequence of collagen is about 1050 amino acids in length, where three linear peptide sequences are required to form a triple helical collagen molecule. At present, there exist no crystallographic structures of collagenase bound to native triple helical collagen; nor has it been shown that collagenase recognizes the triple helical conformation of collagen. In our study, we have used an inhibitor design structure-activity based approach to show that collagenase recognizes and cleaves triple helical collagen conformations in preference to non-triple helical collagen conformations.


Asunto(s)
Colágeno/química , Inhibidores Enzimáticos/farmacología , Metaloproteinasa 1 de la Matriz/química , Inhibidores de la Metaloproteinasa de la Matriz , Animales , Artritis/metabolismo , Catálisis , Colagenasas/química , Biología Computacional/métodos , Cristalografía por Rayos X , Bases de Datos de Proteínas , Diseño de Fármacos , Modelos Moleculares , Conformación Molecular , Péptidos/química , Conformación Proteica , Programas Informáticos , Porcinos
9.
J Biomol Screen ; 9(2): 122-31, 2004 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-15006135

RESUMEN

A high-throughput assay for tau phosphorylation by cdk5/p25 is described. Full-length recombinant tau was used as a substrate in the presence of saturating adenosine triphosphate (ATP). Using PHF-1, an antibody directed specifically against 2 tau phosphorylation epitopes (serine 396 and serine 404), an enzyme-linked immunosorbent assay (ELISA)-based colorimetric assay was formatted in 384-well plates. The assay was validated by measuring kinetic parameters for cdk5/p25 catalysis and known inhibitors. Rate constants for the site-specific phosphorylations at the PHF-1 epitopes were determined and suggested preferential phosphorylation at these sites. The performance of this assay in a high-throughput format was demonstrated and used to identify inhibitors of tau phosphorylation at specific epitopes phosphorylated by cdk5/p25.


Asunto(s)
Quinasas Ciclina-Dependientes/metabolismo , Proteínas tau/metabolismo , Adenosina Trifosfato/análisis , Adenosina Trifosfato/metabolismo , Anticuerpos Monoclonales/metabolismo , Sitios de Unión , Biotinilación , Células Cultivadas , Quinasa 5 Dependiente de la Ciclina , Humanos , Cinética , Cinetina , Miniaturización , Fosforilación , Unión Proteica , Purinas/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Factores de Tiempo
10.
J Mol Neurosci ; 19(3): 261-6, 2002 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-12540051

RESUMEN

Tau is certainly a reasonable target for the development of compounds to prevent neurofibrillary pathology, particularly in the fronto-temporal dementias. Although the mechanism of the filamentous accumulations remains unclear, sufficient knowledge is in place to move forward with high throughput screens. In fact, the development of compounds from such screens will ultimately be the only way to validate the target. The dichotomy for such screens is that in vitro screens are easier to design, but require more assumptions as to the mechanism, in contrast to cell-based screens that are more difficult to design, but make fewer assumptions about mechanism. We have designed a moderate throughput for tau binding that relies on fluorescence detection in living cells and an in vitro cdk5/p25 tau phosphorylation high throughput screen.


Asunto(s)
Diseño de Fármacos , Evaluación Preclínica de Medicamentos/métodos , Tauopatías/prevención & control , Proteínas tau/metabolismo , Animales , Quinasa 5 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Humanos , Mutación , Proteínas del Tejido Nervioso/metabolismo , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Proteínas tau/genética
11.
Assay Drug Dev Technol ; 2(6): 609-19, 2004 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-15674019

RESUMEN

A high throughput assay for measuring tau aggregation using fluorescent resonance energy transfer (FRET) is described. Full-length recombinant tau labeled with Cy3 or Cy5 dye is used as ligand, and the induction of aggregation is accomplished by the addition of arachidonic acid. In the presence of this fatty acid, tau aggregation is measured by FRET in a 384-well format. The nature of tau aggregation is further characterized by competition with unlabeled tau and cross-linking experiments. It is concluded that the FRET observed under the experimental condition is due to the accumulation of tau dimers and tetramers. A model for tau aggregation is presented. The performance of this assay in a high throughput format is demonstrated and can be used to identify inhibitors of tau aggregation.


Asunto(s)
Proteínas tau/química , Proteínas tau/efectos de los fármacos , Ácido Araquidónico/farmacología , Unión Competitiva/efectos de los fármacos , Reactivos de Enlaces Cruzados , ADN Complementario/biosíntesis , ADN Complementario/genética , Dimetilsulfóxido/química , Evaluación Preclínica de Medicamentos , Escherichia coli/metabolismo , Ácidos Grasos/química , Colorantes Fluorescentes , Humanos , Cinética , Unión Proteica , Proteínas Recombinantes , Espectrometría de Fluorescencia , Proteínas tau/aislamiento & purificación
12.
Clin Cancer Res ; 20(13): 3521-30, 2014 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-24780296

RESUMEN

PURPOSE: Irinotecan (CPT-11) induced diarrhea occurs frequently in patients with cancer and limits its usage. Bacteria ß-glucuronidase (GUS) enzymes in intestines convert the nontoxic metabolite of CPT-11, SN-38G, to toxic SN-38, and finally lead to damage of intestinal epithelial cells and diarrhea. We previously reported amoxapine as a potent GUS inhibitor in vitro. To further understand the molecular mechanism of amoxapine and its potential for treatment of CPT-11-induced diarrhea, we studied the binding modes of amoxapine and its metabolites by docking and molecular dynamics simulation, and tested the in vivo efficacy on mice in combination with CPT-11. EXPERIMENTAL DESIGN: The binding of amoxapine, its metabolites, 7-hydroxyamoxapine and 8-hydroxyamoxapine, and a control drug loxapine with GUS was explored by computational protocols. The in vitro potencies of metabolites were measured by Escherichia coli GUS enzyme and cell-based assay. Low-dosage daily oral administration was designed to use along with CPT-11 to treat tumor-bearing mice. RESULTS: Computational modeling results indicated that amoxapine and its metabolites bound in the active site of GUS and satisfied critical pharmacophore features: aromatic features near bacterial loop residue F365' and hydrogen bond toward E413. Amoxapine and its metabolites were demonstrated as potent in vitro. Administration of low dosages of amoxapine with CPT-11 in mice achieved significant suppression of diarrhea and reduced tumor growth. CONCLUSIONS: Amoxapine has great clinical potential to be rapidly translated to human subjects for irinotecan-induced diarrhea.


Asunto(s)
Amoxapina/farmacología , Antineoplásicos/toxicidad , Glicoproteínas/farmacología , Sustancias Protectoras/farmacología , Amoxapina/análogos & derivados , Amoxapina/química , Animales , Antineoplásicos Fitogénicos/toxicidad , Camptotecina/análogos & derivados , Camptotecina/toxicidad , Línea Celular Tumoral , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Femenino , Glicoproteínas/química , Irinotecán , Ratones , Conformación Molecular , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Neoplasias/tratamiento farmacológico , Neoplasias/mortalidad , Neoplasias/patología , Sustancias Protectoras/química , Unión Proteica , Ensayos Antitumor por Modelo de Xenoinjerto
13.
J Biomol Screen ; 17(7): 957-65, 2012 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-22535688

RESUMEN

The active metabolite of the chemotherapeutic irinotecan, SN-38, is detoxified through glucuronidation and then excreted into the gastrointestinal tract. Intestinal bacteria convert the glucuronidated metabolite back to the toxic SN-38 using ß-glucuronidase (GUS), resulting in debilitating diarrhea. Inhibiting GUS activity may relieve this side effect of irinotecan. In this study, we sought to determine whether any known drugs have GUS inhibitory activity. We screened a library of Food and Drug Administration-approved drugs with a cell-free biochemical enzyme assay using purified bacterial GUS. After triage, five drugs were confirmed to inhibit purified bacterial GUS. Three of these were the monoamine oxidase inhibitors nialamide, isocarboxazid, and phenelzine with average IC(50) values for inhibiting GUS of 71, 128, and 2300 nM, respectively. The tricyclic antidepressant amoxapine (IC(50) = 388 nM) and the antimalarial mefloquine (IC(50) = 1.2 µM) also had activity. Nialamide, isocarboxazid, and amoxapine had no significant activity against purified mammalian GUS but showed potent activity for inhibiting endogenous GUS activity in a cell-based assay using living intact Escherichia coli with average IC(50) values of 17, 336, and 119 nM, respectively. Thus, nialamide, isocarboxazid, and amoxapine have potential to be repurposed as therapeutics to reduce diarrhea associated with irinotecan chemotherapy and warrant further investigation for this use.


Asunto(s)
Camptotecina/análogos & derivados , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/farmacología , Escherichia coli/enzimología , Glucuronidasa/antagonistas & inhibidores , Amoxapina/farmacología , Antineoplásicos Fitogénicos/metabolismo , Camptotecina/metabolismo , Descubrimiento de Drogas , Escherichia coli/metabolismo , Proteínas de Escherichia coli/antagonistas & inhibidores , Irinotecán , Isocarboxazida/farmacología , Mefloquina/farmacología , Inhibidores de la Monoaminooxidasa/farmacología , Nialamida/farmacología , Fenelzina/farmacología
14.
Curr Chem Genomics ; 5: 13-20, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21643506

RESUMEN

CPT-11 is a widely-used anti-cancer drug that is converted in vivo to its active metabolite, SN-38. In the liver, enzymes detoxify SN-38 by coupling it to a glucuronidate moiety and this inactive compound (SN-38G) is excreted into the gastrointestinal tract. In the intestine, commensal bacteria convert the SN-38G back to the active and toxic SN-38 using bacterial ß-glucuronidase enzyme (GUS). This intestinal SN-38 causes debilitating diarrhea that prevents dose-intensification and efficacy in a significant fraction of patients undergoing CPT-11 treatment for cancer. This CPT-11 metabolic pathway suggests that small molecule inhibitors of GUS may have utility as novel therapeutics for prevention of dose-limiting diarrhea resulting from CPT-11 therapy. To identify chemical inhibitors of GUS activity, we employed and validated a high throughput, fluorescence-based biochemical assay and used this assay to screen a compound library. Novel inhibitors of GUS were identified with IC(50) values ranging from 50 nM to 4.8 µM. These compounds may be useful as chemical probes for use in proof-of-concept experiments designed to determine the efficacy of GUS inhibitors in altering the intestinal metabolism of drugs. Our results demonstrate that this high throughput assay can be used to identify small molecule inhibitors of GUS.

15.
Curr Chem Genomics ; 5: 30-41, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21760877

RESUMEN

Glycolytic isozymes that are restricted to the male germline are potential targets for the development of reversible, non-hormonal male contraceptives. GAPDHS, the sperm-specific isoform of glyceraldehyde-3-phosphate dehydrogenase, is an essential enzyme for glycolysis making it an attractive target for rational drug design. Toward this goal, we have optimized and validated a high-throughput spectrophotometric assay for GAPDHS in 384-well format. The assay was stable over time and tolerant to DMSO. Whole plate validation experiments yielded Z' values >0.8 indicating a robust assay for HTS. Two compounds were identified and confirmed from a test screen of the Prestwick collection. This assay was used to screen a diverse chemical library and identified fourteen small molecules that modulated the activity of recombinant purified GAPDHS with confirmed IC50 values ranging from 1.8 to 42 µM. These compounds may provide useful scaffolds as molecular tools to probe the role of GAPDHS in sperm motility and long term to develop potent and selective GAPDHS inhibitors leading to novel contraceptive agents.

16.
Curr Chem Genomics ; 4: 34-42, 2010 May 26.
Artículo en Inglés | MEDLINE | ID: mdl-20648224

RESUMEN

The bacterial RecA protein has been implicated as a bacterial drug target not as an antimicrobial target, but as an adjuvant target with the potential to suppress the mechanism by which bacteria gain drug resistance. In order to identify small molecules that inhibit RecA/ssDNA nucleoprotein filament formation, we have adapted the phosphomolybdate-blue ATPase assay for high throughput screening to determine RecA ATPase activity against a library of 33,600 compounds, which is a selected representation of diverse structure of 350,000. Four distinct chemotypes were represented among the 40 validated hits. SAR and further chemical synthesis is underway to optimize this set of inhibitors to be used as antimicrobial adjuvant agents.

17.
Science ; 330(6005): 831-5, 2010 Nov 05.
Artículo en Inglés | MEDLINE | ID: mdl-21051639

RESUMEN

The dose-limiting side effect of the common colon cancer chemotherapeutic CPT-11 is severe diarrhea caused by symbiotic bacterial ß-glucuronidases that reactivate the drug in the gut. We sought to target these enzymes without killing the commensal bacteria essential for human health. Potent bacterial ß-glucuronidase inhibitors were identified by high-throughput screening and shown to have no effect on the orthologous mammalian enzyme. Crystal structures established that selectivity was based on a loop unique to bacterial ß-glucuronidases. Inhibitors were highly effective against the enzyme target in living aerobic and anaerobic bacteria, but did not kill the bacteria or harm mammalian cells. Finally, oral administration of an inhibitor protected mice from CPT-11-induced toxicity. Thus, drugs may be designed to inhibit undesirable enzyme activities in essential microbial symbiotes to enhance chemotherapeutic efficacy.


Asunto(s)
Antineoplásicos Fitogénicos/toxicidad , Camptotecina/análogos & derivados , Inhibidores Enzimáticos/farmacología , Glucuronidasa/antagonistas & inhibidores , Glucuronidasa/farmacología , Animales , Antineoplásicos Fitogénicos/metabolismo , Bacterias Anaerobias/efectos de los fármacos , Camptotecina/metabolismo , Camptotecina/toxicidad , Línea Celular Tumoral , Colon/efectos de los fármacos , Colon/microbiología , Colon/patología , Cristalografía por Rayos X , Diarrea/prevención & control , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Escherichia coli/enzimología , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/aislamiento & purificación , Proteínas de Escherichia coli/metabolismo , Femenino , Glucuronidasa/química , Glucuronidasa/aislamiento & purificación , Glucuronidasa/metabolismo , Humanos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Irinotecán , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Profármacos/metabolismo , Profármacos/toxicidad , Conformación Proteica
18.
J Biomol Screen ; 14(9): 1092-101, 2009 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-19675313

RESUMEN

The phenomenon of antibiotic resistance has created a need for the development of novel antibiotic classes with nonclassical cellular targets. Unfortunately, target-based drug discovery against proteins considered essential for in vitro bacterial viability has yielded few new therapeutic classes of antibiotics. Targeting the large proportion of genes considered nonessential that have yet to be explored by high-throughput screening, for example, RecA, can complement these efforts. Recent evidence suggests that RecA-controlled processes are responsible for tolerance to antibiotic chemotherapy and are involved in pathways that ultimately lead to full-fledged antibiotic resistance. Therefore inhibitors of RecA may serve as therapeutic adjuvants in combination chemotherapy of bacterial infectious diseases. Toward the goal of validating RecA as a novel target in the chemotherapy of bacterial infections, the authors have screened 35,780 small molecules against RecA. In total, 80 small molecules were identified as primary hits and could be clustered in 6 distinct chemotype clades. The most potent class of hits was further examined, and 1 member compound was found to inhibit RecA-mediated strand exchange and prevent ciprofloxacin-induced SOS expression in Escherichia coli. This compound represents the first small molecule demonstrating an ability to inhibit the bacterial SOS response in live bacterial cell cultures.


Asunto(s)
Inhibidores Enzimáticos/farmacología , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento/métodos , Rec A Recombinasas/antagonistas & inhibidores , Respuesta SOS en Genética/efectos de los fármacos , Adenosina Trifosfatasas/metabolismo , Antiinfecciosos/farmacología , Permeabilidad de la Membrana Celular , Ciprofloxacina/farmacología , Farmacorresistencia Bacteriana , Inhibidores Enzimáticos/química , Escherichia coli/metabolismo , Estructura Molecular , Rec A Recombinasas/metabolismo , Reproducibilidad de los Resultados , Bibliotecas de Moléculas Pequeñas
19.
PLoS One ; 4(4): e5274, 2009.
Artículo en Inglés | MEDLINE | ID: mdl-19384407

RESUMEN

BACKGROUND: Hypocatabolism of the amyloid beta-protein (Abeta) by insulin-degrading enzyme (IDE) is implicated in the pathogenesis of Alzheimer disease (AD), making pharmacological activation of IDE an attractive therapeutic strategy. However, it has not been established whether the proteolytic activity of IDE can be enhanced by drug-like compounds. METHODOLOGY/PRINCIPAL FINDINGS: Based on the finding that ATP and other nucleotide polyphosphates modulate IDE activity at physiological concentrations, we conducted parallel high-throughput screening campaigns in the absence or presence of ATP and identified two compounds--designated Ia1 and Ia2--that significantly stimulate IDE proteolytic activity. Both compounds were found to interfere with the crosslinking of a photoaffinity ATP analogue to IDE, suggesting that they interact with a bona fide ATP-binding domain within IDE. Unexpectedly, we observed highly synergistic activation effects when the activity of Ia1 or Ia2 was tested in the presence of ATP, a finding that has implications for the mechanisms underlying ATP-mediated activation of IDE. Notably, Ia1 and Ia2 activated the degradation of Abeta by approximately 700% and approximately 400%, respectively, albeit only when Abeta was presented in a mixture also containing shorter substrates. CONCLUSIONS/SIGNIFICANCE: This study describes the first examples of synthetic small-molecule activators of IDE, showing that pharmacological activation of this important protease with drug-like compounds is achievable. These novel activators help to establish the putative ATP-binding domain as a key modulator of IDE proteolytic activity and offer new insights into the modulatory action of ATP. Several larger lessons abstracted from this screen will help inform the design of future screening campaigns and facilitate the eventual development of IDE activators with therapeutic utility.


Asunto(s)
Química Farmacéutica , Activadores de Enzimas/farmacología , Insulina/metabolismo , Insulisina/metabolismo , Péptidos beta-Amiloides/metabolismo , Hidrólisis , Etiquetas de Fotoafinidad
20.
Anal Biochem ; 338(2): 237-44, 2005 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-15745743

RESUMEN

Tissue transglutaminase (TGase) is a Ca(2+)-dependent enzyme that catalyzes cross-linking of intracellular proteins through a mechanism that involves isopeptide bond formation between Gln and Lys residues. In addition to its transamidation activity, TGase can bind guanosine 5'-triphosphate (GTP) and does so in a manner that is antagonized by calcium. Once bound, GTP undergoes hydrolysis to form guanosine 5'-diphosphate and inorganic phosphate. TGase is thought to play a pathogenic role in neurodegenerative diseases by promoting aggregation of disease-specific proteins that accumulate in these disorders. Thus, this enzyme represents a viable target for drug discovery. We now report the development of a mechanism-based assay for TGase and the results of a screen using this assay in which we tested 56,500 drug-like molecules for their ability to inhibit TGase. In this assay, the Gln- and Lys-donating substrates are N,N-dimethylated casein (NMC) and N-Boc-Lys-NH-CH(2)-CH(2)-NH-dansyl (KXD), respectively. Through a combination of steady state kinetic experiments and reaction progress curve simulations, we were able to calculate values for the initial concentrations of NMC, KXD, and Ca(2+) that would produce a steady state situation in which all thermodynamically significant forms of substrate-bound TGase exist in equal concentration. Under these conditions, the assay is sensitive to both competitive and mixed active-site inhibitors and to inhibitors that bind to the GTP site. The assay was optimized for automated screening in 384-well format and was then used to test our compound library. From among these compounds, 104 authentic hits that represent several mechanistic classes were identified.


Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/farmacología , Proteínas de Unión al GTP/antagonistas & inhibidores , Transglutaminasas/antagonistas & inhibidores , Caseínas/química , Compuestos de Dansilo/química , Lisina/análogos & derivados , Lisina/química , Proteína Glutamina Gamma Glutamiltransferasa 2
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