Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 11 de 11
Filtrar
1.
Bioorg Chem ; 139: 106747, 2023 10.
Artículo en Inglés | MEDLINE | ID: mdl-37531819

RESUMEN

Ceramides impact a diverse array of biological functions and have been implicated in disease pathogenesis. The enzyme neutral ceramidase (nCDase) is a zinc-containing hydrolase and mediates the metabolism of ceramide to sphingosine (Sph), both in cells and in the intestinal lumen. nCDase inhibitors based on substrate mimetics, for example C6-urea ceramide, have limited potency, aqueous solubility, and micelle-free fraction. To identify non-ceramide mimetic nCDase inhibitors, hit compounds from an HTS campaign were evaluated in biochemical, cell based and in silico modeling approaches. A majority of small molecule nCDase inhibitors contained pharmacophores capable of zinc interaction but retained specificity for nCDase over zinc-containing acid and alkaline ceramidases, as well as matrix metalloprotease-3 and histone deacetylase-1. nCDase inhibitors were refined by SAR, were shown to be substrate competitive and were active in cellular assays. nCDase inhibitor compounds were modeled by in silico DOCK screening and by molecular simulation. Modeling data supports zinc interaction and a similar compound binding pose with ceramide. nCDase inhibitors were identified with notably improved activity and solubility in comparison with the reference lipid-mimetic C6-urea ceramide.


Asunto(s)
Ceramidas , Ceramidasa Neutra , Dominio Catalítico , Ceramidas/química , Ceramidasa Neutra/antagonistas & inhibidores , Esfingosina/química
2.
Anal Biochem ; 643: 114577, 2022 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-35134389

RESUMEN

Neutral ceramidase is a hydrolase of ceramide that has been implicated in multiple biologic processes, including inflammation and oncogenesis. Ceramides and other sphingolipids, belong to a family of N-acyl linked lipids that are biologically active in signaling, despite their limited structural functions. Ceramides are generally pro-apoptotic, while sphingosine and sphingosine-1-phosphate (S1P) exert proliferative and pro-oncogenic effects. Ceramidases are important regulators of ceramide levels that hydrolyze ceramide to sphingosine. Thus, ceramidase inhibition significantly increases the quantities of ceramide and its associated signaling. To better understand the function of ceramide, biochemical and cellular assays for enzymatic activity were developed and validated to identify inhibitors of human neutral ceramidase (nCDase). Here we review the measurement of nCDase activity both in vitro and in vivo.


Asunto(s)
Ceramidasa Neutra/análisis , Humanos , Ceramidasa Neutra/genética , Ceramidasa Neutra/metabolismo , Pseudomonas aeruginosa/enzimología
3.
Nat Commun ; 14(1): 3204, 2023 06 02.
Artículo en Inglés | MEDLINE | ID: mdl-37268630

RESUMEN

Lipid droplets (LDs) are dynamic organelles that contain an oil core mainly composed of triglycerides (TAG) that is surrounded by a phospholipid monolayer and LD-associated proteins called perilipins (PLINs). During LD biogenesis, perilipin 3 (PLIN3) is recruited to nascent LDs as they emerge from the endoplasmic reticulum. Here, we analyze how lipid composition affects PLIN3 recruitment to membrane bilayers and LDs, and the structural changes that occur upon membrane binding. We find that the TAG precursors phosphatidic acid and diacylglycerol (DAG) recruit PLIN3 to membrane bilayers and define an expanded Perilipin-ADRP-Tip47 (PAT) domain that preferentially binds DAG-enriched membranes. Membrane binding induces a disorder to order transition of alpha helices within the PAT domain and 11-mer repeats, with intramolecular distance measurements consistent with the expanded PAT domain adopting a folded but dynamic structure upon membrane binding. In cells, PLIN3 is recruited to DAG-enriched ER membranes, and this requires both the PAT domain and 11-mer repeats. This provides molecular details of PLIN3 recruitment to nascent LDs and identifies a function of the PAT domain of PLIN3 in DAG binding.


Asunto(s)
Diglicéridos , Perilipina-3 , Diglicéridos/metabolismo , Retículo Endoplásmico/metabolismo , Gotas Lipídicas/metabolismo , Metabolismo de los Lípidos/fisiología , Perilipina-1/metabolismo , Perilipina-3/metabolismo , Triglicéridos/metabolismo
4.
Bioorg Med Chem Lett ; 22(9): 3296-300, 2012 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-22464456

RESUMEN

Beginning with a screening hit, unique thienopyrazole-indole inhibitors of Itk (interleukin-2-inducible tyrosine kinase) were designed, synthesized, and crystallized in the target kinase. Although initial compounds were highly active in Itk, they were not selective. Increasing the steric bulk around a tertiary alcohol at the 5-indole position dramatically improved selectivity toward Lyk and Syk, but not Txk. Substitutions at the 3- and 4-indole positions gave less active compounds that remained poorly selective. A difluoromethyl substitution at the 5-position of the thienopyrazole led to a highly potent and selective compound. Phenyl at this position reduced activity and selectivity while pushing the side-chains of Lys-391 and Asp-500 away from the binding pocket. Novel and selective thienopyrazole inhibitors of Itk were designed as a result of combining structure-based design and medicinal chemistry.


Asunto(s)
Diseño de Fármacos , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirazoles/química , Pirazoles/farmacología , Cristalografía por Rayos X , Humanos , Relación Estructura-Actividad
5.
Nat Commun ; 12(1): 4718, 2021 08 05.
Artículo en Inglés | MEDLINE | ID: mdl-34354069

RESUMEN

Phospholipid synthesis and fat storage as triglycerides are regulated by lipin phosphatidic acid phosphatases (PAPs), whose enzymatic PAP function requires association with cellular membranes. Using hydrogen deuterium exchange mass spectrometry, we find mouse lipin 1 binds membranes through an N-terminal amphipathic helix, the Ig-like domain and HAD phosphatase catalytic core, and a middle lipin (M-Lip) domain that is conserved in mammalian and mammalian-like lipins. Crystal structures of the M-Lip domain reveal a previously unrecognized protein fold that dimerizes. The isolated M-Lip domain binds membranes both in vitro and in cells through conserved basic and hydrophobic residues. Deletion of the M-Lip domain in lipin 1 reduces PAP activity, membrane association, and oligomerization, alters subcellular localization, diminishes acceleration of adipocyte differentiation, but does not affect transcriptional co-activation. This establishes the M-Lip domain as a dimeric protein fold that binds membranes and is critical for full functionality of mammalian lipins.


Asunto(s)
Fosfatidato Fosfatasa/química , Células 3T3-L1 , Adipogénesis , Secuencia de Aminoácidos , Animales , Membrana Celular/metabolismo , Secuencia Conservada , Cristalografía por Rayos X , Células HEK293 , Humanos , Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Modelos Moleculares , Simulación de Dinámica Molecular , Fosfatidato Fosfatasa/genética , Fosfatidato Fosfatasa/metabolismo , Unión Proteica , Dominios Proteicos , Pliegue de Proteína , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Transcripción Genética
6.
SLAS Discov ; 26(1): 113-121, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-32734807

RESUMEN

There is interest in developing inhibitors of human neutral ceramidase (nCDase) because this enzyme plays a critical role in colon cancer. There are currently no potent or clinically effective inhibitors for nCDase reported to date, so we adapted a fluorescence-based enzyme activity method to a high-throughput screening format. We opted to use an assay whereby nCDase hydrolyzes the substrate RBM 14-16, and the addition of NaIO4 acts as an oxidant that releases umbelliferone, resulting in a fluorescent signal. As designed, test compounds that act as ceramidase inhibitors will prevent the hydrolysis of RBM 14-16, thereby decreasing fluorescence. This assay uses a 1536-well plate format with excitation in the blue spectrum of light energy, which could be a liability, so we incorporated a counterscreen that allows for rapid selection against fluorescence artifacts to minimize false-positive hits. The high-throughput screen of >650,000 small molecules found several lead series of hits. Multiple rounds of chemical optimization ensued with improved potency in terms of IC50 and selectivity over counterscreen assays. This study describes the first large-scale high-throughput optical screening assay for nCDase inhibitors that has resulted in leads that are now being pursued in crystal docking studies and in vitro drug metabolism and pharmacokinetics (DMPK).


Asunto(s)
Descubrimiento de Drogas/métodos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Ensayos Analíticos de Alto Rendimiento , Ceramidasa Neutra/antagonistas & inhibidores , Ceramidasa Neutra/química , Animales , Línea Celular , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales/métodos , Activación Enzimática/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Bibliotecas de Moléculas Pequeñas
7.
Bioorg Med Chem Lett ; 20(6): 1821-4, 2010 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-20185308

RESUMEN

In the course of a fragment screening campaign by in silico docking followed by X-ray crystallography, a novel binding site for migration inhibitory factor (MIF) inhibitors was demonstrated. The site is formed by rotation of the side-chain of Tyr-36 to reveal a surface binding site in MIF that is hydrophobic and surrounded by aromatic side-chain residues. The crystal structures of two small inhibitors that bind to this site and of a quinolinone inhibitor, that spans the canonical deep pocket near Pro-1 and the new surface binding site, have been solved. These results suggest new opportunities for structure-based design of MIF inhibitors.


Asunto(s)
Inhibidores Enzimáticos/farmacología , Factores Inhibidores de la Migración de Macrófagos/antagonistas & inhibidores , Sitios de Unión , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Factores Inhibidores de la Migración de Macrófagos/química , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Modelos Moleculares , Estructura Molecular
9.
Curr Med Chem ; 14(23): 2471-81, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-17979700

RESUMEN

Factor Xa (fXa) is a critical serine protease situated at the confluence of the intrinsic and extrinsic pathways of the blood coagulation cascade. FXa catalyses the conversion of prothrombin to thrombin via the prothrombinase complex. Its singular role in thrombin generation, coupled with its potentiating effects on clot formation render it an attractive target for therapeutic intervention. Otamixaban is a synthetically derived parenteral fXa inhibitor currently in late stage clinical development at Sanofi-Aventis for the management of acute coronary syndrome. Otamixaban is a potent (Ki = 0.5 nM), selective, rapid acting, competitive and reversible fXa inhibitor that effectively inhibits both free and prothrombinase-bound fXa. In vivo experiments have demonstrated that Otamixaban is highly efficacious in rodent, canine and porcine models of thrombosis. In addition, recent clinical findings indicate that Otamixaban is efficacious, safe and well tolerated in humans and therefore has considerable potential for the treatment of acute coronary syndrome. This review article chronicles the discovery and pre-clinical data surrounding the fXa inhibitor Otamixaban as well as the recent clinical findings in humans.


Asunto(s)
Óxidos N-Cíclicos/farmacología , Inhibidores del Factor Xa , Piridinas/farmacología , Animales , Ensayos Clínicos como Asunto , Óxidos N-Cíclicos/química , Perros , Diseño de Fármacos , Ésteres/química , Haplorrinos , Humanos , Ratones , Modelos Biológicos , Modelos Químicos , Conformación Molecular , Piridinas/química , Conejos , Serina Endopeptidasas/química , Transducción de Señal , Resultado del Tratamiento
10.
PLoS One ; 11(12): e0168019, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27936107

RESUMEN

Kaposi's sarcoma-associated herpesvirus (KSHV) is a highly infectious human herpesvirus that causes Kaposi's sarcoma. KSHV encodes functional thymidylate synthase, which is a target for anticancer drugs such as raltitrexed or 5-fluorouracil. Thymidylate synthase catalyzes the conversion of 2'-deoxyuridine-5'-monophosphate (dUMP) to thymidine-5'-monophosphate (dTMP) using 5,10-methylenetetrahydrofolate (mTHF) as a co-substrate. The crystal structures of thymidylate synthase from KSHV (apo), complexes with dUMP (binary), and complexes with both dUMP and raltitrexed (ternary) were determined at 1.7 Å, 2.0 Å, and 2.4 Å, respectively. While the ternary complex structures of human thymidylate synthase and E. coli thymidylate synthase had a closed conformation, the ternary complex structure of KSHV thymidylate synthase was observed in an open conformation, similar to that of rat thymidylate synthase. The complex structures of KSHV thymidylate synthase did not have a covalent bond between the sulfhydryl group of Cys219 and C6 atom of dUMP, unlike the human thymidylate synthase. The catalytic Cys residue demonstrated a dual conformation in the apo structure, and its sulfhydryl group was oriented toward the C6 atom of dUMP with no covalent bond upon ligand binding in the complex structures. These structural data provide the potential use of antifolates such as raltitrexed as a viral induced anticancer drug and structural basis to design drugs for targeting the thymidylate synthase of KSHV.


Asunto(s)
Antimetabolitos Antineoplásicos/química , Herpesvirus Humano 8/enzimología , Nucleósido-Fosfato Quinasa/química , Quinazolinas/química , Tiofenos/química , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Ligandos , Modelos Moleculares , Estructura Molecular , Homología de Secuencia de Aminoácido
11.
J Pharm Anal ; 4(3): 197-204, 2014 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-29403883

RESUMEN

Otamixaban is a potent (Ki=0.5 nM) fXa inhibitor currently in late-stage clinical development at Sanofi for the management of acute coronary syndrome. Being unproductive in obtaining a suitable crystal of Otamixaban, the required enantiomeric characterization has been accomplished using vibrational circular dichroism (VCD) spectroscopy. Selected by a spectrum similarity index, the calculated spectra of several higher energy conformers were found to match well with the observed spectra. The characteristic IR bands of these conformers were also identified and attributed to the solvation effect. Combined with both the single crystal x-ray diffraction results for an intermediate and the proton NMR study, the absolute configuration of Otamixaban is unambiguously determined to be (R,R).

SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA