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1.
Nat Commun ; 12(1): 4718, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354069

RESUMO

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.


Assuntos
Fosfatidato Fosfatase/química , Células 3T3-L1 , Adipogenia , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Sequência Conservada , Cristalografia por Raios X , Células HEK293 , Humanos , Espectrometria de Massa com Troca Hidrogênio-Deutério , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Modelos Moleculares , Simulação de Dinâmica Molecular , Fosfatidato Fosfatase/genética , Fosfatidato Fosfatase/metabolismo , Ligação Proteica , Domínios Proteicos , Dobramento de Proteína , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Transcrição Genética
2.
SLAS Discov ; 26(1): 113-121, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32734807

RESUMO

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).

3.
PLoS One ; 11(12): e0168019, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27936107

RESUMO

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.


Assuntos
Antimetabólitos Antineoplásicos/química , Herpesvirus Humano 8/enzimologia , Núcleosídeo-Fosfato Quinase/química , Quinazolinas/química , Tiofenos/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Ligantes , Modelos Moleculares , Estrutura Molecular , Homologia de Sequência de Aminoácidos
4.
J Pharm Anal ; 4(3): 197-204, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29403883

RESUMO

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).

5.
Bioorg Med Chem Lett ; 22(9): 3296-300, 2012 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-22464456

RESUMO

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.


Assuntos
Desenho de Fármacos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirazóis/química , Pirazóis/farmacologia , Cristalografia por Raios X , Humanos , Relação Estrutura-Atividade
6.
Bioorg Med Chem Lett ; 20(6): 1821-4, 2010 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-20185308

RESUMO

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.


Assuntos
Inibidores Enzimáticos/farmacologia , Fatores Inibidores da Migração de Macrófagos/antagonistas & inibidores , Sítios de Ligação , Cristalografia por Raios X , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Fatores Inibidores da Migração de Macrófagos/química , Fatores Inibidores da Migração de Macrófagos/metabolismo , Modelos Moleculares , Estrutura Molecular
8.
Curr Med Chem ; 14(23): 2471-81, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17979700

RESUMO

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.


Assuntos
Óxidos N-Cíclicos/farmacologia , Inibidores do Fator Xa , Piridinas/farmacologia , Animais , Ensaios Clínicos como Assunto , Óxidos N-Cíclicos/química , Cães , Desenho de Fármacos , Ésteres/química , Haplorrinos , Humanos , Camundongos , Modelos Biológicos , Modelos Químicos , Conformação Molecular , Piridinas/química , Coelhos , Serina Endopeptidases/química , Transdução de Sinais , Resultado do Tratamento
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