Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 18 de 18
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Bioanalysis ; 15(15): 861-903, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37584363

RESUMO

The 16th Workshop on Recent Issues in Bioanalysis (16th WRIB) took place in Atlanta, GA, USA on September 26-30, 2022. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 16th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on ICH M10 BMV final guideline (focused on this guideline training, interpretation, adoption and transition); mass spectrometry innovation (focused on novel technologies, novel modalities, and novel challenges); and flow cytometry bioanalysis (rising of the 3rd most common/important technology in bioanalytical labs) were the special features of the 16th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2022 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2022 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations on LBA, Biomarkers/CDx and Cytometry. Part 1 (Mass Spectrometry and ICH M10) and Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) are published in volume 15 of Bioanalysis, issues 16 and 14 (2023), respectively.


Assuntos
Bioensaio , Relatório de Pesquisa , Citometria de Fluxo/métodos , Ligantes , Biomarcadores/análise , Bioensaio/métodos
2.
ACS Omega ; 6(15): 10288-10305, 2021 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-34056183

RESUMO

The nematode Haemonchus contortus (the barber's pole worm) is an endoparasite infecting wild and domesticated ruminants worldwide. Widespread anthelmintic resistance of H. contortus requires alternative strategies to control this parasite. Neuropeptide signaling represents a promising target for anthelmintic drugs. Identification and relative quantification of nematode neuropeptides are, therefore, required for the development of such therapeutic targets. In this work, we undertook the profiling of the whole H. contortus larvae at different stages for the direct sequencing of the neuropeptides expressed at low levels in these tissues. We set out a peptide extraction protocol and a peptidomic workflow to biochemically characterize bioactive peptides from both first-stage (L1) and third-stage larvae (L3) of H. contortus. This work led to the identification and quantification at the peptidomic level of more than 180 mature neuropeptides, including amidated and nonamidated peptides, arising from 55 precursors of H. contortus. The differential peptidomic approach provided evidence that both life stages express most FMRFamide-like peptides (FLPs) and neuropeptide-like proteins (NLPs). The H. contortus peptidome resource, established in this work, could add the discovery of neuropeptide system-targeting drugs for ruminants.

3.
PLoS One ; 15(6): e0234780, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32579589

RESUMO

Obesity epidemic continues to spread and obesity rates are increasing in the world. In addition to public health effort to reduce obesity, there is a need to better understand the underlying biology to enable more effective treatment and the discovery of new pharmacological agents. Abhydrolase domain-containing protein 11 (ABHD11) is a serine hydrolase enzyme, localized in mitochondria, that can synthesize the endocannabinoid 2-arachidonoyl glycerol (2AG) in vitro. In vivo preclinical studies demonstrated that knock-out ABHD11 mice have a similar 2AG level as WT mice and exhibit a lean metabolic phenotype. Such mice resist to weight gain in Diet Induced Obesity studies (DIO) and display normal biochemical plasma parameters. Metabolic and transcriptomic analyses on serum and tissues of ABHD11 KO mice from DIO studies show a modulation in bile salts associated with reduced fat intestinal absorption. These data suggest that modulating ABHD11 signaling pathway could be of therapeutic value for the treatment of metabolic disorders.


Assuntos
Serina Proteases/metabolismo , Aumento de Peso , Animais , Fezes/enzimologia , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Células MCF-7 , Camundongos , Mitocôndrias/metabolismo , Serina Proteases/deficiência , Serina Proteases/genética , Transdução de Sinais
4.
Diabetes Obes Metab ; 22(8): 1328-1338, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32196896

RESUMO

AIMS: To test specific mono-agonists to the glucagon-like peptide-1 receptor (GLP-1R), glucagon receptor (GCGR) and glucose-dependent insulinotropic peptide receptor (GIPR), individually and in combination, in a mouse model of diet-induced non-alcoholic steatohepatitis (NASH) and fibrosis in order to decipher the contribution of their activities and potential additive effects to improving systemic and hepatic metabolism. MATERIALS AND METHODS: We induced NASH by pre-feeding C57BL/6J mice a diet rich in fat, fructose and cholesterol for 36 weeks. This was followed by 8 weeks of treatment with the receptor-specific agonists 1-GCG (20 µg/kg twice daily), 2-GLP1 (3 µg/kg twice daily) or 3-GIP (30 µg/kg twice daily), or the dual (1 + 2) or triple (1 + 2 + 3) combinations thereof. A dual GLP-1R/GCGR agonistic peptide, 4-dual-GLP1/GCGR (30 µg/kg twice daily), and liraglutide (100 µg/kg twice daily) were included as references. RESULTS: Whereas low-dose 1-GCG or 3-GIP alone did not influence body weight, liver lipids and histology, their combination with 2-GLP1 provided additional weight loss, reduction in liver triglycerides and improvement in histological disease activity score. Notably, 4-dual-GLP-1R/GCGR and the triple combination of selective mono-agonists led to a significantly stronger reduction in the histological non-alcoholic fatty liver disease activity score compared to high-dose liraglutide, at the same extent of body weight loss. CONCLUSIONS: GCGR and GIPR agonism provide additional, body weight-independent improvements on top of GLP-1R agonism in a murine model of manifest NASH with fibrosis.


Assuntos
Incretinas , Hepatopatia Gordurosa não Alcoólica , Animais , Receptor do Peptídeo Semelhante ao Glucagon 1 , Incretinas/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Receptores de Glucagon
5.
J Clin Transl Hepatol ; 8(4): 359-370, 2020 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-33447518

RESUMO

Background and Aims: To better understand nonalcoholic steatohepatitis (NASH) disease progression and to evaluate drug targets and compound activity, we undertook the development of an in vitro 3D model to mimic liver architecture and the NASH environment. Methods: We have developed an in vitro preclinical 3D NASH model by coculturing primary human hepatocytes, human stellate cells, liver endothelial cells and Kupffer cells embedded in a hydrogel of rat collagen on a 96-well plate. A NASH-like environment was induced by addition of medium containing free fatty acids and tumor necrosis factor-α. This model was then characterized by biochemical, imaging and transcriptomics analyses. Results: We succeeded in defining suitable culture conditions to maintain the 3D coculture for up to 10 days in vitro, with the lowest level of steatosis and reproducible low level of inflammation and fibrosis. NASH disease was induced with a custom medium mimicking NASH features. The cell model exhibited the key NASH disease phenotypes of hepatocyte injury, steatosis, inflammation, and fibrosis. Hepatocyte injury was highlighted by a decrease of CYP3A4 expression and activity, without loss of viability up to day 10. Moreover, the model was able to stimulate a stable inflammatory and early fibrotic environment, with expression and secretion of several cytokines. A global gene expression analysis confirmed the NASH induction. Conclusions: This is a new in vitro model of NASH disease consisting of four human primary cell-types that exhibits most features of the disease. The 10-day cell viability and cost effectiveness of the model make it suitable for medium throughput drug screening and provide attractive avenues to better understand disease physiology and to identify and characterize new drug targets.

6.
Nat Med ; 25(9): 1402-1407, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31501610

RESUMO

Natalizumab (NZM), a humanized monoclonal IgG4 antibody to α4 integrins, is used to treat patients with relapsing-remitting multiple sclerosis (MS)1,2, but in about 6% of the cases persistent neutralizing anti-drug antibodies (ADAs) are induced leading to therapy discontinuation3,4. To understand the basis of the ADA response and the mechanism of ADA-mediated neutralization, we performed an in-depth analysis of the B and T cell responses in two patients. By characterizing a large panel of NZM-specific monoclonal antibodies, we found that, in both patients, the response was polyclonal and targeted different epitopes of the NZM idiotype. The neutralizing activity was acquired through somatic mutations and correlated with a slow dissociation rate, a finding that was supported by structural data. Interestingly, in both patients, the analysis of the CD4+ T cell response, combined with mass spectrometry-based peptidomics, revealed a single immunodominant T cell epitope spanning the FR2-CDR2 region of the NZM light chain. Moreover, a CDR2-modified version of NZM was not recognized by T cells, while retaining binding to α4 integrins. Collectively, our integrated analysis identifies the basis of T-B collaboration that leads to ADA-mediated therapeutic resistance and delineates an approach to design novel deimmunized antibodies for autoimmune disease and cancer treatment.


Assuntos
Anticorpos Neutralizantes/administração & dosagem , Epitopos de Linfócito T/imunologia , Esclerose Múltipla/tratamento farmacológico , Natalizumab/administração & dosagem , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Neutralizantes/química , Formação de Anticorpos/efeitos dos fármacos , Formação de Anticorpos/imunologia , Linfócitos B/efeitos dos fármacos , Humanos , Imunoglobulina G/química , Imunoglobulina G/imunologia , Integrina alfa4/antagonistas & inibidores , Integrina alfa4/imunologia , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Conformação Proteica/efeitos dos fármacos , Linfócitos T/química , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia
7.
Stem Cell Reports ; 13(2): 380-393, 2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31378672

RESUMO

Here, we have used patient-derived induced pluripotent stem cell (iPSC) and gene-editing technology to study the cardiac-related molecular and functional consequences of mutations in GLA causing the lysosomal storage disorder Fabry disease (FD), for which heart dysfunction is a major cause of mortality. Our in vitro model recapitulated clinical data with FD cardiomyocytes accumulating GL-3 and displaying an increased excitability, with altered electrophysiology and calcium handling. Quantitative proteomics enabled the identification of >5,500 proteins in the cardiomyocyte proteome and secretome, and revealed accumulation of the lysosomal protein LIMP-2 and secretion of cathepsin F and HSPA2/HSP70-2 in FD. Genetic correction reversed these changes. Overexpression of LIMP-2 directly induced the secretion of cathepsin F and HSPA2/HSP70-2, implying causative relationship, and led to massive vacuole accumulation. In summary, our study has revealed potential new cardiac biomarkers for FD, and provides valuable mechanistic insight into the earliest pathological events in FD cardiomyocytes.


Assuntos
Doença de Fabry/patologia , Proteínas de Membrana Lisossomal/metabolismo , Modelos Biológicos , Miócitos Cardíacos/metabolismo , Receptores Depuradores/metabolismo , Potenciais de Ação , Biomarcadores/metabolismo , Catepsina F/metabolismo , Edição de Genes , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/fisiologia , Mutação Puntual , Mapas de Interação de Proteínas , Proteômica , Vacúolos/metabolismo , alfa-Galactosidase/genética
8.
Allergy ; 74(3): 549-559, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-29987849

RESUMO

BACKGROUND: Chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) is a chronic inflammatory disease often accompanied by impairment of sense of smell. This symptom has been somewhat overlooked, and its relationship to inflammatory cytokines, tissue compression, neuronal loss, and neurogenesis is still unclear. METHODS: In order to elucidate potential mechanisms leading to CRS in humans, we have established a type 2/T helper type 2 cell (Th2)-mediated allergic CRS mouse model, based on house dust mite (HDM) and Staphylococcus aureus enterotoxin B (SEB) sensitization. The inflammatory status of the olfactory epithelium (OE) was assessed using histology, biochemistry, and transcriptomics. The sense of smell was evaluated by studying olfactory behavior and recording electro-olfactograms (EOGs). RESULTS: After 22 weeks, a typical type 2/Th2-mediated inflammatory profile was obtained, as demonstrated by increased interleukin (IL)-4, IL-5, and IL-13 in the OE. The number of mast cells and eosinophils was increased, and infiltration of these cells into the olfactory mucosa was also observed. In parallel, transcriptomic and histology analyses indicated a decreased number of immature olfactory neurons, possibly due to decreased renewal. However, the number of mature sensory neurons was not affected and neither the EOG nor olfactory behavior was impaired. CONCLUSION: Our mouse model of CRS displayed an allergic response to HDM + SEB administration, including the type 2/Th2 inflammatory profile characteristic of human eosinophilic CRSwNP. Although the sense of smell did not appear to be altered in these conditions, the data reveal the influence of chronic inflammation on olfactory neurogenesis, suggesting that factors unique to humans may be involved in CRSwNP-associated anosmia.


Assuntos
Neurogênese , Mucosa Olfatória/metabolismo , Rinite/etiologia , Rinite/metabolismo , Sinusite/etiologia , Sinusite/metabolismo , Células Th2/imunologia , Células Th2/metabolismo , Animais , Biomarcadores , Doença Crônica , Modelos Animais de Doenças , Camundongos , Neurogênese/genética , Neurogênese/imunologia , Mucosa Olfatória/fisiopatologia , Neurônios Receptores Olfatórios/metabolismo , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Rinite/fisiopatologia , Sinusite/fisiopatologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo
9.
JCI Insight ; 3(11)2018 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-29875313

RESUMO

Multiple sclerosis (MS) is an autoimmune disease characterized by CNS inflammation leading to demyelination and axonal damage. IFN-ß is an established treatment for MS; however, up to 30% of IFN-ß-treated MS patients develop neutralizing antidrug antibodies (nADA), leading to reduced drug bioactivity and efficacy. Mechanisms driving antidrug immunogenicity remain uncertain, and reliable biomarkers to predict immunogenicity development are lacking. Using high-throughput flow cytometry, NOTCH2 expression on CD14+ monocytes and increased frequency of proinflammatory monocyte subsets were identified as baseline predictors of nADA development in MS patients treated with IFN-ß. The association of this monocyte profile with nADA development was validated in 2 independent cross-sectional MS patient cohorts and a prospective cohort followed before and after IFN-ß administration. Reduced monocyte NOTCH2 expression in nADA+ MS patients was associated with NOTCH2 activation measured by increased expression of Notch-responsive genes, polarization of monocytes toward a nonclassical phenotype, and increased proinflammatory IL-6 production. NOTCH2 activation was T cell dependent and was only triggered in the presence of serum from nADA+ patients. Thus, nADA development was driven by a proinflammatory environment that triggered activation of the NOTCH2 signaling pathway prior to first IFN-ß administration.


Assuntos
Hipersensibilidade a Drogas/diagnóstico , Interferon beta/efeitos adversos , Monócitos/metabolismo , Esclerose Múltipla/tratamento farmacológico , Receptor Notch2/metabolismo , Adulto , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Biomarcadores/análise , Biomarcadores/metabolismo , Estudos Transversais , Hipersensibilidade a Drogas/sangue , Hipersensibilidade a Drogas/imunologia , Feminino , Humanos , Interferon beta/imunologia , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/sangue , Esclerose Múltipla/imunologia , Valor Preditivo dos Testes , Prognóstico , Estudos Prospectivos , Receptor Notch2/análise
10.
MAbs ; 8(5): 867-78, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-26984268

RESUMO

Bispecific immunoglobulins (Igs) typically contain at least two distinct variable domains (Fv) that bind to two different target proteins. They are conceived to facilitate clinical development of biotherapeutic agents for diseases where improved clinical outcome is obtained or expected by combination therapy compared to treatment by single agents. Almost all existing formats are linear in their concept and differ widely in drug-like and manufacture-related properties. To overcome their major limitations, we designed cross-over dual variable Ig-like proteins (CODV-Ig). Their design is akin to the design of circularly closed repeat architectures. Indeed, initial results showed that the traditional approach of utilizing (G4S)x linkers for biotherapeutics design does not identify functional CODV-Igs. Therefore, we applied an unprecedented molecular modeling strategy for linker design that consistently results in CODV-Igs with excellent biochemical and biophysical properties. CODV architecture results in a circular self-contained structure functioning as a self-supporting truss that maintains the parental antibody affinities for both antigens without positional effects. The format is universally suitable for therapeutic applications targeting both circulating and membrane-localized proteins. Due to the full functionality of the Fc domains, serum half-life extension as well as antibody- or complement-dependent cytotoxicity may support biological efficiency of CODV-Igs. We show that judicious choice in combination of epitopes and paratope orientations of bispecific biotherapeutics is anticipated to be critical for clinical outcome. Uniting the major advantages of alternative bispecific biotherapeutics, CODV-Igs are applicable in a wide range of disease areas for fast-track multi-parametric drug optimization.


Assuntos
Anticorpos Biespecíficos/biossíntese , Desenho de Fármacos , Modelos Moleculares , Humanos , Engenharia de Proteínas/métodos
11.
Sci Rep ; 5: 7642, 2015 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-25560837

RESUMO

Monoacylglycerol lipase (MAGL) represents a primary degradation enzyme of the endogenous cannabinoid (eCB), 2-arachidonoyglycerol (2-AG). This study reports a potent covalent MAGL inhibitor, SAR127303. The compound behaves as a selective and competitive inhibitor of mouse and human MAGL, which potently elevates hippocampal levels of 2-AG in mice. In vivo, SAR127303 produces antinociceptive effects in assays of inflammatory and visceral pain. In addition, the drug alters learning performance in several assays related to episodic, working and spatial memory. Moreover, long term potentiation (LTP) of CA1 synaptic transmission and acetylcholine release in the hippocampus, two hallmarks of memory function, are both decreased by SAR127303. Although inactive in acute seizure tests, repeated administration of SAR127303 delays the acquisition and decreases kindled seizures in mice, indicating that the drug slows down epileptogenesis, a finding deserving further investigation to evaluate the potential of MAGL inhibitors as antiepileptics. However, the observation that 2-AG hydrolysis blockade alters learning and memory performance, suggests that such drugs may have limited value as therapeutic agents.


Assuntos
Analgésicos/farmacologia , Ácidos Araquidônicos/metabolismo , Carbamatos/farmacologia , Endocanabinoides/metabolismo , Glicerídeos/metabolismo , Aprendizagem/efeitos dos fármacos , Memória de Curto Prazo/efeitos dos fármacos , Monoacilglicerol Lipases/metabolismo , Sulfonamidas/farmacologia , Acetilcolina/metabolismo , Administração Oral , Analgésicos/química , Analgésicos/uso terapêutico , Animais , Ácidos Araquidônicos/química , Sítios de Ligação , Encéfalo/metabolismo , Antagonistas de Receptores de Canabinoides/farmacologia , Carbamatos/química , Carbamatos/uso terapêutico , Cromatografia Líquida de Alta Pressão , Cristalografia por Raios X , Modelos Animais de Doenças , Estimulação Elétrica , Endocanabinoides/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Glicerídeos/química , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Humanos , Hidrólise , Técnicas In Vitro , Potenciação de Longa Duração/efeitos dos fármacos , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Monoacilglicerol Lipases/antagonistas & inibidores , Dor/tratamento farmacológico , Dor/patologia , Piperidinas/farmacologia , Estrutura Terciária de Proteína , Pirazóis/farmacologia , Rimonabanto , Convulsões/tratamento farmacológico , Convulsões/patologia , Sulfonamidas/química , Sulfonamidas/uso terapêutico
12.
J Chem Inf Model ; 52(8): 2204-14, 2012 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-22788756

RESUMO

The implementation of a structure based virtual affinity maturation protocol and evaluation of its predictivity are presented. The in silico protocol is based on conformational sampling of the interface residues (using the Dead End Elimination/A* algorithm), followed by the estimation of the change of free energy of binding due to a point mutation, applying MM/PBSA calculations. Several implementations of the protocol have been evaluated for 173 mutations in 7 different protein complexes for which experimental data were available: the use of the Boltzamnn averaged predictor based on the free energy of binding (ΔΔG(*)) combined with the one based on its polar component only (ΔΔE(pol*)) led to the proposal of a subset of mutations out of which 45% would have successfully enhanced the binding. When focusing on those mutations that are less likely to be introduced by natural in vivo maturation methods (99 mutations with at least two base changes in the codon), the success rate is increased to 63%. In another evaluation, focusing on 56 alanine scanning mutations, the in silico protocol was able to detect 89% of the hot-spots.


Assuntos
Biologia Computacional/métodos , Engenharia de Proteínas/métodos , Proteínas/metabolismo , Proteínas/uso terapêutico , Interface Usuário-Computador , Modelos Moleculares , Mutação Puntual , Conformação Proteica , Proteínas/química , Proteínas/genética , Termodinâmica
13.
J Med Chem ; 54(20): 7206-19, 2011 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-21972823

RESUMO

A novel class of heat shock protein 90 (Hsp90) inhibitors was developed after a low throughput screen (LTS) of a focused library containing approximately 21K compounds selected by virtual screening. The initial [1-{3-H-imidazo[4-5-c]pyridin-2-yl}-3,4-dihydro-2H-pyrido[2,1-a]isoindole-6-one] (1) compound showed moderate activity (IC(50) = 7.6 µM on Hsp82, the yeast homologue of Hsp90). A high-resolution X-ray structure shows that compound 1 binds into an "induced" hydrophobic pocket, 10-15 Å away from the ATP/resorcinol binding site. Iterative cycles of structure-based drug design (SBDD) and chemical synthesis led to the design and preparation of analogues with improved affinity. These optimized molecules make productive interactions within the ATP binding site as reported by other Hsp90 inhibitors. This resulted in compound 8, which is a highly potent inhibitor in biochemical and cellular assays (K(d) = 0.35 nM on Hsp90; IC(50) = 30 nM on SKBr3 mammary carcinoma cells) and in an in vivo leukemia model.


Assuntos
Antineoplásicos/síntese química , Fluorenos/síntese química , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Compostos Heterocíclicos com 3 Anéis/síntese química , Imidazóis/síntese química , Piridinas/síntese química , Trifosfato de Adenosina/química , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Fluorenos/química , Fluorenos/farmacologia , Compostos Heterocíclicos com 3 Anéis/química , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Imidazóis/química , Imidazóis/farmacologia , Leucemia/tratamento farmacológico , Camundongos , Modelos Moleculares , Ligação Proteica , Piridinas/química , Piridinas/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade
14.
Proc Natl Acad Sci U S A ; 106(33): 13775-9, 2009 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-19666559

RESUMO

Structural changes occur in the alphabeta-tubulin heterodimer during the microtubule assembly/disassembly cycle. Their most prominent feature is a transition from a straight, microtubular structure to a curved structure. There is a broad range of small molecule compounds that disturbs the microtubule cycle, a class of which targets the colchicine-binding site and prevents microtubule assembly. This class includes compounds with very different chemical structures, and it is presently unknown whether they prevent tubulin polymerization by the same mechanism. To address this issue, we have determined the structures of tubulin complexed with a set of such ligands and show that they interfere with several of the movements of tubulin subunits structural elements upon its transition from curved to straight. We also determined the structure of tubulin unliganded at the colchicine site; this reveals that a beta-tubulin loop (termed T7) flips into this site. As with colchicine site ligands, this prevents a helix which is at the interface with alpha-tubulin from stacking onto a beta-tubulin beta sheet as in straight protofilaments. Whereas in the presence of these ligands the interference with microtubule assembly gets frozen, by flipping in and out the beta-subunit T7 loop participates in a reversible way in the resistance to straightening that opposes microtubule assembly. Our results suggest that it thereby contributes to microtubule dynamic instability.


Assuntos
Colchicina/química , Tubulina (Proteína)/química , Animais , Antineoplásicos/farmacologia , Encéfalo/metabolismo , Dimerização , Ligantes , Microtúbulos/metabolismo , Modelos Químicos , Conformação Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ovinos
15.
J Biol Chem ; 280(19): 18916-22, 2005 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-15705579

RESUMO

In some bacteria, such as Escherichia coli, the addition of L-glutamate to dihydropteroate (dihydrofolate synthetase activity) and the subsequent additions of L-glutamate to tetrahydrofolate (folylpolyglutamate synthetase (FPGS) activity) are catalyzed by the same enzyme, FolC. The crystal structure of E. coli FolC is described in this paper. It showed strong similarities to that of the FPGS enzyme of Lactobacillus casei within the ATP binding site and the catalytic site, as do all other members of the Mur synthethase superfamily. FolC structure revealed an unexpected dihydropteroate binding site very different from the folate site identified previously in the FPGS structure. The relevance of this site is exemplified by the presence of phosphorylated dihydropteroate, a reaction intermediate in the DHFS reaction. L. casei FPGS is considered a relevant model for human FPGS. As such, the presence of a folate binding site in E. coli FolC, which is different from the one seen in FPGS enzymes, provides avenues for the design of specific inhibitors of this enzyme in antimicrobial therapy.


Assuntos
Escherichia coli/enzimologia , Complexos Multienzimáticos/química , Peptídeo Sintases/química , Trifosfato de Adenosina/química , Anti-Infecciosos/farmacologia , Sítios de Ligação , Catálise , Domínio Catalítico , Cristalografia por Raios X , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Lactobacillus/enzimologia , Modelos Químicos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Pterinas/química
16.
J Med Chem ; 46(5): 681-4, 2003 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-12593648

RESUMO

The discovery and SAR of ketopiperazino methylazaindole factor Xa inhibitors are described. Structure-activity data suggesting that this class of inhibitors does not bind in the canonical mode were confirmed by an X-ray crystal structure showing the neutral haloaromatic bound in the S(1) subsite. The most potent azaindole, 33 (RPR209685), is selective against related serine proteases and attains higher levels of exposure upon oral dosing than comparable benzamidines and benzamidine isosteres. Compound 33 was efficacious in the canine AV model of thrombosis.


Assuntos
Compostos Aza/síntese química , Inibidores do Fator Xa , Indóis/síntese química , Piperazinas/síntese química , Inibidores de Serina Proteinase/síntese química , Sulfonamidas/síntese química , Administração Oral , Animais , Compostos Aza/química , Compostos Aza/farmacologia , Disponibilidade Biológica , Cristalografia por Raios X , Cães , Técnicas In Vitro , Indóis/química , Indóis/farmacologia , Ligantes , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Piperazinas/química , Piperazinas/farmacologia , Ratos , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/farmacologia , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacologia
17.
J Med Chem ; 46(5): 685-90, 2003 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-12593649

RESUMO

The structures of the noncovalent complex of human factor Xa (fXa) with four non-peptide inhibitors containing a central sulfonylpiperazinone scaffold have been determined to about 2.1 A resolution. Highly potent fXa inhibitors containing both neutral groups such as chlorobenzothiophene or chlorothiophene and basic groups such as benzamidine were shown to interact in the S1 pocket through the neutral group whereas the S4 pocket is occupied by the basic moiety. The scaffold comprising the sulfonyl keto piperazine moiety might play a pivotal role in the orientation of substituents, since there is a strong hydrogen bond between Gly219 of fXa and the carbonyl oxygen of the piperazine. This unique "reverse" binding mode is heretofore unreported in fXa and shows that electrostatic interactions in the S1 subsite are not an absolute requirement to maintain high affinity. Selectivity against other serine proteases can be readily explained in light of these structural results. It has opened up new prospects for designing fXa inhibitors with increased oral bioavailability.


Assuntos
Fator Xa/química , Piperazinas/química , Inibidores de Serina Proteinase/química , Sítios de Ligação , Humanos , Modelos Moleculares , Estrutura Molecular , Ligação Proteica
18.
J Mol Biol ; 320(5): 951-62, 2002 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-12126617

RESUMO

Bacterial peptide deformylase (PDF) belongs to a sub-family of metalloproteases that catalyse the removal of the N-terminal formyl group from newly synthesised proteins. PDF is essential in prokaryotes and conserved throughout the eubacteria. It is therefore considered an attractive target for developing new antibacterial agents. Here, we report the crystal structures of four bacterial deformylases, free or bound to the naturally occurring antibiotic actinonin, including two from the major bacterial pathogens Pseudomonas aeruginosa and Staphylococcus aureus. The overall tertiary structure is essentially conserved but shows significant differences, namely at the C terminus, which are directly related to the deformylase type (i.e. I or II) they belong to. The geometry around the catalytic metal ion exhibits a high level of similarity within the different enzymes, as does the binding mode of actinonin to the various deformylases. However, some significant structural differences are found in the vicinity of the active site, highlighting the structural and molecular requirements for the design of a deformylase inhibitor active against a broad spectrum of bacterial strains.


Assuntos
Amidoidrolases , Aminopeptidases/química , Antibacterianos/química , Ácidos Hidroxâmicos/química , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Escherichia coli/enzimologia , Geobacillus stearothermophilus/enzimologia , Modelos Moleculares , Dados de Sequência Molecular , Níquel/química , Estrutura Terciária de Proteína , Pseudomonas aeruginosa/enzimologia , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Staphylococcus aureus/enzimologia , Zinco/química
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...