Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 23
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
SLAS Discov ; 27(1): 20-28, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-35058172

RESUMO

Screening campaigns, especially those aimed at modulating enzyme activity, often rely on measuring substrate→product conversions. Unfortunately, the presence of endogenous substrates and/or products can limit one's ability to measure conversions. As well, coupled detection systems, often used to facilitate optical readouts, are subject to interference. Stable isotope labeled substrates can overcome background contamination and yield a direct readout of enzyme activity. Not only can isotope kinetic assays enable early screening, but they can also be used to follow hit progression in translational (pre)clinical studies. Herein, we consider a case study surrounding lipid biology to exemplify how metabolic flux analyses can connect stages of drug development, caveats are highlighted to ensure reliable data interpretations. For example, when measuring enzyme activity in early biochemical screening it may be enough to quantify the formation of a labeled product. In contrast, cell-based and in vivo studies must account for variable exposure to a labeled substrate (or precursor) which occurs via tracer dilution and/or isotopic exchange. Strategies are discussed to correct for these complications. We believe that measures of metabolic flux can help connect structure-activity relationships with pharmacodynamic mechanisms of action and determine whether mechanistically differentiated biophysical interactions lead to physiologically relevant outcomes. Adoption of this logic may allow research programs to (i) build a critical bridge between primary screening and (pre)clinical development, (ii) elucidate biology in parallel with screening and (iii) suggest a strategy aimed at in vivo biomarker development.


Assuntos
Isótopos , Marcação por Isótopo
2.
Anal Chem ; 93(15): 6071-6079, 2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33819010

RESUMO

The primary goal of high-throughput screening (HTS) is to rapidly survey a broad collection of compounds, numbering from tens of thousands to millions of members, and identify those that modulate the activity of a therapeutic target of interest. For nearly two decades, mass spectrometry has been used as a label-free, direct-detection method for HTS and is widely acknowledged as being less susceptible to interferences than traditional optical techniques. Despite these advantages, the throughput of conventional MS-based platforms like RapidFire or parallel LC-MS, which typically acquire data at speeds of 6-30 s/sample, can still be limiting for large HTS campaigns. To overcome this bottleneck, the field has recently turned to chromatography-free approaches including MALDI-TOF-MS and acoustic droplet ejection-MS, both of which are capable of throughputs of 1 sample/second or faster. In keeping with these advances, we report here on our own characterization of an acoustic droplet ejection, open port interface (ADE-OPI)-MS system as a platform for HTS using the membrane-associated, lipid metabolizing enzyme diacylglycerol acyltransferase 2 (DGAT2) as a model system. We demonstrate for the first time that the platform is capable of ejecting droplets from phase-separated samples, allowing direct coupling of liquid-liquid extraction with OPI-MS analysis. By applying the platform to screen a 6400-member library, we further demonstrate that the ADE-OPI-MS assay is suitable for HTS and also performs comparably to LC-MS, but with an efficiency gain of >20-fold.


Assuntos
Diacilglicerol O-Aciltransferase , Inibidores Enzimáticos/química , Ensaios de Triagem em Larga Escala , Acústica , Cromatografia Líquida , Diacilglicerol O-Aciltransferase/antagonistas & inibidores , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
3.
Metabolites ; 10(11)2020 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-33233825

RESUMO

Stable isotope tracers can be used to quantify the activity of metabolic pathways. Specifically, 2H-water is quite versatile, and its incorporation into various products can enable measurements of carbohydrate, lipid, protein and nucleic acid kinetics. However, since there are limits on how much 2H-water can be administered and since some metabolic processes may be slow, it is possible that one may be challenged with measuring small changes in isotopic enrichment. We demonstrate an advantage of the isotope fractionation that occurs during gas chromatography, namely, setting tightly bounded integration regions yields a powerful approach for determining isotope ratios. We determined how the degree of isotope fractionation, chromatographic peak width and mass spectrometer dwell time can increase the apparent isotope labeling. Relatively simple changes in the logic surrounding data acquisition and processing can enhance gas chromatography-mass spectrometry measures of low levels of 2H-labeling, this is especially useful when asymmetrical peaks are recorded at low signal:background. Although we have largely focused attention on alanine (which is of interest in studies of protein synthesis), it should be possible to extend the concepts to other analytes and/or hardware configurations.

4.
ACS Chem Biol ; 15(10): 2623-2635, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-32930572

RESUMO

The ability to target specific proteins for degradation may open a new door toward developing therapeutics. Although effort in chemistry is essential for advancing this modality, i.e., one needs to generate proteolysis targeting chimeras (bifunctional molecules, also referred to as PROTACS) or "molecular glues" to accelerate protein degradation, we suspect that investigations could also benefit by directing attention toward physiological regulation surrounding protein homeostasis, including the methods that can be used to examine changes in protein kinetics. This perspective will first consider some metabolic scenarios that might be of importance when one aims to change protein abundance by increasing protein degradation. Specifically, could protein turnover impact the apparent outcome? We will then outline how to study protein dynamics by coupling stable isotope tracer methods with mass spectrometry-based detection; since the experimental conditions could have a dramatic effect on protein turnover, special attention is directed toward the application of methods for quantifying protein kinetics using in vitro and in vivo models. Our goal is to present key concepts that should enable mechanistically informed studies which test targeted protein degradation strategies.


Assuntos
Biossíntese de Proteínas/fisiologia , Proteínas/análise , Proteínas/metabolismo , Proteólise/efeitos dos fármacos , Proteostase/fisiologia , Animais , Humanos , Marcação por Isótopo , Cinética , Espectrometria de Massas , Proteínas/química
5.
J Am Soc Mass Spectrom ; 31(12): 2421-2425, 2020 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-32840373

RESUMO

Spatial characterization of triglyceride metabolism is an area of significant interest which can be enabled by mass spectrometry imaging via recent advances in neutral lipid laser desorption analytical approaches. Here, we extend recent advancements in gold-assisted neutral lipid imaging and demonstrate the potential to map lipid flux in rodents. We address here critical issues surrounding the analytical configuration and interpretation of the data for a group of select triglycerides. Specifically, we examined how the signal intensity and spatial resolution would impact the apparent isotope ratio in a given analyte (which is an important consideration when performing MS based kinetics studies of this kind) with attention given to molecular ions and not fragments. We evaluated the analytics by contrasting lipid flux in well characterized mouse models, including fed vs fed states and different dietary perturbations. In total, the experimental paradigm described here should enable studies of hepatic lipogenesis; presumably, this logic can be enhanced via the inclusion of ion mobility and/or fragmentation. Although this study was carried out in robust models of liver lipogenesis, we expect that the model system could be expanded to a variety of tissues where zonated (or heterogeneous) lipid synthesis may occur, including solid tumor metabolism.


Assuntos
Lipídeos/análise , Animais , Ouro/análise , Cinética , Masculino , Espectrometria de Massas/métodos , Camundongos Endogâmicos C57BL
6.
J Mol Biol ; 429(7): 1030-1044, 2017 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-28232034

RESUMO

The exotoxins toxin A (TcdA) and toxin B (TcdB) are produced by the bacterial pathogen Clostridium difficile and are responsible for the pathology associated with C. difficile infection (CDI). The antitoxin antibodies actoxumab and bezlotoxumab bind to and neutralize TcdA and TcdB, respectively. Bezlotoxumab was recently approved by the FDA for reducing the recurrence of CDI. We have previously shown that a single molecule of bezlotoxumab binds to two distinct epitopes within the TcdB combined repetitive oligopeptide (CROP) domain, preventing toxin binding to host cells. In this study, we characterize the binding of actoxumab to TcdA and examine its mechanism of toxin neutralization. Using a combination of approaches including a number of biophysical techniques, we show that there are two distinct actoxumab binding sites within the CROP domain of TcdA centered on identical amino acid sequences at residues 2162-2189 and 2410-2437. Actoxumab binding caused the aggregation of TcdA especially at higher antibody:toxin concentration ratios. Actoxumab prevented the association of TcdA with target cells demonstrating that actoxumab neutralizes toxin activity by inhibiting the first step of the intoxication cascade. This mechanism of neutralization is similar to that observed with bezlotoxumab and TcdB. Comparisons of the putative TcdA epitope sequences across several C. difficile ribotypes and homologous repeat sequences within TcdA suggest a structural basis for observed differences in actoxumab binding and/or neutralization potency. These data provide a mechanistic basis for the protective effects of the antibody in vitro and in vivo, including in various preclinical models of CDI.


Assuntos
Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/metabolismo , Toxinas Bacterianas/antagonistas & inibidores , Enterotoxinas/antagonistas & inibidores , Epitopos/metabolismo , Sítios de Ligação , Anticorpos Amplamente Neutralizantes , Agregados Proteicos , Ligação Proteica
7.
Anal Biochem ; 518: 9-15, 2017 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-27815077

RESUMO

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) has created an urgent need for new therapeutic agents capable of combating this threat. We have previously reported on the discovery of novel inhibitors targeting enzymes involved in the biosynthesis of wall teichoic acid (WTA) and demonstrated that these agents can restore ß-lactam efficacy against MRSA. In those previous reports pathway engagement of inhibitors was demonstrated by reduction in WTA levels measured by polyacrylamide gel electrophoresis. To enable a more rigorous analysis of these inhibitors we sought to develop a quantitative method for measuring whole-cell reductions in WTA. Herein we describe a robust methodology for hydrolyzing polymeric WTA to the monomeric component ribitol-N-acetylglucosamine coupled with measurement by LC-MS/MS. Critical elements of the protocol were found to include the time and temperature of hydrofluoric acid-mediated hydrolysis of polymeric WTA and optimization of these parameters is fully described. Most significantly, the assay enabled accurate and reproducible measurement of depletion EC50s for tunicamycin and representatives from the novel class of TarO inhibitors, the tarocins. The method described can readily be adapted to quantifying levels of WTA in tissue homogenates from a murine model of infection, highlighting the applicability for both in vitro and in vivo characterizations.


Assuntos
Espectrometria de Massas/métodos , Staphylococcus aureus Resistente à Meticilina/metabolismo , Ácidos Teicoicos/metabolismo , Cromatografia Líquida/métodos , Staphylococcus aureus Resistente à Meticilina/química , Ácidos Teicoicos/química , Tunicamicina/farmacologia
8.
Bioorg Med Chem Lett ; 26(19): 4743-4747, 2016 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-27575474

RESUMO

A series of benzimidazole analogs have been synthesized to improve the profile of the previous lead compounds tarocin B and 1. The syntheses, structure-activity relationships, and selected biochemical data of these analogs are described. The optimization efforts allowed the identification of 21, a fluoro-substituted benzimidazole, exhibiting potent TarO inhibitory activity and typical profile for a wall teichoic acid (WTA) biosynthesis inhibitor. Compound 21 displayed a potent synergistic and bactericidal effect in combination with imipenem against diverse methicillin-resistant Staphylococci.


Assuntos
Antibacterianos/farmacologia , Benzimidazóis/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Ácidos Teicoicos/antagonistas & inibidores , Animais , Antibacterianos/química , Benzimidazóis/química , Testes de Sensibilidade Microbiana , Ratos , Relação Estrutura-Atividade , Ácidos Teicoicos/biossíntese
9.
PLoS Pathog ; 12(5): e1005585, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-27144276

RESUMO

Here we describe a chemical biology strategy performed in Staphylococcus aureus and Staphylococcus epidermidis to identify MnaA, a 2-epimerase that we demonstrate interconverts UDP-GlcNAc and UDP-ManNAc to modulate substrate levels of TarO and TarA wall teichoic acid (WTA) biosynthesis enzymes. Genetic inactivation of mnaA results in complete loss of WTA and dramatic in vitro ß-lactam hypersensitivity in methicillin-resistant S. aureus (MRSA) and S. epidermidis (MRSE). Likewise, the ß-lactam antibiotic imipenem exhibits restored bactericidal activity against mnaA mutants in vitro and concomitant efficacy against 2-epimerase defective strains in a mouse thigh model of MRSA and MRSE infection. Interestingly, whereas MnaA serves as the sole 2-epimerase required for WTA biosynthesis in S. epidermidis, MnaA and Cap5P provide compensatory WTA functional roles in S. aureus. We also demonstrate that MnaA and other enzymes of WTA biosynthesis are required for biofilm formation in MRSA and MRSE. We further determine the 1.9Å crystal structure of S. aureus MnaA and identify critical residues for enzymatic dimerization, stability, and substrate binding. Finally, the natural product antibiotic tunicamycin is shown to physically bind MnaA and Cap5P and inhibit 2-epimerase activity, demonstrating that it inhibits a previously unanticipated step in WTA biosynthesis. In summary, MnaA serves as a new Staphylococcal antibiotic target with cognate inhibitors predicted to possess dual therapeutic benefit: as combination agents to restore ß-lactam efficacy against MRSA and MRSE and as non-bioactive prophylactic agents to prevent Staphylococcal biofilm formation.


Assuntos
Proteínas de Bactérias/metabolismo , Racemases e Epimerases/metabolismo , Staphylococcus aureus/metabolismo , Staphylococcus epidermidis/metabolismo , Ácidos Teicoicos/biossíntese , Animais , Proteínas de Bactérias/química , Biofilmes/crescimento & desenvolvimento , Parede Celular/metabolismo , Cristalografia por Raios X , Modelos Animais de Doenças , Staphylococcus aureus Resistente à Meticilina , Camundongos , Testes de Sensibilidade Microbiana , Microscopia de Fluorescência , Ressonância Magnética Nuclear Biomolecular , Racemases e Epimerases/química , Infecções Estafilocócicas/metabolismo
10.
Sci Transl Med ; 8(329): 329ra32, 2016 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-26962156

RESUMO

The widespread emergence of methicillin-resistant Staphylococcus aureus (MRSA) has dramatically eroded the efficacy of current ß-lactam antibiotics and created an urgent need for new treatment options. We report an S. aureus phenotypic screening strategy involving chemical suppression of the growth inhibitory consequences of depleting late-stage wall teichoic acid biosynthesis. This enabled us to identify early-stage pathway-specific inhibitors of wall teichoic acid biosynthesis predicted to be chemically synergistic with ß-lactams. We demonstrated by genetic and biochemical means that each of the new chemical series discovered, herein named tarocin A and tarocin B, inhibited the first step in wall teichoic acid biosynthesis (TarO). Tarocins do not have intrinsic bioactivity but rather demonstrated potent bactericidal synergy in combination with broad-spectrum ß-lactam antibiotics against diverse clinical isolates of methicillin-resistant staphylococci as well as robust efficacy in a murine infection model of MRSA. Tarocins and other inhibitors of wall teichoic acid biosynthesis may provide a rational strategy to develop Gram-positive bactericidal ß-lactam combination agents active against methicillin-resistant staphylococci.


Assuntos
Proteínas de Bactérias/metabolismo , Vias Biossintéticas/efeitos dos fármacos , Parede Celular/metabolismo , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Ácidos Teicoicos/biossíntese , beta-Lactamas/farmacologia , Animais , Parede Celular/efeitos dos fármacos , Dicloxacilina/farmacologia , Dicloxacilina/uso terapêutico , Feminino , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Modelos Moleculares , Fenótipo , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Resultado do Tratamento
11.
Nature ; 526(7575): 672-7, 2015 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-26416753

RESUMO

Riboswitches are non-coding RNA structures located in messenger RNAs that bind endogenous ligands, such as a specific metabolite or ion, to regulate gene expression. As such, riboswitches serve as a novel, yet largely unexploited, class of emerging drug targets. Demonstrating this potential, however, has proven difficult and is restricted to structurally similar antimetabolites and semi-synthetic analogues of their cognate ligand, thus greatly restricting the chemical space and selectivity sought for such inhibitors. Here we report the discovery and characterization of ribocil, a highly selective chemical modulator of bacterial riboflavin riboswitches, which was identified in a phenotypic screen and acts as a structurally distinct synthetic mimic of the natural ligand, flavin mononucleotide, to repress riboswitch-mediated ribB gene expression and inhibit bacterial cell growth. Our findings indicate that non-coding RNA structural elements may be more broadly targeted by synthetic small molecules than previously expected.


Assuntos
Pirimidinas/química , Pirimidinas/farmacologia , RNA Bacteriano/química , RNA Bacteriano/efeitos dos fármacos , Riboswitch/efeitos dos fármacos , Animais , Aptâmeros de Nucleotídeos/química , Bactérias/citologia , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Sequência de Bases , Cristalografia por Raios X , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Feminino , Mononucleotídeo de Flavina/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico/genética , Transferases Intramoleculares/genética , Ligantes , Camundongos , Camundongos Endogâmicos DBA , Modelos Moleculares , Dados de Sequência Molecular , Pirimidinas/isolamento & purificação , Pirimidinas/uso terapêutico , RNA Bacteriano/genética , Reprodutibilidade dos Testes , Riboflavina/biossíntese , Riboswitch/genética , Especificidade por Substrato
12.
Antimicrob Agents Chemother ; 59(2): 1052-60, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25451052

RESUMO

Clostridium difficile infections (CDIs) are the leading cause of hospital-acquired infectious diarrhea and primarily involve two exotoxins, TcdA and TcdB. Actoxumab and bezlotoxumab are human monoclonal antibodies that neutralize the cytotoxic/cytopathic effects of TcdA and TcdB, respectively. In a phase II clinical study, the actoxumab-bezlotoxumab combination reduced the rate of CDI recurrence in patients who were also treated with standard-of-care antibiotics. However, it is not known whether the antibody combination will be effective against a broad range of C. difficile strains. As a first step toward addressing this, we tested the ability of actoxumab and bezlotoxumab to neutralize the activities of toxins from a number of clinically relevant and geographically diverse strains of C. difficile. Neutralization potencies, as measured in a cell growth/survival assay with purified toxins from various C. difficile strains, correlated well with antibody/toxin binding affinities. Actoxumab and bezlotoxumab neutralized toxins from culture supernatants of all clinical isolates tested, including multiple isolates of the BI/NAP1/027 and BK/NAP7/078 strains, at antibody concentrations well below plasma levels observed in humans. We compared the bezlotoxumab epitopes in the TcdB receptor binding domain across known TcdB sequences and found that key substitutions within the bezlotoxumab epitopes correlated with the relative differences in potencies of bezlotoxumab against TcdB of some strains, including ribotypes 027 and 078. Combined with in vitro neutralization data, epitope modeling will enhance our ability to predict the coverage of new and emerging strains by actoxumab-bezlotoxumab in the clinic.


Assuntos
Antibacterianos/farmacologia , Anticorpos Monoclonais/farmacologia , Clostridioides difficile/efeitos dos fármacos , Proteínas de Bactérias/genética , Linhagem Celular , Clostridioides difficile/imunologia , Clostridioides difficile/patogenicidade , Infecções por Clostridium/tratamento farmacológico , Infecções por Clostridium/microbiologia , Epitopos/imunologia , Feminino , Humanos , Masculino
13.
J Biol Chem ; 289(26): 18008-21, 2014 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-24821719

RESUMO

The symptoms of Clostridium difficile infections are caused by two exotoxins, TcdA and TcdB, which target host colonocytes by binding to unknown cell surface receptors, at least in part via their combined repetitive oligopeptide (CROP) domains. A combination of the anti-TcdA antibody actoxumab and the anti-TcdB antibody bezlotoxumab is currently under development for the prevention of recurrent C. difficile infections. We demonstrate here through various biophysical approaches that bezlotoxumab binds to specific regions within the N-terminal half of the TcdB CROP domain. Based on this information, we solved the x-ray structure of the N-terminal half of the TcdB CROP domain bound to Fab fragments of bezlotoxumab. The structure reveals that the TcdB CROP domain adopts a ß-solenoid fold consisting of long and short repeats and that bezlotoxumab binds to two homologous sites within the CROP domain, partially occluding two of the four putative carbohydrate binding pockets located in TcdB. We also show that bezlotoxumab neutralizes TcdB by blocking binding of TcdB to mammalian cells. Overall, our data are consistent with a model wherein a single molecule of bezlotoxumab neutralizes TcdB by binding via its two Fab regions to two epitopes within the N-terminal half of the TcdB CROP domain, partially blocking the carbohydrate binding pockets of the toxin and preventing toxin binding to host cells.


Assuntos
Anticorpos Antibacterianos/imunologia , Anticorpos Neutralizantes/imunologia , Proteínas de Bactérias/química , Proteínas de Bactérias/imunologia , Toxinas Bacterianas/química , Toxinas Bacterianas/imunologia , Clostridioides difficile/imunologia , Epitopos/imunologia , Sequência de Aminoácidos , Anticorpos Antibacterianos/química , Anticorpos Monoclonais , Anticorpos Neutralizantes/química , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Sítios de Ligação , Anticorpos Amplamente Neutralizantes , Clostridioides difficile/química , Clostridioides difficile/genética , Cristalografia por Raios X , Mapeamento de Epitopos , Epitopos/química , Epitopos/genética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína
14.
Anal Biochem ; 418(1): 10-8, 2011 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-21726521

RESUMO

Affinity characterization by mass spectrometry (AC-MS) is a novel LC-MS methodology for quantitative determination of small molecule ligand binding to macromolecules. Its most distinguishing feature is the direct determination of all three concentration terms of the equilibrium binding equation, i.e., (M), (L), and (ML), which denote the macromolecule, ligand, and the corresponding complex, respectively. Although it is possible to obtain the dissociation constant from a single mixing experiment, saturation analyses are still valuable for assessing the overall binding phenomenon based on an established formalism. In addition to providing the prerequisite dissociation constant and binding stoichiometry, the technique also provides valuable information about the actual solubility of both macromolecule and ligand upon dilution and mixing in binding buffers. The dissociation constants and binding mode for interactions of DNA primase and thymidylate synthetase (TS) with high and low affinity small molecule ligands were obtained using the AC-MS method. The data were consistent with the expected affinity of TS for these ligands based on dissociation constants determined by alternative thermal-denaturation techniques: TdF or TdCD, and also consistent enzyme inhibition constants reported in the literature. The validity of AC-MS was likewise extended to a larger set of soluble protein-ligand systems. It was established as a valuable resource for counter screen and structure-activity relationship studies in drug discovery, especially when other classical techniques could only provide ambiguous results.


Assuntos
Espectrometria de Massas/métodos , Proteínas/química , Sítios de Ligação , DNA Primase/química , DNA Primase/metabolismo , Cinética , Ligantes , Desnaturação Proteica , Proteínas/metabolismo , Quinazolinas/química , Quinazolinas/metabolismo , Soluções , Relação Estrutura-Atividade , Temperatura , Tiofenos/química , Tiofenos/metabolismo , Timidilato Sintase/química , Timidilato Sintase/metabolismo
15.
Biochemistry ; 50(37): 7964-76, 2011 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-21793567

RESUMO

Kinases catalyze the transfer of γ-phosphate from ATP to substrate protein residues triggering signaling pathways responsible for a plethora of cellular events. Isolation and production of homogeneous preparations of kinases in their fully active forms is important for accurate in vitro measurements of activity, stability, and ligand binding properties of these proteins. Previous studies have shown that MEK1 can be produced in its active phosphorylated form by coexpression with RAF1 in insect cells. In this study, using activated MEK1 produced by in vitro activation by RAF1 (pMEK1(in vitro)), we demonstrate that the simultaneous expression of RAF1 for production of activated MEK1 does not result in stoichiometric phosphorylation of MEK1. The pMEK1(in vitro) showed higher specific activity toward ERK2 protein substrate compared to the pMEK1 that was activated via coexpression with RAF1 (pMEK1(in situ)). The two pMEK1 preparations showed quantitative differences in the phosphorylation of T-loop residue serine 222 by Western blotting and mass spectrometry. Finally, pMEK1(in vitro) showed marked differences in the ligand binding properties compared to pMEK1(in situ). Contrary to previous findings, pMEK1(in vitro) bound allosteric inhibitors U0126 and PD0325901 with a significantly lower affinity than pMEK1(in situ) as well as its unphosphorylated counterpart (npMEK1) as demonstrated by thermal-shift, AS-MS, and calorimetric studies. The differences in inhibitor binding affinity provide direct evidence that unphosphorylated and RAF1-phosphorylated MEK1 form distinct inhibitor sites.


Assuntos
Benzamidas/metabolismo , Butadienos/metabolismo , Difenilamina/análogos & derivados , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Nitrilas/metabolismo , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/fisiologia , Animais , Benzamidas/farmacologia , Butadienos/farmacologia , Linhagem Celular , Difenilamina/metabolismo , Difenilamina/farmacologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Insetos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Nitrilas/farmacologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia
16.
FEBS Lett ; 585(1): 104-10, 2011 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-21093442

RESUMO

Protein kinase CK2 (CK2), a constitutively active serine/threonine kinase, is involved in a variety of roles essential to the maintenance of cellular homeostasis. Elevated levels of CK2 expression results in the dysregulation of key signaling pathways that regulate transcription, and has been implicated in cancer. The adenosine-5'-triphosphate-competitive inhibitor CX-4945 has been reported to show broad spectrum anti-proliferative activity in multiple cancer cell lines. Although the enzymatic IC(50) of CX-4945 has been reported, the thermodynamics and structural basis of binding to CK2α remained elusive. Presented here are the crystal structures of human CK2α in complex with CX-4945 and adenylyl phosphoramidate at 2.7 and 1.3 Å, respectively. Biophysical analysis of CX-4945 binding is also described. This data provides the structural rationale for the design of more potent inhibitors against this emerging cancer target.


Assuntos
Caseína Quinase II/química , Modelos Moleculares , Naftiridinas/química , Estrutura Terciária de Proteína , Adenilil Imidodifosfato/química , Adenilil Imidodifosfato/metabolismo , Sítios de Ligação , Calorimetria , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/metabolismo , Domínio Catalítico , Dicroísmo Circular , Cristalografia por Raios X , Humanos , Naftiridinas/metabolismo , Naftiridinas/farmacologia , Fenazinas , Ligação Proteica , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Temperatura , Termodinâmica
17.
Mol Cancer Ther ; 9(11): 2993-3002, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20978164

RESUMO

Kinesin spindle protein (KSP) is a mitotic kinesin required for the formation of the bipolar mitotic spindle, and inhibition of this motor protein results in mitotic arrest and cell death. KSP inhibitors show preclinical antitumor activity and are currently undergoing testing in clinical trials. These agents have been dosed intravenously using various dosing schedules. We sought to identify a KSP inhibitor that could be delivered orally and thus provide convenience of dosing as well as the ability to achieve more continuous exposure via the use of dose-dense administration. We discovered SCH 2047069, a potent KSP inhibitor with oral bioavailability across species and the ability to cross the blood-brain barrier. The compound induces mitotic arrest characterized by a monaster spindle and is associated with an increase in histone H3 and mitotic protein monoclonal 2 phosphorylation both in vitro and in vivo. SCH 2047069 showed antitumor activity in a variety of preclinical models as a single agent and in combination with paclitaxel, gemcitabine, or vincristine.


Assuntos
Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Benzopiranos/administração & dosagem , Benzopiranos/farmacologia , Cinesinas/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Tiadiazóis/administração & dosagem , Tiadiazóis/farmacologia , Administração Oral , Animais , Cães , Avaliação Pré-Clínica de Medicamentos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Células HCT116 , Haplorrinos , Humanos , Camundongos , Camundongos Nus , Neoplasias/patologia , Ratos , Resultado do Tratamento , Células Tumorais Cultivadas
18.
Arch Biochem Biophys ; 503(2): 191-201, 2010 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-20699085

RESUMO

Aurora B kinase plays a critical role in regulating mitotic progression, and its dysregulation has been linked to tumorigenesis. The structure of the kinase domain of human Aurora B and the complementary information of binding thermodynamics of known Aurora inhibitors is lacking. Towards that effort, we sought to identify a human Aurora B construct that would be amenable for large-scale protein production for biophysical and structural studies. Although the designed AurB(69-333) construct expressed at high levels in Escherichia coli, the purified protein was largely unstable and prone to aggregation. We employed thermal-shift assay for high-throughput screening of 192 conditions to identify optimal pH and salt conditions that increased the stability and minimized aggregation of AurB(69-333). Direct ligand binding analyses using temperature-dependent circular dichroism (TdCD) and TR-FRET-based Lanthascreen™ binding assay showed that the purified protein was folded and functional. The affinity rank-order obtained using TdCD and Lanthascreen™ binding assay correlated with enzymatic IC50 values measured using full-length Aurora B protein for all the inhibitors tested except for AZD1152. The direct binding results support the hypothesis that the purified human AurB(69-333) fragment is a good surrogate for its full-length counterpart for biophysical and structural analyses.


Assuntos
Proteínas Serina-Treonina Quinases/isolamento & purificação , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína/genética , Sequência de Aminoácidos , Aurora Quinase B , Aurora Quinases , Dicroísmo Circular , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/metabolismo , Transferência Ressonante de Energia de Fluorescência , Humanos , Concentração Inibidora 50 , Espectrometria de Massas , Dados de Sequência Molecular , Peso Molecular , Dobramento de Proteína , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Termodinâmica
19.
Biochemistry ; 49(38): 8350-8, 2010 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-20718440

RESUMO

Affinity selection-mass spectrometry (AS-MS) screening of kinesin spindle protein (KSP) followed by enzyme inhibition studies and temperature-dependent circular dichroism (TdCD) characterization was utilized to identify a series of benzimidazole compounds. This series also binds in the presence of Ispinesib, a known anticancer KSP inhibitor in phase I/II clinical trials for breast cancer. TdCD and AS-MS analyses support simultaneous binding implying existence of a novel non-Ispinesib binding pocket within KSP. Additional TdCD analyses demonstrate direct binding of these compounds to Ispinesib-resistant mutants (D130V, A133D, and A133D + D130V double mutant), further strengthening the hypothesis that the compounds bind to a distinct binding pocket. Also importantly, binding to this pocket causes uncompetitive inhibition of KSP ATPase activity. The uncompetitive inhibition with respect to ATP is also confirmed by the requirement of nucleotide for binding of the compounds. After preliminary affinity optimization, the benzimidazole series exhibited distinctive antimitotic activity as evidenced by blockade of bipolar spindle formation and appearance of monoasters. Cancer cell growth inhibition was also demonstrated either as a single agent or in combination with Ispinesib. The combination was additive as predicted by the binding studies using TdCD and AS-MS analyses. The available data support the existence of a KSP inhibitory site hitherto unknown in the literature. The data also suggest that targeting this novel site could be a productive strategy for eluding Ispinesib-resistant tumors. Finally, AS-MS and TdCD techniques are general in scope and may enable screening other targets in the presence of known drugs, clinical candidates, or tool compounds that bind to the protein of interest in an effort to identify potency-enhancing small molecules that increase efficacy and impede resistance in combination therapy.


Assuntos
Benzimidazóis/farmacologia , Cinesinas/metabolismo , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/metabolismo , Antineoplásicos/antagonistas & inibidores , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Benzamidas/metabolismo , Benzimidazóis/antagonistas & inibidores , Sítios de Ligação , Dicroísmo Circular , Humanos , Cinesinas/antagonistas & inibidores , Cinesinas/química , Espectrometria de Massas , Nucleotídeos/antagonistas & inibidores , Nucleotídeos/química , Estrutura Terciária de Proteína , Quinazolinas/metabolismo
20.
Protein Expr Purif ; 70(1): 13-22, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19836452

RESUMO

AMP-activated protein kinase (AMPK) is considered an important target for treatment of type II diabetes and the metabolic syndrome. The muscle-specific isoform of the regulatory gamma-subunit, gamma 3, within the context of AMPK alpha 2 beta 2 gamma 3 complex, is involved in glucose and fat metabolism in skeletal muscle. In an effort to identify gamma 3-specific activators of AMPK, we have produced truncated human recombinant AMPK alpha 2 beta 2 gamma 3 (hu alpha 2 beta 2 gamma 3(trunc)) for biochemical characterization. Infection of insect cells with three baculoviral stocks encoding the individual subunits resulted in soluble expression of a stable hu alpha 2 beta 2 gamma 3(trunc) heterotrimeric complex. Co-expression of the three subunits was essential for solubility since the individual protein components, when expressed separately, were almost completely insoluble. The hu alpha 2 beta 2 gamma 3(trunc) heterotrimer was purified to apparent homogeneity from baculovirus-infected insect cells in a 1:1:1 stoichiometric complex. The hu alpha 2 beta 2 gamma 3(trunc) heterotrimer had significant circular dichroism signal that was lost as a function of temperature, implying that the purified protein was folded. The hu alpha 2 beta 2 gamma 3(trunc) complex was capable of binding AMP and ATP, although the heterotrimer showed preference for AMP, in particular, as seen by thermal denaturation circular dichroism analyses. Further experiments showed that the truncated complex bound ZMP (AICAR-monophosphate) albeit with much lower affinity than AMP. To investigate whether there were isoform-specific differences in the nucleotide binding affinities, a well-characterized truncated mammalian alpha 1 beta 2 gamma 1 (m alpha 1 beta 2 gamma 1(trunc)) equivalent of hu alpha 2 beta 2 gamma 3(trunc) was also purified. The gamma 1 and gamma 3 isoforms showed comparable nucleotide binding affinities and solution behavior properties.


Assuntos
Proteínas Quinases Ativadas por AMP/isolamento & purificação , Baculoviridae/genética , Proteínas Quinases Ativadas por AMP/química , Sequência de Aminoácidos , Animais , Baculoviridae/metabolismo , Sítios de Ligação , Linhagem Celular , Humanos , Isoenzimas/química , Isoenzimas/isolamento & purificação , Dados de Sequência Molecular , Conformação Proteica , Alinhamento de Sequência , Spodoptera/citologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...