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1.
Nature ; 584(7822): 614-618, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32612233

RESUMO

Oral antiretroviral agents provide life-saving treatments for millions of people living with HIV, and can prevent new infections via pre-exposure prophylaxis1-5. However, some people living with HIV who are heavily treatment-experienced have limited or no treatment options, owing to multidrug resistance6. In addition, suboptimal adherence to oral daily regimens can negatively affect the outcome of treatment-which contributes to virologic failure, resistance generation and viral transmission-as well as of pre-exposure prophylaxis, leading to new infections1,2,4,7-9. Long-acting agents from new antiretroviral classes can provide much-needed treatment options for people living with HIV who are heavily treatment-experienced, and additionally can improve adherence10. Here we describe GS-6207, a small molecule that disrupts the functions of HIV capsid protein and is amenable to long-acting therapy owing to its high potency, low in vivo systemic clearance and slow release kinetics from the subcutaneous injection site. Drawing on X-ray crystallographic information, we designed GS-6207 to bind tightly at a conserved interface between capsid protein monomers, where it interferes with capsid-protein-mediated interactions between proteins that are essential for multiple phases of the viral replication cycle. GS-6207 exhibits antiviral activity at picomolar concentrations against all subtypes of HIV-1 that we tested, and shows high synergy and no cross-resistance with approved antiretroviral drugs. In phase-1 clinical studies, monotherapy with a single subcutaneous dose of GS-6207 (450 mg) resulted in a mean log10-transformed reduction of plasma viral load of 2.2 after 9 days, and showed sustained plasma exposure at antivirally active concentrations for more than 6 months. These results provide clinical validation for therapies that target the functions of HIV capsid protein, and demonstrate the potential of GS-6207 as a long-acting agent to treat or prevent infection with HIV.


Assuntos
Fármacos Anti-HIV/farmacologia , Fármacos Anti-HIV/uso terapêutico , Proteínas do Capsídeo/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Adolescente , Adulto , Fármacos Anti-HIV/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Células Cultivadas , Farmacorresistência Viral/genética , Feminino , HIV-1/crescimento & desenvolvimento , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Replicação Viral/efeitos dos fármacos , Adulto Jovem
2.
J Biol Chem ; 292(16): 6810-6820, 2017 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-28235803

RESUMO

Matrix metalloproteinase 9 (MMP9) is a member of a large family of proteases that are secreted as inactive zymogens. It is a key regulator of the extracellular matrix, involved in the degradation of various extracellular matrix proteins. MMP9 plays a pathological role in a variety of inflammatory and oncology disorders and has long been considered an attractive therapeutic target. GS-5745, a potent, highly selective humanized monoclonal antibody inhibitor of MMP9, has shown promise in treating ulcerative colitis and gastric cancer. Here we describe the crystal structure of GS-5745·MMP9 complex and biochemical studies to elucidate the mechanism of inhibition of MMP9 by GS-5745. GS-5745 binds MMP9 distal to the active site, near the junction between the prodomain and catalytic domain, and inhibits MMP9 by two mechanisms. Binding to pro-MMP9 prevents MMP9 activation, whereas binding to active MMP9 allosterically inhibits activity.


Assuntos
Anticorpos Monoclonais Humanizados/química , Colite Ulcerativa/tratamento farmacológico , Metaloproteinase 9 da Matriz/química , Inibidores de Metaloproteinases de Matriz/química , Neoplasias Gástricas/tratamento farmacológico , Sítio Alostérico , Anticorpos/química , Domínio Catalítico , Cristalografia por Raios X , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Gelatina/química , Deleção de Genes , Células HEK293 , Humanos , Concentração Inibidora 50 , Ligação Proteica , Proteínas Recombinantes/química , Ressonância de Plasmônio de Superfície
3.
J Biol Chem ; 290(13): 8439-46, 2015 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-25631052

RESUMO

Idelalisib (also known as GS-1101, CAL-101, IC489666, and Zydelig) is a PI3Kδ inhibitor that has recently been approved for the treatment of several hematological malignancies. Given its use in human diseases, we needed a clear picture of how idelalisib binds to and inhibits PI3Kδ. Our data show that idelalisib is a potent and selective inhibitor of the kinase activity of PI3Kδ. A kinetic characterization clearly demonstrated ATP-competitive inhibition, and several additional biochemical and biophysical assays showed that the compound binds reversibly and noncovalently to the kinase. A crystal structure of idelalisib bound to the p110δ subunit of PI3Kδ furthers our understanding of the binding interactions that confer the potency and selectivity of idelalisib.


Assuntos
Fosfatidilinositol 3-Quinases/química , Purinas/química , Quinazolinonas/química , Trifosfato de Adenosina/química , Androstadienos/química , Animais , Ligação Competitiva , Domínio Catalítico , Classe I de Fosfatidilinositol 3-Quinases , Classe Ia de Fosfatidilinositol 3-Quinase/química , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Cinética , Camundongos , Modelos Moleculares , Inibidores de Fosfoinositídeo-3 Quinase , Ligação Proteica , Wortmanina
4.
Biochemistry ; 54(13): 2240-8, 2015 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-25774576

RESUMO

HIV capsid protein is an important target for antiviral drug design. High-throughput screening campaigns have identified two classes of compounds (PF74 and BI64) that directly target HIV capsid, resulting in antiviral activity against HIV-1 and HIV-2 laboratory strains. Using recombinant proteins, we developed a suite of label-free assays to mechanistically understand how these compounds modulate capsid activity. PF74 preferentially binds to the preassembled hexameric capsid form and prevents disruption of higher-order capsid structures by stabilizing capsid intersubunit interactions. BI64 binds only the monomeric capsid and locks the protein in the assembly incompetent monomeric form by disrupting capsid intersubunit interactions. We also used these assays to characterize the interaction between capsid and the host protein cleavage and polyadenylation specific factor 6 (CPSF6). Consistent with recently published results, our assays revealed CPSF6 activates capsid polymerization and preferentially binds to the preassembled hexameric capsid form similar to the small molecule compound, PF74. Furthermore, these label-free assays provide a robust method for facilitating the identification of a different class of small molecule modulators of capsid function.


Assuntos
Fármacos Anti-HIV/farmacologia , Técnicas Biossensoriais/métodos , Capsídeo/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Sequência de Aminoácidos , Fármacos Anti-HIV/química , Fármacos Anti-HIV/metabolismo , Benzimidazóis/farmacologia , Capsídeo/química , HIV-1 , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Indóis/química , Indóis/metabolismo , Indóis/farmacologia , Dados de Sequência Molecular , Fenilalanina/análogos & derivados , Fenilalanina/química , Fenilalanina/metabolismo , Fenilalanina/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Fatores de Poliadenilação e Clivagem de mRNA/genética
5.
Anal Biochem ; 429(1): 58-69, 2012 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-22766435

RESUMO

To characterize a proprietary therapeutic monoclonal antibody (mAb) candidate, a rigorous biophysical study consisting of 53 Biacore and kinetic exclusion assay (KinExA) experiments was undertaken on the therapeutic mAb complexing with its target antigen. Unexpectedly, the observed binding kinetics depended on the chip used, suggesting that the negatively charged carboxyl groups on CM5, CM4, and C1 chips were adversely affecting the Biacore kinetic results. To study this hypothesis, Biacore solution-phase and KinExA equilibrium titrations, as well as KinExA kinetic measurements, were performed to establish accurate values for the affinity and kinetic rate constants of the binding reaction between antigen and mAb. The results revealed that as the negative charge on the biosensor surface decreased, the binding kinetics and K(D) approached the accurate binding parameters more closely when measured in solution. Two potential causes for the artifactual Biacore surface-based measurements are (i) steric hindrance of antigen binding arising from an interaction of the negatively charged carboxymethyldextran matrix with the mAb, which is a highly basic protein with a pI of 9.4, and (ii) an electrostatic repulsion between the negatively charged antigen and the carboxymethyldextran matrix. Importantly, simple diagnostic tests can be performed early in the measurement process to identify these types of matrix-mediated artifacts.


Assuntos
Anticorpos Monoclonais/imunologia , Antígenos/imunologia , Ressonância de Plasmônio de Superfície/métodos , Artefatos , Dextranos/química , Humanos , Cinética , Propriedades de Superfície
6.
Anal Biochem ; 385(2): 309-13, 2009 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-19059374

RESUMO

We have developed a novel referencing technique for surface plasmon resonance imaging systems referred to as "spot and hop." The technique enables internal referencing for individual flow cells in a parallel processing microfluidic network. Internal referencing provides the ability to correct for nonspecific binding and instrument drift, significantly improving data quality at each region of interest. The performance of a 48-flow-cell device was demonstrated through a series of studies, including "rise and fall" time, ligand preconcentration, ligand immobilization, analyte binding, and regeneration tests. Interfacing parallel processing fluidics with imaging systems will significantly expand the throughput and applications of array-based optical biosensors while retaining high data quality.


Assuntos
Técnicas Analíticas Microfluídicas , Ressonância de Plasmônio de Superfície/normas , Técnicas Biossensoriais/métodos , Ligantes , Padrões de Referência
7.
Anal Biochem ; 386(2): 194-216, 2009 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-19133223

RESUMO

To explore the variability in biosensor studies, 150 participants from 20 countries were given the same protein samples and asked to determine kinetic rate constants for the interaction. We chose a protein system that was amenable to analysis using different biosensor platforms as well as by users of different expertise levels. The two proteins (a 50-kDa Fab and a 60-kDa glutathione S-transferase [GST] antigen) form a relatively high-affinity complex, so participants needed to optimize several experimental parameters, including ligand immobilization and regeneration conditions as well as analyte concentrations and injection/dissociation times. Although most participants collected binding responses that could be fit to yield kinetic parameters, the quality of a few data sets could have been improved by optimizing the assay design. Once these outliers were removed, the average reported affinity across the remaining panel of participants was 620 pM with a standard deviation of 980 pM. These results demonstrate that when this biosensor assay was designed and executed appropriately, the reported rate constants were consistent, and independent of which protein was immobilized and which biosensor was used.


Assuntos
Técnicas Biossensoriais/métodos , Proteínas/análise , Anticorpos Catalíticos/análise , Benchmarking , Sítios de Ligação , Técnicas Biossensoriais/estatística & dados numéricos , Glutationa Transferase/análise , Cinética , Ligantes
8.
Mol Immunol ; 45(5): 1241-7, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17983654

RESUMO

Early work examining the interactions of IL-2 and the urinary glycoprotein uromodulin led to the suggestion that IL-2 was a lectin with specificity for high-mannose and mannan ligands. Subsequent studies have attributed various roles to these properties, some critical to the cell proliferative activity of IL-2. In an attempt to verify the reported interaction between IL-2 and mannose containing carbohydrate ligands we studied two biologically active forms of IL-2 using various techniques including affinity chromatography, equilibrium dialysis, and NMR methods. Despite previous reports we have not been able to demonstrate that IL-2 possesses the ability to bind carbohydrate.


Assuntos
Interleucina-2/metabolismo , Lectinas/metabolismo , Mucoproteínas/metabolismo , Antígenos de Grupos Sanguíneos/química , Antígenos de Grupos Sanguíneos/metabolismo , Cromatografia de Afinidade , Ensaio de Imunoadsorção Enzimática , Humanos , Interleucina-2/química , Lectinas/química , Ligantes , Espectroscopia de Ressonância Magnética , Mananas , Manose , Mucoproteínas/química , Ligação Proteica , Uromodulina
9.
J Mol Biol ; 431(7): 1440-1459, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30753871

RESUMO

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional serine/threonine protein kinase that transmits calcium signals in various cellular processes. CaMKII is activated by calcium-bound calmodulin (Ca2+/CaM) through a direct binding mechanism involving a regulatory C-terminal α-helix in CaMKII. The Ca2+/CaM binding triggers transphosphorylation of critical threonine residues proximal to the CaM-binding site leading to the autoactivated state of CaMKII. The demonstration of its critical roles in pathophysiological processes has elevated CaMKII to a key target in the management of numerous diseases. The molecule KN-93 is the most widely used inhibitor for studying the cellular and in vivo functions of CaMKII. It is widely believed that KN-93 binds directly to CaMKII, thus preventing kinase activation by competing with Ca2+/CaM. Herein, we employed surface plasmon resonance, NMR, and isothermal titration calorimetry to characterize this presumed interaction. Our results revealed that KN-93 binds directly to Ca2+/CaM and not to CaMKII. This binding would disrupt the ability of Ca2+/CaM to interact with CaMKII, effectively inhibiting CaMKII activation. Our findings also indicated that KN-93 can specifically compete with a CaMKIIδ-derived peptide for binding to Ca2+/CaM. As indicated by the surface plasmon resonance and isothermal titration calorimetry data, apparently at least two KN-93 molecules can bind to Ca2+/CaM. Our findings provide new insight into how in vitro and in vivo data obtained with KN-93 should be interpreted. They further suggest that other Ca2+/CaM-dependent, non-CaMKII activities should be considered in KN-93-based mechanism-of-action studies and drug discovery efforts.


Assuntos
Benzilaminas/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Cálcio/metabolismo , Calmodulina/metabolismo , Sulfonamidas/farmacologia , Benzilaminas/metabolismo , Sinalização do Cálcio , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Calorimetria , Humanos , Fosforilação , Sulfonamidas/metabolismo , Ressonância de Plasmônio de Superfície
10.
Nat Med ; 25(9): 1377-1384, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31501601

RESUMO

People living with HIV (PLWH) have expressed concern about the life-long burden and stigma associated with taking pills daily and can experience medication fatigue that might lead to suboptimal treatment adherence and the emergence of drug-resistant viral variants, thereby limiting future treatment options1-3. As such, there is strong interest in long-acting antiretroviral (ARV) agents that can be administered less frequently4. Herein, we report GS-CA1, a new archetypal small-molecule HIV capsid inhibitor with exceptional potency against HIV-2 and all major HIV-1 types, including viral variants resistant to the ARVs currently in clinical use. Mechanism-of-action studies indicate that GS-CA1 binds directly to the HIV-1 capsid and interferes with capsid-mediated nuclear import of viral DNA, HIV particle production and ordered capsid assembly. GS-CA1 selects in vitro for unfit GS-CA1-resistant capsid variants that remain fully susceptible to other classes of ARVs. Its high metabolic stability and low solubility enabled sustained drug release in mice following a single subcutaneous dosing. GS-CA1 showed high antiviral efficacy as a long-acting injectable monotherapy in a humanized mouse model of HIV-1 infection, outperforming long-acting rilpivirine. Collectively, these results demonstrate the potential of ultrapotent capsid inhibitors as new long-acting agents for the treatment of HIV-1 infection.


Assuntos
Fármacos Anti-HIV/farmacologia , Proteínas do Capsídeo/antagonistas & inibidores , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Indazóis/farmacologia , Piridinas/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Fármacos Anti-HIV/uso terapêutico , Capsídeo/efeitos dos fármacos , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , DNA Viral/efeitos dos fármacos , Preparações de Ação Retardada , Farmacorresistência Viral/efeitos dos fármacos , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/genética , HIV-1/patogenicidade , HIV-2/efeitos dos fármacos , HIV-2/patogenicidade , Humanos , Indazóis/uso terapêutico , Adesão à Medicação , Camundongos , Piridinas/uso terapêutico
11.
Anal Biochem ; 383(2): 255-64, 2008 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-18774767

RESUMO

Biacore T100 technology was used in conjunction with a van't Hoff analysis to characterize the thermodynamic binding parameters of 85 small-molecule inhibitors of adenosine triphosphate (ATP) binding to p38 mitogen-activated protein (MAP) kinase. The compounds were selected from a large panel of azaindole and pyrazole derivatives for which IC(50) data exist. We showed a strong relationship between the K(D) and IC(50) of a compound, but only a modest relationship between k(off) and IC(50) was detected and an apparent relationship between a compound's k(on) and its IC(50) could not be discerned. Similarly, a correlation between a compound's IC(50) and its thermodynamic parameters DeltaH degrees and DeltaS degrees could not be established. The lack of a predominant kinetic or thermodynamic signature associated with the inhibitory potential of these compounds demonstrates that there exists, even within a single well-defined system, a library of kinetic routes or, alternatively, a library of initial and final enthalpic and entropic states from which to effect inhibition. As a complement to these studies, selected double mutant thermodynamic cycles were performed to probe the energetic coupling, if any, between common sites of fluorination in both the azaindole and pyrazole classes and two different substituents. Although both cycles indicated negligible coupling free energies, both revealed significant coupling enthalpies, an observation made in other similarly dissected systems. The possible significance and caveats associated with these findings along with the advantages of using Biacore technology to derive thermodynamic parameters in drug discovery efforts are discussed.


Assuntos
Indóis/química , Indóis/metabolismo , Pirazóis/química , Pirazóis/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Concentração Inibidora 50 , Cinética , Ligação Proteica , Ressonância de Plasmônio de Superfície , Temperatura , Termodinâmica
12.
J Clin Invest ; 128(10): 4485-4500, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30024858

RESUMO

Oxidative stress is an underlying component of acute and chronic kidney disease. Apoptosis signal-regulating kinase 1 (ASK1) is a widely expressed redox-sensitive serine threonine kinase that activates p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase kinases, and induces apoptotic, inflammatory, and fibrotic signaling in settings of oxidative stress. We describe the discovery and characterization of a potent and selective small-molecule inhibitor of ASK1, GS-444217, and demonstrate the therapeutic potential of ASK1 inhibition to reduce kidney injury and fibrosis. Activation of the ASK1 pathway in glomerular and tubular compartments was confirmed in renal biopsies from patients with diabetic kidney disease (DKD) and was decreased by GS-444217 in several rodent models of kidney injury and fibrosis that collectively represented the hallmarks of DKD pathology. Treatment with GS-444217 reduced progressive inflammation and fibrosis in the kidney and halted glomerular filtration rate decline. Combination of GS-444217 with enalapril, an angiotensin-converting enzyme inhibitor, led to a greater reduction in proteinuria and regression of glomerulosclerosis. These results identify ASK1 as an important target for renal disease and support the clinical development of an ASK1 inhibitor for the treatment of DKD.


Assuntos
Nefropatias Diabéticas/enzimologia , Fibroblastos/enzimologia , Glomérulos Renais/enzimologia , MAP Quinase Quinase Quinase 5/metabolismo , Sistema de Sinalização das MAP Quinases , Animais , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Modelos Animais de Doenças , Feminino , Fibroblastos/patologia , Fibrose , Humanos , Glomérulos Renais/patologia , MAP Quinase Quinase Quinase 5/antagonistas & inibidores , MAP Quinase Quinase Quinase 5/genética , Masculino , Camundongos , Camundongos Knockout , Inibidores de Proteínas Quinases/farmacologia , Distribuição Aleatória , Ratos Sprague-Dawley
13.
Anal Biochem ; 373(1): 141-6, 2008 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-17868635

RESUMO

Arraying proteins is often more challenging than creating oligonucleotide arrays. Protein concentration and purity can severely limit the capacity of spots created by traditional pin and ink jet printing techniques. To improve protein printing methods, we have developed a three-dimensional microfluidic system to deposit protein samples within discrete spots (250-microm squares) on a target surface. Our current technology produces a 48-spot array within a 0.5 x 1 cm target area. A chief advantage of this method is that samples may be introduced in continuous flow, which makes it possible to expose each spot to a larger volume of sample than would be possible with standard printing methods. Using Biacore Flexchip (Biacore AB) surface plasmon resonance array-based biosensor as a chip reader, we demonstrate that the microfluidic printer is capable of spotting proteins that are dilute (<0.1 microg/ml) and contain high concentrations of contaminating protein (>10,000-fold molar excess). We also show that the spots created by the microfluidic printer are more uniform and have better-defined borders than what can be achieved with pin printing. The ability to readily print proteins using continuous flow will help expand the application of protein arrays.


Assuntos
Microfluídica/métodos , Proteínas/isolamento & purificação , Ressonância de Plasmônio de Superfície/métodos , Análise Serial de Proteínas
14.
J Biol Chem ; 282(41): 30120-30, 2007 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-17716977

RESUMO

Formins are multidomain proteins that regulate numerous cytoskeleton-dependent cellular processes. These effects are mediated by the presence of two regions of homology, formin homology 1 and FH2. The diaphanous-related formins (DRFs) are distinguished by the presence of interacting N- and C-terminal regulatory domains. The GTPase binding domain and diaphanous inhibitory domain (DID) are found in the N terminus and bind to the diaphanous autoregulatory domain (DAD) found in the C terminus. Adjacent to the DID is an N-terminal dimerization motif (DD) and coiled-coil region (CC). The N terminus of Dia1 is also proposed to contain a Rho-independent membrane-targeting motif. We undertook an extensive structure/function analysis of the mDia1 N terminus to further our understanding of its role in vivo. We show here that both DID and DD are required for efficient autoinhibition in the context of full-length mDia1 and that the DD of mDia1 and mDia2, like formin homology 2, mediates homo- but not heterodimerization with other DRF family members. In contrast, our results suggest that the DID/DAD interaction mediates heterodimerization of full-length mDia1 and mDia2 and that the auto-inhibited conformation of DRFs is oligomeric. In addition, we also show that the DD/CC region is required for the Rho-independent membrane targeting of the isolated N terminus.


Assuntos
Proteínas de Transporte/química , NADPH Desidrogenase/química , Actinas/química , Motivos de Aminoácidos , Animais , Linhagem Celular , Citoplasma/metabolismo , Dimerização , Forminas , Regulação da Expressão Gênica , Imuno-Histoquímica/métodos , Camundongos , Proteínas Associadas aos Microtúbulos , Modelos Biológicos , Células NIH 3T3 , Ligação Proteica , Estrutura Terciária de Proteína
15.
Anal Biochem ; 364(1): 67-77, 2007 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-17362870

RESUMO

A total of 22 individuals participated in this benchmark study to characterize the thermodynamics of small-molecule inhibitor-enzyme interactions using Biacore instruments. Participants were provided with reagents (the enzyme carbonic anhydrase II, which was immobilized onto the sensor surface, and four sulfonamide-based inhibitors) and were instructed to collect response data from 6 to 36 degrees C. van't Hoff enthalpies and entropies were calculated from the temperature dependence of the binding constants. The equilibrium dissociation and thermodynamic constants determined from the Biacore analysis matched the values determined using isothermal titration calorimetry. These results demonstrate that immobilization of the enzyme onto the sensor surface did not alter the thermodynamics of these interactions. This benchmark study also provides insights into the opportunities and challenges in carrying out thermodynamic studies using optical biosensors.


Assuntos
Técnicas Biossensoriais/instrumentação , Calorimetria/instrumentação , Calorimetria/normas , Inibidores da Anidrase Carbônica/classificação , Inibidores da Anidrase Carbônica/metabolismo , Sulfonamidas/antagonistas & inibidores , Benchmarking , Pesquisa Biomédica , Técnicas Biossensoriais/normas , Anidrase Carbônica II/química , Anidrase Carbônica II/metabolismo , Variações Dependentes do Observador , Ligação Proteica , Sulfonamidas/classificação , Ressonância de Plasmônio de Superfície/instrumentação , Ressonância de Plasmônio de Superfície/normas , Termodinâmica
16.
Anal Biochem ; 359(1): 112-9, 2006 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-17027901

RESUMO

A Biacore T100 optical biosensor was used to characterize the binding kinetics of a panel of antigen binding fragments (Fabs) directed against the PcrV protein from Pseudomonas aeruginosa. PcrV protein forms part of the type III secretion system complex of this opportunistic pathogen. We demonstrate that the biosensor response data for each Fab collected from three different surface densities of the antigen could be fit globally to a simple 1:1 interaction model. Importantly, we found that the Fabs with the slowest dissociation rate provided the best protection in cell cytotoxicity studies. To further characterize the Fab interactions, binding data were automatically acquired at different temperatures and under different buffer conditions. The comprehensive characterization of these Fabs shows how Biacore T100 can be used to complement protein therapeutic discovery programs from basic research to the selection of therapeutic candidates.


Assuntos
Reações Antígeno-Anticorpo/imunologia , Antígenos de Bactérias/biossíntese , Toxinas Bacterianas/biossíntese , Técnicas Biossensoriais/métodos , Fragmentos Fab das Imunoglobulinas/biossíntese , Fragmentos Fab das Imunoglobulinas/imunologia , Proteínas Citotóxicas Formadoras de Poros/imunologia , Pseudomonas aeruginosa/patogenicidade , Animais , Antígenos de Bactérias/química , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/química , Toxinas Bacterianas/imunologia , Humanos , Camundongos , Proteínas Citotóxicas Formadoras de Poros/biossíntese , Proteínas Citotóxicas Formadoras de Poros/química , Pseudomonas aeruginosa/imunologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Projetos de Pesquisa
17.
Anal Biochem ; 358(2): 281-8, 2006 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-16962556

RESUMO

A ProteOn XPR36 parallel array biosensor was used to characterize the binding kinetics of a set of small molecule/enzyme interactions. Using one injection with the ProteOn's crisscrossing flow path system, we collected response data for six different concentrations of each analyte over six different target protein surfaces. This "one-shot" approach to kinetic analysis significantly improves throughput while generating high-quality data even for low-molecular-mass analytes. We found that the affinities determined for nine sulfonamide-based inhibitors of the enzyme carbonic anhydrase II were highly correlated with the values determined using isothermal titration calorimetry. We also measured the temperature dependence (from 15 to 35 degrees C) of the kinetics for four of the inhibitor/enzyme interactions. Our results illustrate the potential of this new parallel-processing biosensor to increase the speed of kinetic analysis in drug discovery and expand the applications of real-time protein interaction arrays.


Assuntos
Técnicas Biossensoriais , Anidrase Carbônica II/efeitos dos fármacos , Anidrase Carbônica II/metabolismo , Inibidores da Anidrase Carbônica/farmacologia , Cinética , Peso Molecular , Ligação Proteica , Sulfonamidas/farmacologia , Temperatura
18.
Anal Biochem ; 359(1): 94-105, 2006 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-17007806

RESUMO

In this benchmark study, 26 investigators were asked to characterize the kinetics and affinities of 10 sulfonamide inhibitors binding to the enzyme carbonic anhydrase II using Biacore optical biosensors. A majority of the participants collected data that could be fit to a 1:1 interaction model, but a subset of the data sets obtained from some instruments were of poor quality. The experimental errors in the k(a), k(d), and K(D) parameters determined for each of the compounds averaged 34, 24, and 37%, respectively. As expected, the greatest variation in the reported constants was observed for compounds with exceptionally weak affinity and/or fast association rates. The binding constants determined using the biosensor correlated well with solution-based titration calorimetry measurements. The results of this study provide insight into the challenges, as well as the level of experimental variation, that one would expect to observe when using Biacore technology for small molecule analyses.


Assuntos
Anidrase Carbônica II/química , Anidrase Carbônica II/metabolismo , Inibidores da Anidrase Carbônica/metabolismo , Sulfonamidas/antagonistas & inibidores , Técnicas Biossensoriais , Calorimetria , Inibidores da Anidrase Carbônica/classificação , Variações Dependentes do Observador , Ligação Proteica , Pesquisadores , Sulfonamidas/classificação , Ressonância de Plasmônio de Superfície/instrumentação , Ressonância de Plasmônio de Superfície/normas
19.
Anal Biochem ; 330(1): 98-113, 2004 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-15183767

RESUMO

To gauge the experimental variability associated with Biacore analysis, 36 different investigators analyzed a small molecule/enzyme interaction under similar conditions. Acetazolamide (222 g/mol) binding to carbonic anhydrase II (CAII; 30000 Da) was chosen as a model system. Both reagents were stable and their interaction posed a challenge to measure because of the low molecular weight of the analyte and the fast association rate constant. Each investigator created three different density surfaces of CAII and analyzed an identical dilution series of acetazolamide (ranging from 4.1 to 1000 nM). The greatest variability in the results was observed during the enzyme immobilization step since each investigator provided their own surface activating reagents. Variability in the quality of the acetazolamide binding responses was likely a product of how well the investigators' instruments had been maintained. To determine the reaction kinetics, the responses from the different density surfaces were fit globally to a 1:1 interaction model that included a term for mass transport. The averaged association and dissociation rate constants were 3.1+/-1.6 x 10(6)M(-1)s(-1) and 6.7+/-2.5 x 10(-2)s(-1), respectively, which corresponded to an average equilibrium dissociation constant (K(D) of 2.6+/-1.4 x 10(-8)M. The results provide a benchmark of variability in interpreting binding constants from the biosensor and highlight keys areas that should be considered when analyzing small molecule interactions.


Assuntos
Acetazolamida/química , Anidrase Carbônica II/química , Ressonância de Plasmônio de Superfície , Acetazolamida/metabolismo , Anidrase Carbônica II/metabolismo , Cinética , Variações Dependentes do Observador , Ligação Proteica , Pesquisadores , Ressonância de Plasmônio de Superfície/instrumentação , Ressonância de Plasmônio de Superfície/normas
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