RESUMO
Lysosomal acid lipase (LAL) is a serine hydrolase that hydrolyzes cholesteryl ester (CE) and TGs delivered to the lysosomes into free cholesterol and fatty acids. LAL deficiency due to mutations in the LAL gene (LIPA) results in accumulation of TGs and cholesterol esters in various tissues of the body leading to pathological conditions such as Wolman's disease and CE storage disease (CESD). Here, we present the first crystal structure of recombinant human LAL (HLAL) to 2.6 Å resolution in its closed form. The crystal structure was enabled by mutating three of the six potential glycosylation sites. The overall structure of HLAL closely resembles that of the evolutionarily related human gastric lipase (HGL). It consists of a core domain belonging to the classical α/ß hydrolase-fold family with a classical catalytic triad (Ser-153, His-353, Asp-324), an oxyanion hole, and a "cap" domain, which regulates substrate entry to the catalytic site. Most significant structural differences between HLAL and HGL exist at the lid region. Deletion of the short helix, 238NLCFLLC244, at the lid region implied a possible role in regulating the highly hydrophobic substrate binding site from self-oligomerization during interfacial activation. We also performed molecular dynamic simulations of dog gastric lipase (lid-open form) and HLAL to gain insights and speculated a possible role of the human mutant, H274Y, leading to CESD.
Assuntos
Doença do Armazenamento de Colesterol Éster/enzimologia , Esterol Esterase/química , Esterol Esterase/metabolismo , Doença do Armazenamento de Colesterol Éster/genética , Cristalografia por Raios X , Glicosilação , Humanos , Modelos Moleculares , Mutação , Domínios Proteicos , Esterol Esterase/genéticaRESUMO
Diabetic nephropathy remains an area of high unmet medical need, with current therapies that slow down, but do not prevent, the progression of disease. A reduced phosphorylation state of adenosine monophosphate-activated protein kinase (AMPK) has been correlated with diminished kidney function in both humans and animal models of renal disease. Here, we describe the identification of novel, potent, small molecule activators of AMPK that selectively activate AMPK heterotrimers containing the ß1 subunit. After confirming that human and rodent kidney predominately express AMPK ß1, we explore the effects of pharmacological activation of AMPK in the ZSF1 rat model of diabetic nephropathy. Chronic administration of these direct activators elevates the phosphorylation of AMPK in the kidney, without impacting blood glucose levels, and reduces the progression of proteinuria to a greater degree than the current standard of care, angiotensin-converting enzyme inhibitor ramipril. Further analyses of urine biomarkers and kidney tissue gene expression reveal AMPK activation leads to the modulation of multiple pathways implicated in kidney injury, including cellular hypertrophy, fibrosis, and oxidative stress. These results support the need for further investigation into the potential beneficial effects of AMPK activation in kidney disease.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Aminopiridinas/farmacologia , Nefropatias Diabéticas/tratamento farmacológico , Ativadores de Enzimas/farmacologia , Indóis/farmacologia , Rim/efeitos dos fármacos , Aminopiridinas/uso terapêutico , Animais , Tamanho Celular , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Ativação Enzimática , Fibrose , Humanos , Indóis/uso terapêutico , Isoenzimas/metabolismo , Rim/metabolismo , Rim/patologia , Testes de Função Renal , Macaca fascicularis , Camundongos Endogâmicos C57BL , Estresse Oxidativo , Fosforilação , Proteinúria/tratamento farmacológico , Proteinúria/metabolismo , Ratos , Especificidade da EspécieRESUMO
AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that serves as a pleotropic regulator of whole body energy homoeostasis. AMPK exists as a heterotrimeric complex, composed of a catalytic subunit (α) and two regulatory subunits (ß and γ), each present as multiple isoforms. In the present study, we compared the enzyme kinetics and allosteric modulation of six recombinant AMPK isoforms, α1ß1γ1, α1ß2γ1, α1ß2γ3, α2ß1γ1, α2ß2γ1 and α2ß2γ3 using known activators, A769662 and AMP. The α1-containing complexes exhibited higher specific activities and lower Km values for a widely used peptide substrate (SAMS) compared with α2-complexes. Surface plasmon resonance (SPR)-based direct binding measurements revealed biphasic binding modes with two distinct equilibrium binding constants for AMP, ADP and ATP across all isoforms tested. The α2-complexes were â¼25-fold more sensitive than α1-complexes to dephosphorylation of a critical threonine on their activation loop (pThr(172/174)). However, α2-complexes were more readily activated by AMP than α1-complexes. Compared with ß1-containing heterotrimers, ß2-containing AMPK isoforms are less sensitive to activation by A769662, a synthetic activator. These data demonstrate that ligand induced activation of AMPK isoforms may vary significantly based on their AMPK subunit composition. Our studies provide insights for the design of isoform-selective AMPK activators for the treatment of metabolic diseases.
Assuntos
Proteínas Quinases Ativadas por AMP/química , Monofosfato de Adenosina/química , Regulação Alostérica , Compostos de Bifenilo , Ativação Enzimática , Ativadores de Enzimas/química , Ensaios Enzimáticos , Humanos , Isoenzimas/química , Cinética , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Pironas/química , Proteínas Recombinantes/química , Tiofenos/químicaRESUMO
Adenosine monophosphate-activated protein kinase (AMPK), a serine/threonine heterotrimeric protein kinase, is a critical regulator of cellular and whole body energy homeostasis. There are twelve known AMPK isoforms that are differentially expressed in tissues and species. Dysregulation of AMPK signaling is associated with a multitude of human pathologies. Hence isoform-selective activators of AMPK are actively being sought for the treatment of cardiovascular and metabolic diseases. The present review summarizes the status of direct AMPK activators from the patent and published literature.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Ativadores de Enzimas/farmacologia , Proteínas Quinases Ativadas por AMP/química , Animais , Ativação Enzimática , Humanos , Fosforilação , Conformação ProteicaRESUMO
Glycogen synthase kinase-3 (GSK-3) regulates multiple cellular processes in diabetes, oncology, and neurology. N-(3-(1H-1,2,4-triazol-1-yl)propyl)-5-(3-chloro-4-methoxyphenyl)oxazole-4-carboxamide (PF-04802367 or PF-367) has been identified as a highly potent inhibitor, which is among the most selective antagonists of GSK-3 to date. Its efficacy was demonstrated in modulation of tau phosphorylation inâ vitro and inâ vivo. Whereas the kinetics of PF-367 binding in brain tissues are too fast for an effective therapeutic agent, the pharmacokinetic profile of PF-367 is ideal for discovery of radiopharmaceuticals for GSK-3 in the central nervous system. A (11) C-isotopologue of PF-367 was synthesized and preliminary PET imaging studies in non-human primates confirmed that we have overcome the two major obstacles for imaging GSK-3, namely, reasonable brain permeability and displaceable binding.
Assuntos
Encéfalo/efeitos dos fármacos , Encéfalo/diagnóstico por imagem , Neuroimagem , Oxazóis/farmacologia , Tomografia por Emissão de Pósitrons , Inibidores de Proteínas Quinases/farmacologia , Triazóis/farmacologia , Proteínas tau/antagonistas & inibidores , Encéfalo/metabolismo , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Modelos Moleculares , Estrutura Molecular , Oxazóis/síntese química , Oxazóis/química , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Triazóis/síntese química , Triazóis/química , Proteínas tau/metabolismoRESUMO
Lysosomal acid lipase (LAL) is a serine hydrolase which hydrolyzes cholesteryl ester and triglycerides delivered to the lysosomes into free cholesterol and free fatty acids. Mutations in the LAL gene (LIPA) result in accumulation of triglycerides and cholesterol esters in various tissues of the body, leading to pathological conditions such as Wolman's disease (WD) and cholesteryl ester storage disease (CESD). CESD patients homozygous for His295Tyr (H295Y) mutation have less than 5% of normal LAL activity. To shed light on the molecular basis for this loss-of-function phenotype, we have generated the recombinant H295Y enzyme and studied its biophysical and biochemical properties. No significant differences were observed in the expression levels or glycosylation patterns between the mutant and the wild type LAL. However, the H295Y mutant displayed only residual enzymatic activity (<5%) compared to the wild type. While wild type LAL is mostly a monomer at pH 5.0, the vast majority H295Y exists as a high molecular soluble aggregate. Besides, the H295Y mutant has a 20°C lower melting temperature compared to the wild type. Transient expression studies in WD fibroblasts showed that mutation of His295 to other amino acids resulted in a significant loss of enzymatic activity. A homology model of LAL revealed that His295 is located on an α-helix of the cap domain and could be important for tethering it to its core domain. The observed loss-of-function phenotype in CESD patients might arise from a combination of protein destabilization and the shift to a non-functional soluble aggregate.
Assuntos
Lisossomos/enzimologia , Esterol Esterase/genética , Doença de Wolman/enzimologia , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Ésteres do Colesterol/química , Ésteres do Colesterol/metabolismo , Clonagem Molecular , Fibroblastos/metabolismo , Fibroblastos/patologia , Expressão Gênica , Glicosilação , Humanos , Cinética , Metabolismo dos Lipídeos , Lisossomos/patologia , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Plasmídeos/química , Plasmídeos/metabolismo , Agregados Proteicos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Células Sf9 , Spodoptera , Esterol Esterase/isolamento & purificação , Esterol Esterase/metabolismo , Doença de Wolman/genética , Doença de Wolman/patologiaRESUMO
ITK (interleukin-2-inducible T-cell kinase) is a critical component of signal transduction in T-cells and has a well-validated role in their proliferation, cytokine release and chemotaxis. ITK is an attractive target for the treatment of T-cell-mediated inflammatory diseases. In the present study we describe the discovery of kinase inhibitors that preferentially bind to an allosteric pocket of ITK. The novel ITK allosteric site was characterized by NMR, surface plasmon resonance, isothermal titration calorimetry, enzymology and X-ray crystallography. Initial screening hits bound to both the allosteric pocket and the ATP site. Successful lead optimization was achieved by improving the contribution of the allosteric component to the overall inhibition. NMR competition experiments demonstrated that the dual-site binders showed higher affinity for the allosteric site compared with the ATP site. Moreover, an optimized inhibitor displayed non-competitive inhibition with respect to ATP as shown by steady-state enzyme kinetics. The activity of the isolated kinase domain and auto-activation of the full-length enzyme were inhibited with similar potency. However, inhibition of the activated full-length enzyme was weaker, presumably because the allosteric site is altered when ITK becomes activated. An optimized lead showed exquisite kinome selectivity and is efficacious in human whole blood and proximal cell-based assays.
Assuntos
Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Trifosfato de Adenosina/farmacologia , Regulação Alostérica , Sítio Alostérico , Cristalização , Cristalografia por Raios X , Humanos , Modelos Moleculares , Conformação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Ressonância de Plasmônio de SuperfícieRESUMO
Leucine rich repeat kinase 2 (LRRK2) has been genetically linked to Parkinson's disease (PD). The most common mutant, G2019S, increases kinase activity, thus LRRK2 kinase inhibitors are potentially useful in the treatment of PD. We herein disclose the structure, potential ligand-protein binding interactions, and pharmacological profiling of potent and highly selective kinase inhibitors based on a triazolopyridazine chemical scaffold.
Assuntos
Compostos Heterocíclicos com 2 Anéis/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/química , Piridazinas/farmacologia , Triazóis/farmacologia , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Compostos Heterocíclicos com 2 Anéis/síntese química , Compostos Heterocíclicos com 2 Anéis/química , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína/efeitos dos fármacos , Piridazinas/síntese química , Piridazinas/química , Relação Estrutura-Atividade , Triazóis/síntese química , Triazóis/químicaRESUMO
Installation of sites for metabolism in the lead compound PHA-767408 was the key focus of the IKK-2 inhaled program. This paper reports our efforts to identify a novel series of aminopyridinecarboxamide-based IKK-2 inhibitors, which display low nanomolar potency against IKK-2 with long duration of action (DOA), and metabolically labile to phase I and/or phase II metabolizing enzymes with potential capability for multiple routes of clearance. Several compounds have demonstrated their potential usefulness in the treatment of asthma and chronic obstructive pulmonary disease (COPD).
Assuntos
Aminopiridinas/síntese química , Asma/tratamento farmacológico , Quinase I-kappa B/antagonistas & inibidores , Niacinamida/análogos & derivados , Inibidores de Proteínas Quinases/síntese química , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Pirazóis/síntese química , Administração por Inalação , Aminopiridinas/química , Aminopiridinas/farmacologia , Ligação Competitiva , Desenho de Fármacos , Células HEK293 , Humanos , Indazóis/química , Indazóis/metabolismo , Indazóis/farmacologia , Ácidos Isonicotínicos/química , Ácidos Isonicotínicos/metabolismo , Ácidos Isonicotínicos/farmacologia , Microssomos Hepáticos/efeitos dos fármacos , Modelos Moleculares , Estrutura Molecular , Terapia de Alvo Molecular , Niacinamida/síntese química , Niacinamida/química , Niacinamida/metabolismo , Niacinamida/farmacologia , Fenetilaminas/metabolismo , Bloqueadores dos Canais de Potássio/metabolismo , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/química , Pirazóis/metabolismo , Pirazóis/farmacologia , Relação Estrutura-Atividade , Sulfonamidas/metabolismoRESUMO
AMP-activated protein kinase (AMPK) is an energy-sensing serine/threonine protein kinase that plays a central role in whole-body energy homeostasis. AMPK is a heterotrimeric enzyme with a catalytic (alpha) subunit and two regulatory (beta and gamma) subunits. The muscle-specific AMPK heterotrimeric complex (alpha2beta2gamma3) is involved in glucose and fat metabolism in skeletal muscle and therefore has emerged as an attractive target for drug development for diabetes and metabolic syndrome. To date, expression of recombinant full-length human AMPK alpha2beta2gamma3 has not been reported. Here we describe the expression, purification and biochemical characterization of functional full-length AMPK alpha2beta2gamma3 heterotrimeric complex using an Escherichia coli expression system. All three subunits of AMPK alpha2beta2gamma3 were transcribed as a single tricistronic transcript driven by the T7 RNA polymerase promoter, allowing spontaneous formation of the heterotrimeric complex in the bacterial cytosol. The self-assembled trimeric complex was purified from the cell lysate by nickel-ion chromatography using the hexahistidine tag fused exclusively at the N-terminus of the alpha 2 domain. The un-assembled beta 2 and gamma 3 domains were removed by extensive washing of the column. Further purification of the heterotrimer was performed using size exclusion chromatography. The final yield of the recombinant AMPK alpha2beta2gamma3 complex was 1.1mg/L culture in shaker flasks. The E. coli expressed enzyme was catalytically inactive after purification, but was activated in vitro by upstream kinases such as CaMKKbeta and LKB1. The kinase activity of activated AMPK alpha2beta2gamma3 complex was significantly enhanced by AMP (an allosteric activator) but not by thienopyridone A-769662, a known small molecule activator of AMPK. Mass spectrometric characterization of recombinant AMPK alpha2beta2gamma3 showed significant heterogeneity before and after activation that could potentially hamper crystallographic studies of this complex.
Assuntos
Proteínas Quinases Ativadas por AMP/fisiologia , Escherichia coli/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Monofosfato de Adenosina/metabolismo , Compostos de Bifenilo , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/genética , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/fisiologia , Domínio Catalítico , Subunidade RIIbeta da Proteína Quinase Dependente de AMP Cíclico/genética , Subunidade RIIbeta da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Escherichia coli/genética , Homeostase , Humanos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Pironas/farmacologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Tiofenos/farmacologiaRESUMO
NF-kappaB signaling plays a pivotal role in a variety of pathological conditions. Because of its central role in the overall NF-kappaB regulation, IKK-2 is a viable target for drug discovery. In order to enable structure-based design of IKK-2 inhibitors, we carried out a rational generation of IKK-2 mutants based on induced-fit docking of a selective IKK-2 inhibitor, PHA-408, into the homology model of IKK-2. One mutant we have characterized is a catalytically inactive form of IKK-2, D145A IKK-2, wherein the catalytic aspartic acid, D145 was replaced with alanine. Unlike the WT enzyme, D145A IKK-2 is devoid of kinase activity despite its ability to bind ATP with high affinity and is not phosphorylated at the T loop. In addition, this mutant binds a diverse collection of inhibitors with comparable binding affinities to WT IKK-2. Another interesting mutant we have characterized is F26A IKK-2 (F26 is an aromatic residue located at the very tip of the Gly-rich loop). Pre-incubation of F26A IKK-2 with PHA-408 revealed the role of F26 in the time-dependent binding of this inhibitor. Thus, functional characterization of these mutants provides the first evidence showing the role of a Gly-rich loop residue of a kinase in binding kinetics. These two mutants along with others that we have identified could be used to validate homology models and probe the interactions of IKK-2 with a variety of inhibitors.
Assuntos
Quinase I-kappa B/metabolismo , Animais , Células Cultivadas , Simulação por Computador , Descoberta de Drogas/métodos , Humanos , Quinase I-kappa B/biossíntese , Quinase I-kappa B/química , Quinase I-kappa B/genética , Indazóis/química , Indazóis/metabolismo , Ácidos Isonicotínicos/química , Ácidos Isonicotínicos/metabolismo , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Fosforilação , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Spodoptera , Homologia Estrutural de ProteínaRESUMO
In this manuscript, we report the discovery of the substituted 2-trifluoromethyl-2H-benzopyran-3-carboxylic acids as a novel series of potent and selective cyclooxygenase-2 (COX-2) inhibitors. 5c-(S) (SD-8381) was advanced into clinical studies due to its superior in vivo potency. The high plasma protein binding (>99% bound) of 5c-(S) has resulted in a surprisingly long human half life t(1/2)=360 h.
Assuntos
Benzopiranos/química , Benzopiranos/farmacocinética , Inibidores de Ciclo-Oxigenase 2/química , Proteínas Sanguíneas/metabolismo , Ácidos Carboxílicos , Inibidores de Ciclo-Oxigenase 2/farmacocinética , Meia-Vida , Humanos , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
In this Letter, we provide the structure-activity relationships, optimization of design, testing criteria, and human half-life data for a series of selective COX-2 inhibitors. During the course of our structure-based drug design efforts, we discovered two distinct binding modes within the COX-2 active site for differently substituted members of this class. The challenge of a undesirably long human half-life for the first clinical candidate 1t(1/2)=360 h was addressed by multiple strategies, leading to the discovery of 29b-(S) (SC-75416) with t(1/2)=34 h.
Assuntos
Benzopiranos/química , Benzopiranos/farmacocinética , Inibidores de Ciclo-Oxigenase 2/farmacocinética , Sítios de Ligação , Domínio Catalítico , Inibidores de Ciclo-Oxigenase 2/química , Meia-Vida , Humanos , Relação Estrutura-AtividadeRESUMO
PH-797804 is a diarylpyridinone inhibitor of p38alpha mitogen-activated protein (MAP) kinase derived from a racemic mixture as the more potent atropisomer (aS), first proposed by molecular modeling and subsequently confirmed by experiments. On the basis of structural comparison with a different biaryl pyrazole template and supported by dozens of high-resolution crystal structures of p38alpha inhibitor complexes, PH-797804 is predicted to possess a high level of specificity across the broad human kinase genome. We used a structural bioinformatics approach to identify two selectivity elements encoded by the TXXXG sequence motif on the p38alpha kinase hinge: (i) Thr106 that serves as the gatekeeper to the buried hydrophobic pocket occupied by 2,4-difluorophenyl of PH-797804 and (ii) the bidentate hydrogen bonds formed by the pyridinone moiety with the kinase hinge requiring an induced 180 degrees rotation of the Met109-Gly110 peptide bond. The peptide flip occurs in p38alpha kinase due to the critical glycine residue marked by its conformational flexibility. Kinome-wide sequence mining revealed rare presentation of the selectivity motif. Corroboratively, PH-797804 exhibited exceptionally high specificity against MAP kinases and the related kinases. No cross-reactivity was observed in large panels of kinase screens (selectivity ratio of >500-fold). In cellular assays, PH-797804 demonstrated superior potency and selectivity consistent with the biochemical measurements. PH-797804 has met safety criteria in human phase I studies and is under clinical development for several inflammatory conditions. Understanding the rationale for selectivity at the molecular level helps elucidate the biological function and design of specific p38alpha kinase inhibitors.
Assuntos
Benzamidas/farmacologia , Biologia Computacional , Inibidores de Proteínas Quinases/farmacologia , Pironas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Benzamidas/química , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Estrutura Molecular , Fosforilação , Inibidores de Proteínas Quinases/química , Piridonas , Pironas/química , Especificidade por Substrato , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Optimization of kinase selectivity for a set of benzothiophene MK2 inhibitors provided analogs with potencies of less than 500 nM in a cell based assay. The selectivity of the inhibitors can be rationalized by examination of X-ray crystal structures of inhibitors bound to MK2.
Assuntos
MAP Quinase Quinase 2/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Tiofenos/química , Sítios de Ligação , Linhagem Celular Tumoral , Cristalografia por Raios X , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 2 Dependente de Ciclina/metabolismo , Desenho de Fármacos , Humanos , MAP Quinase Quinase 2/metabolismo , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Tiofenos/síntese química , Tiofenos/farmacologiaRESUMO
Identification of potent benzothiophene inhibitors of mitogen activated protein kinase-activated protein kinase 2 (MK2), structure-activity relationship (SAR) studies, selectivity assessments against CDK2, cellular potency and mechanism of action are presented. Crystallographic data provide a rationale for the observed MK2 potency as well as selectivity over CDK2 for this class of inhibitors.
Assuntos
MAP Quinase Quinase 2/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Tiofenos/química , Sítios de Ligação , Linhagem Celular Tumoral , Cristalografia por Raios X , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 2 Dependente de Ciclina/metabolismo , Humanos , MAP Quinase Quinase 2/metabolismo , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Relação Estrutura-Atividade , Tiofenos/síntese química , Tiofenos/farmacologiaRESUMO
Inducible, microsomal prostaglandin E synthase 1 (mPGES-1), the terminal enzyme in the prostaglandin (PG) biosynthetic pathway, constitutes a promising therapeutic target for the development of new anti-inflammatory drugs. To elucidate structure-function relationships and to enable structure-based design, an mPGES-1 homology model was developed using the three-dimensional structure of the closest homologue of the MAPEG family (Membrane Associated Proteins in Eicosanoid and Glutathione metabolism), mGST-1. The ensuing model of mPGES-1 is a homo-trimer, with each monomer consisting of four membrane-spanning segments. Extensive structure refinement revealed an inter-monomer salt bridge (K26-E77) as well as inter-helical interactions within each monomer, including polar hydrogen bonds (e.g. T78-R110-T129) and hydrophobic pi-stacking (F82-F103-F106), all contributing to the overall stability of the homo-trimer of mPGES-1. Catalytic co-factor glutathione (GSH) was docked into the mPGES-1 model by flexible optimization of both the ligand and the protein conformations, starting from the initial location ascertained from the mGST-1 structure. Possible binding site for the substrate, prostaglandin H(2) (PGH(2)), was identified by systematically probing the refined molecular structure of mPGES-1. A binding model was generated by induced fit docking of PGH(2) in the presence of GSH. The homology model prescribes three potential inhibitor binding sites per mPGES-1 trimer. This was further confirmed experimentally by equilibrium dialysis study which generated a binding stoichiometric ratio of approximately three inhibitor molecules to three mPGES-1 monomers. The structural model that we have derived could serve as a useful tool for structure-guided design of inhibitors for this emergently important therapeutic target.
Assuntos
Inibidores Enzimáticos/química , Oxirredutases Intramoleculares/química , Microssomos/enzimologia , Sequência de Aminoácidos , Biopolímeros , Inibidores Enzimáticos/farmacologia , Humanos , Oxirredutases Intramoleculares/antagonistas & inibidores , Oxirredutases Intramoleculares/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Prostaglandina-E Sintases , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
Using structure-guided design, several cell based assays, and microdosed positron emission tomography (PET) imaging, we identified a series of highly potent, selective, and brain-penetrant oxazole-4-carboxamide-based inhibitors of glycogen synthase kinase-3 (GSK-3). An isotopologue of our first-generation lead, [3H]PF-367, demonstrates selective and specific target engagement in vitro, irrespective of the activation state. We discovered substantial ubiquitous GSK-3-specific radioligand binding in Tg2576 Alzheimer's disease (AD), suggesting application for these compounds in AD diagnosis and identified [11C]OCM-44 as our lead GSK-3 radiotracer, with optimized brain uptake by PET imaging in nonhuman primates. GSK-3ß-isozyme selectivity was assessed to reveal OCM-51, the most potent (IC50 = 0.030 nM) and selective (>10-fold GSK-3ß/GSK-3α) GSK-3ß inhibitor known to date. Inhibition of CRMP2T514 and tau phosphorylation, as well as favorable therapeutic window against WNT/ß-catenin signaling activation, was observed in cells.
Assuntos
Encéfalo/metabolismo , Descoberta de Drogas , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Tomografia por Emissão de Pósitrons/métodos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Animais , Barreira Hematoencefálica/metabolismo , Encéfalo/diagnóstico por imagem , Domínio Catalítico , Glicogênio Sintase Quinase 3 beta/química , Células HEK293 , Humanos , Camundongos , Modelos Moleculares , Neuroimagem , Oxazóis/química , Oxazóis/metabolismo , Oxazóis/farmacologia , Inibidores de Proteínas Quinases/metabolismo , Triazóis/química , Triazóis/metabolismo , Triazóis/farmacologiaRESUMO
Protein-ligand interactions can be evaluated by a number of different biophysical methods. Here we describe some of the experimental methods that we have used to generate AMPK protein reagents and characterize its interactions with direct synthetic activators. Recombinant heterotrimeric AMPK complexes were generated using standard molecular biology methods by expression either in insect cells via infection with three different viruses or more routinely in Escherichia coli with a tricistronic expression vector. Hydrogen/deuterium exchange (HDX) coupled with mass spectrometry was used to probe protein conformational changes and potential binding sites of activators on AMPK. X-ray crystallographic studies were carried out on crystals of AMPK with bound ligands to reveal detailed molecular interactions formed by AMPK activators at near-atomic resolution. In order to gain insights into the mechanism of enzyme activation and to probe the effects of AMPK activators on kinetic parameters such as Michaelis-Menten constant (K m ) or maximal reaction velocity (V max), we performed classical enzyme kinetic studies using radioactive 33P-ATP-based filter assay. Equilibrium dissociation constants (K D ) and on and off rates of ligand binding were obtained by application of surface plasmon resonance (SPR) technique.
Assuntos
Proteínas Quinases Ativadas por AMP/química , Medição da Troca de Deutério/métodos , Ativadores de Enzimas/química , Ressonância de Plasmônio de Superfície/métodos , Proteínas Quinases Ativadas por AMP/isolamento & purificação , Animais , Sítios de Ligação , Cristalografia por Raios X , Medição da Troca de Deutério/instrumentação , Ativação Enzimática , Ensaios Enzimáticos/instrumentação , Ensaios Enzimáticos/métodos , Cinética , Ligantes , Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Simulação de Acoplamento Molecular , Ligação Proteica , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Células Sf9 , Ressonância de Plasmônio de Superfície/instrumentaçãoRESUMO
Studies on indole-3-carboxylic acid derivatives as direct activators of human adenosine monophosphate-activated protein kinase (AMPK) α1ß1γ1 isoform have culminated in the identification of PF-06409577 (1), PF-06885249 (2), and PF-06679142 (3) as potential clinical candidates. Compounds 1-3 are primarily cleared in animals and humans via glucuronidation. Herein, we describe the biosynthetic preparation, purification, and structural characterization of the glucuronide conjugates of 1-3. Spectral characterization of the purified glucuronides M1, M2, and M3 indicated that they were acyl glucuronide derivatives. In vitro pharmacological evaluation revealed that all three acyl glucuronides retained selective activation of ß1-containing AMPK isoforms. Inhibition of de novo lipogenesis with representative parent carboxylic acids and their respective acyl glucuronide conjugates in human hepatocytes demonstrated their propensity to activate cellular AMPK. Cocrystallization of the AMPK α1ß1γ1 isoform with 1-3 and M1-M3 provided molecular insights into the structural basis for AMPK activation by the glucuronide conjugates.