RESUMO
Human GPR40 receptor (hGPR40), also known as free fatty-acid receptor 1 (FFAR1), is a G-protein-coupled receptor that binds long-chain free fatty acids to enhance glucose-dependent insulin secretion. Novel treatments for type-2 diabetes mellitus are therefore possible by targeting hGPR40 with partial or full agonists. TAK-875, or fasiglifam, is an orally available, potent and selective partial agonist of hGPR40 receptor, which reached phase III clinical trials for the potential treatment of type-2 diabetes mellitus. Data from clinical studies indicate that TAK-875, which is an ago-allosteric modulator of hGPR40 (ref. 3), demonstrates improved glycaemic control and low hypoglycaemic risk in diabetic patients. Here we report the crystal structure of hGPR40 receptor bound to TAK-875 at 2.3 Å resolution. The co-complex structure reveals a unique binding mode of TAK-875 and suggests that entry to the non-canonical binding pocket most probably occurs via the lipid bilayer. The atomic details of the extensive charge network in the ligand binding pocket reveal additional interactions not identified in previous studies and contribute to a clear understanding of TAK-875 binding to the receptor. The hGPR40-TAK-875 structure also provides insights into the plausible binding of multiple ligands to the receptor, which has been observed in radioligand binding and Ca(2+) influx assay studies. Comparison of the transmembrane helix architecture with other G-protein-coupled receptors suggests that the crystallized TAK-875-bound hGPR40 complex is in an inactive-like state.
Assuntos
Benzofuranos/química , Benzofuranos/farmacologia , Agonismo Parcial de Drogas , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/química , Sulfonas/química , Sulfonas/farmacologia , Regulação Alostérica/efeitos dos fármacos , Benzofuranos/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Diabetes Mellitus Tipo 2/tratamento farmacológico , Humanos , Ligantes , Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Receptores Acoplados a Proteínas G/metabolismo , Homologia Estrutural de Proteína , Sulfonas/metabolismo , Propriedades de SuperfícieRESUMO
Inhibition of phosphodiesterase 2A (PDE2A) has been proposed as a potential approach to enhance cognitive functioning and memory through boosting intracellular cGMP/cAMP and enhancing neuroplasticity in memory-related neural circuitry. Previous preclinical studies demonstrated that PDE2A inhibitors could reverse N-methyl-D-aspartate receptor antagonist (5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine or ketamine-induced memory deficit. Here, we report that the potent and selective PDE2A inhibitor 4-(1-azetidinyl)-7-methyl-5-[1-methyl-5-[5-(trifluoromethyl)-2-pyridinyl]-1H-pyrazol-4-yl]-imidazo[5,1-f][1,2,4]triazine (PF-05180999) enhances long-term memory in a contextual fear conditioning model in the rat at the oral dose of 0.3 mg/kg. Target engagement at this efficacious dose was explored using in vivo autoradiography. Converse to the results of a decrease of PDE2A binding (target occupancy) by the PDE2A inhibitor, a paradoxical increase (up to 40%) in PDE2A binding was detected. However, a typical target occupancy curve could be generated by PF-05180999 at much higher doses. In vitro experiments using recombinant PDE2A protein or rat brain homogenate that contains native PDE2A protein demonstrated that increased cGMP after initial PDE2A inhibition could be responsible for the activation of PDE2A enzyme via allosteric binding to the GAF-B domain, leading to positive cooperativity of the dormant PDE2A enzymes. Our results suggest that when evaluating target engagement of PDE2A inhibitors for memory disorder in clinical setting with occupancy assays, the efficacious dose may not fall on the typical receptor/target curve. On the contrary, an increase in PDE2A tracer binding is likely seen. Our results also suggest that when evaluating target occupancy of enzymes, potential regulation of enzyme activities should be considered.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/metabolismo , Memória de Longo Prazo/efeitos dos fármacos , Inibidores de Fosfodiesterase/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/fisiologia , Relação Dose-Resposta a Droga , Ligantes , Masculino , RatosRESUMO
Recent innovative approaches to stabilize and crystallize GPCRs have resulted in an unprecedented breakthrough in GPCR crystal structures as well as application of the purified receptor protein in biophysical and biochemical ligand binding assays. However, the protein optimization process to enable these technologies is lengthy and requires iterative overexpression, solubilization, purification and functional analysis of tens to hundreds of protein variants. Here, we report a new and versatile method to screen in parallel hundreds of GPCR variants in HEK293 produced virus-like particles (VLPs) for protein yield, stability, functionality and ligand binding. This approach reduces the time and resources during GPCR construct optimization by eliminating lengthy protein solubilization and purification steps and by its adaptability to many binding assay formats (label or label-free detection). We exemplified the robustness of our VLP method by screening 210 GALR3-VLP variants in a radiometric agonist-based binding assay and a subset of 88 variants in a label-free antagonist-based assay. The resulting GALR3 agonist or antagonist stabilizing variants were then further used for recombinant protein expression in transfected insect cells. The final purified protein variants were successfully immobilized on a biosensor chip and used in a surface plasmon resonance binding assay.
Assuntos
Expressão Gênica , Receptor Tipo 3 de Galanina , Proteínas Recombinantes de Fusão , Vírion , Células HEK293 , Humanos , Estabilidade Proteica , Receptor Tipo 3 de Galanina/biossíntese , Receptor Tipo 3 de Galanina/química , Receptor Tipo 3 de Galanina/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Vírion/química , Vírion/genética , Vírion/metabolismoRESUMO
Aberrant signaling of ErbB family members human epidermal growth factor 2 (HER2) and epidermal growth factor receptor (EGFR) is implicated in many human cancers, and HER2 expression is predictive of human disease recurrence and prognosis. Small molecule kinase inhibitors of EGFR and of both HER2 and EGFR have received approval for the treatment of cancer. We present the first high resolution crystal structure of the kinase domain of HER2 in complex with a selective inhibitor to understand protein activation, inhibition, and function at the molecular level. HER2 kinase domain crystallizes as a dimer and suggests evidence for an allosteric mechanism of activation comparable with previously reported activation mechanisms for EGFR and HER4. A unique Gly-rich region in HER2 following the α-helix C is responsible for increased conformational flexibility within the active site and could explain the low intrinsic catalytic activity previously reported for HER2. In addition, we solved the crystal structure of the kinase domain of EGFR in complex with a HER2/EGFR dual inhibitor (TAK-285). Comparison with previously reported inactive and active EGFR kinase domain structures gave insight into the mechanism of HER2 and EGFR inhibition and may help guide the design and development of new cancer drugs with improved potency and selectivity.
Assuntos
Receptor ErbB-2/química , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/genética , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Cristalografia por Raios X , Desenho de Fármacos , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Receptores ErbB/química , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/genética , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/uso terapêutico , Estrutura Terciária de Proteína , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptor ErbB-4 , Relação Estrutura-AtividadeRESUMO
Our aim was to discover RAF/vascular endothelial growth factor receptor 2 (VEGFR2) inhibitors that possess strong activity and sufficient oral absorption, and thus, we selected a 5-amino-linked thiazolo[5,4-d]pyrimidine derivative as the lead compound because of its potential kinase inhibitory activities and its desired solubility. The novel tertiary 1-cyano-1-methylethoxy substituent was designed to occupy the hydrophobic region of 'back pocket' of BRAF on the basis of the X-ray co-crystal structure data of BRAF. In addition, we found that N-methylation of the amine linker could control the twisted molecular conformation leading to improved solubility. These approaches produced N-methyl thiazolo[5,4-b]pyridine-5-amine derivative 5. To maximize the in vivo efficacy, we attempted salt formation of 5. Our result indicated that the besylate monohydrate salt form (5c) showed significant improvement of both solubility and oral absorption. Owing to the improved physicochemical properties, compound 5c demonstrated regressive antitumor efficacy in a HT-29 xenograft model.
Assuntos
Antineoplásicos/farmacologia , Desenho de Fármacos , Neoplasias Experimentais/tratamento farmacológico , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Tiazóis/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Células HT29 , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Camundongos , Microssomos/efeitos dos fármacos , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/química , Ratos , Ratos Endogâmicos F344 , Relação Estrutura-Atividade , Tiazóis/síntese química , Tiazóis/química , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Overexpression of biologically functional GPCRs and homogeneous purified protein solutions are required to enable structural studies and protein-based biophysical assay development. Iterative and time-consuming optimization cycles of protein engineering, expression, and purification are often needed to achieve the desired protein quantity and quality. Here, we describe the reconstitution of GPCRs in virus-like particles (VLPs) and their use in biophysical assays to characterize protein yield, stability, and small molecule ligand binding. This approach prevents the need for time-consuming detergent solubilization and protein purification during recombinant GPCR protein optimization.
Assuntos
Receptores Acoplados a Proteínas G , Biofísica , Cromatografia de Afinidade , Expressão Gênica , Ligantes , Receptores Acoplados a Proteínas G/química , Proteínas RecombinantesRESUMO
We have previously discovered nicotinic acid derivative 1 as a structurally novel dipeptidyl peptidase IV (DPP-4) inhibitor. In this study, we obtained the X-ray co-crystal structure between nicotinic acid derivative 1 and DPP-4. From these X-ray co-crystallography results, to achieve more potent inhibitory activity, we targeted Arg125 as a potential amino acid residue because it was located near the pyridine core, and some known DPP-4 inhibitors were reported to interact with this residue. We hypothesized that the guanidino group of Arg125 could interact with two hydrogen-bond acceptors in a bidentate manner. Therefore, we designed a series of 3-pyridylacetamide derivatives possessing an additional hydrogen-bond acceptor that could have the desired bidentate interaction with Arg125. We discovered the dihydrochloride of 1-{[5-(aminomethyl)-2-methyl-4-(4-methylphenyl)-6-(2-methylpropyl)pyridin-3-yl]acetyl}-l-prolinamide (13j) to be a potent and selective DPP-4 inhibitor that could interact with the guanidino group of Arg125 in a unique bidentate manner.
Assuntos
Acetamidas/química , Arginina/química , Inibidores da Dipeptidil Peptidase IV/farmacologia , Desenho de Fármacos , Cristalografia por Raios X , Inibidores da Dipeptidil Peptidase IV/química , Modelos Moleculares , Relação Estrutura-AtividadeRESUMO
The design, synthesis, and structure-activity relationships of a new class of potent and orally active non-peptide dipeptidyl peptidase IV (DPP-4) inhibitors, 3-aminomethyl-1,2-dihydro-4-phenyl-1-isoquinolones, are described. We hypothesized that the 4-phenyl group of the isoquinolone occupies the S1 pocket of the enzyme, the 3-aminomethyl group forms an electrostatic interaction with the S2 pocket, and the introduction of a hydrogen bond donor onto the 6- or 7-substituent provides interaction with the hydrophilic region of the enzyme. Based on this hypothesis, intensive research focused on developing new non-peptide DPP-4 inhibitors has been carried out. Among the compounds designed in this study, we identified 2-[(3-aminomethyl-2-(2-methylpropyl)-1-oxo-4-phenyl-1,2-dihydro-6-isoquinolinyl)oxy]acetamide (35a) as a potent, selective, and orally bioavailable DPP-4 inhibitor, which exhibited in vivo efficacy in diabetic model rats. Finally, X-ray crystallography of 35a in a complex with the enzyme validated our hypothesized binding mode and identified Lys554 as a new target-binding site available for DPP-4 inhibitors.
Assuntos
Dipeptidil Peptidase 4/efeitos dos fármacos , Inibidores da Dipeptidil Peptidase IV/síntese química , Hipoglicemiantes/síntese química , Isoquinolinas/síntese química , Administração Oral , Animais , Glicemia , Células CACO-2 , Diabetes Mellitus Tipo 2/tratamento farmacológico , Dipeptidil Peptidase 4/análise , Inibidores da Dipeptidil Peptidase IV/administração & dosagem , Inibidores da Dipeptidil Peptidase IV/química , Inibidores da Dipeptidil Peptidase IV/farmacologia , Inibidores da Dipeptidil Peptidase IV/uso terapêutico , Dipeptidil Peptidases e Tripeptidil Peptidases/análise , Dipeptidil Peptidases e Tripeptidil Peptidases/antagonistas & inibidores , Dipeptidil Peptidases e Tripeptidil Peptidases/efeitos dos fármacos , Desenho de Fármacos , Feminino , Teste de Tolerância a Glucose , Humanos , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Hipoglicemiantes/uso terapêutico , Isoquinolinas/administração & dosagem , Isoquinolinas/farmacologia , Isoquinolinas/uso terapêutico , Terapia de Alvo Molecular , Peptídeos/metabolismo , Quinolonas/administração & dosagem , Quinolonas/síntese química , Quinolonas/química , Quinolonas/farmacologia , Quinolonas/uso terapêutico , Ratos , Ratos Wistar , Relação Estrutura-AtividadeRESUMO
Dipeptidyl peptidase IV (DPP-4) inhibition is a validated therapeutic option for type 2 diabetes, exhibiting multiple antidiabetic effects with little or no risk of hypoglycemia. In our studies involving non-covalent DPP-4 inhibitors, a novel series of quinoline-based inhibitors were designed based on the co-crystal structure of isoquinolone 2 in complex with DPP-4 to target the side chain of Lys554. Synthesis and evaluation of designed compounds revealed 1-[3-(aminomethyl)-4-(4-methylphenyl)-2-(2-methylpropyl)quinolin-6-yl]piperazine-2,5-dione (1) as a potent, selective, and orally active DPP-4 inhibitor (IC50=1.3 nM) with long-lasting ex vivo activity in dogs and excellent antihyperglycemic effects in rats. A docking study of compound 1 revealed a hydrogen-bonding interaction with the side chain of Lys554, suggesting this residue as a potential target site useful for enhancing DPP-4 inhibition.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV/química , Inibidores da Dipeptidil Peptidase IV/uso terapêutico , Hipoglicemiantes/química , Hipoglicemiantes/uso terapêutico , Quinolinas/química , Quinolinas/uso terapêutico , Animais , Células CACO-2 , Linhagem Celular , Dipeptidil Peptidase 4/química , Inibidores da Dipeptidil Peptidase IV/farmacocinética , Inibidores da Dipeptidil Peptidase IV/farmacologia , Cães , Feminino , Humanos , Hipoglicemiantes/farmacocinética , Hipoglicemiantes/farmacologia , Lisina/metabolismo , Quinolinas/farmacocinética , Quinolinas/farmacologia , Ratos , Ratos WistarRESUMO
The binding of urokinase plaminogen activator (uPA) to its cell surface receptor (uPAR; CD87) promotes cell adhesion by increasing the affinity of the receptor for both vitronectin (VN) and integrins. We provide evidence that plasminogen activator inhibitor (PAI)-1 can detach cells by disrupting uPAR-VN and integrin-VN interactions and that it does so by binding to the uPA present in uPA-uPAR-integrin complexes on the cell surface. The detached cells cannot reattach to VN unless their surface integrins are first activated by treatment with MnCl2. Immunoprecipitation and subcellular fractionation experiments reveal that PAI-1 treatment triggers deactivation and disengagement of uPA-uPAR-integrin complexes and their endocytic clearance by the low density lipoprotein receptor-related protein. Transfection experiments demonstrate that efficient cell detachment by PAI-1 requires an excess of matrix-engaged uPA-uPAR-integrin complexes over free engaged integrins and that changes in this ratio alter the efficacy of PAI-1. Together, these results suggest a VN-independent, uPA-uPAR-dependent mechanism by which PAI-1 induces cell detachment. This pathway may represent a general mechanism, since PAI-1 also can detach cells from fibronectin and type-1 collagen. This novel "deadhesive" activity of PAI-1 toward a variety of cells growing on different extracellular matrices may begin to explain why high PAI-1 levels often are associated with a poor prognosis in human metastatic disease.
Assuntos
Adesão Celular/fisiologia , Membrana Celular/metabolismo , Células Eucarióticas/metabolismo , Matriz Extracelular/metabolismo , Integrinas/metabolismo , Metástase Neoplásica/fisiopatologia , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Animais , Células CHO , Membrana Celular/ultraestrutura , Movimento Celular/fisiologia , Colágeno Tipo I/metabolismo , Cricetinae , Endocitose/fisiologia , Células Eucarióticas/citologia , Matriz Extracelular/ultraestrutura , Fibronectinas/metabolismo , Humanos , Integrina alfaV/metabolismo , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/farmacologia , Receptores de Superfície Celular/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Vitronectina/metabolismoRESUMO
In medicinal chemistry, accurate prediction of additivity-based structure-activity relationship (SAR) analysis rests on three assumptions: (1) a consistent binding pose of the central scaffold, (2) no interaction between the R group substituents, and (3) a relatively rigid binding pocket in which the R group substituents act independently. Previously, examples of nonadditive SAR have been documented in systems that deviate from the first two assumptions. Local protein structural change upon ligand binding, through induced fit or conformational selection, although a well-known phenomenon that invalidates the third assumption, has not been linked to nonadditive SAR conclusively. Here, for the first time, we present clear structural evidence that the formation of a hydrophobic pocket upon ligand binding in PDE2 catalytic site reduces the size of another distinct subpocket and contributes to strong nonadditive SAR between two otherwise distant R groups.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/química , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/metabolismo , Inibidores Enzimáticos/farmacologia , Modelos Teóricos , Conformação Proteica , Quinazolinas/química , Triazóis/química , Sítios de Ligação , Cristalografia por Raios X , Inibidores Enzimáticos/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Galanin Receptor 3 (GALR3) is a G-protein-coupled receptor with a widespread distribution in the brain and plays a role in a variety of physiologic processes including cognition/memory, sensory/pain processing, hormone secretion, and feeding behavior. Therefore, GALR3 is considered an attractive CNS drug target (Freimann et al., 2015) [1]. This dataset contains GALR3 point mutants that improve recombinant protein expression and thermal stability of the receptor contained in virus-like particles (VLPs) or obtained by detergent-purification of baculovirus-infected insect cells. The mutations listed can be grouped in those that improve the stability of the agonist-bound and the antagonist-bound form of the receptor. Protein characteristics in terms of protein expression and thermal stability were comparable between GPCR-VLP and GPCR overexpressing Sf9 cultures. The further analysis and detailed results of these mutants as well as their impact on biophysical assay development for drug discovery can be found in "Method for Rapid Optimization of Recombinant GPCR Protein Expression and Stability using Virus-Like Particles" (Ho et al., 2017) [2].
RESUMO
A series of potent thienotriazolopyrimidinone-based PDE1 inhibitors was discovered. X-ray crystal structures of example compounds from this series in complex with the catalytic domain of PDE1B and PDE10A were determined, allowing optimization of PDE1B potency and PDE selectivity. Reduction of hERG affinity led to greater than a 3000-fold selectivity for PDE1B over hERG. 6-(4-Methoxybenzyl)-9-((tetrahydro-2H-pyran-4-yl)methyl)-8,9,10,11-tetrahydropyrido[4',3':4,5]thieno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidin-5(6H)-one was identified as an orally bioavailable and brain penetrating PDE1B enzyme inhibitor with potent memory-enhancing effects in a rat model of object recognition memory.
Assuntos
Memória/efeitos dos fármacos , Inibidores de Fosfodiesterase/farmacologia , Cristalografia por Raios X , Descoberta de Drogas , Inibidores de Fosfodiesterase/químicaRESUMO
A series of potent and selective [1,2,4]triazolo[1,5-a]pyrimidine PDE2a inhibitors is reported. The design and improvement of the binding properties of this series was achieved using X-ray crystal structures in conjunction with careful analysis of electronic and structural requirements for the PDE2a enzyme. One of the lead compounds, compound 27 (DNS-8254), was identified as a potent and highly selective PDE2a enzyme inhibitor with favorable rat pharmacokinetic properties. Interestingly, the increased potency of compound 27 was facilitated by the formation of a halogen bond with the oxygen of Tyr827 present in the PDE2a active site. In vivo, compound 27 demonstrated significant memory enhancing effects in a rat model of novel object recognition. Taken together, these data suggest that compound 27 may be a useful tool to explore the pharmacology of selective PDE2a inhibition.
Assuntos
Exonucleases/efeitos dos fármacos , Transtornos da Memória/tratamento farmacológico , Inibidores de Fosfodiesterase/síntese química , Inibidores de Fosfodiesterase/farmacologia , Cromatografia Líquida , Humanos , Espectroscopia de Prótons por Ressonância MagnéticaRESUMO
Crystallographic studies of ligands bound to biological macromolecules (proteins and nucleic acids) represent an important source of information concerning drug-target interactions, providing atomic level insights into the physical chemistry of complex formation between macromolecules and ligands. Of the more than 115,000 entries extant in the Protein Data Bank (PDB) archive, â¼75% include at least one non-polymeric ligand. Ligand geometrical and stereochemical quality, the suitability of ligand models for in silico drug discovery and design, and the goodness-of-fit of ligand models to electron-density maps vary widely across the archive. We describe the proceedings and conclusions from the first Worldwide PDB/Cambridge Crystallographic Data Center/Drug Design Data Resource (wwPDB/CCDC/D3R) Ligand Validation Workshop held at the Research Collaboratory for Structural Bioinformatics at Rutgers University on July 30-31, 2015. Experts in protein crystallography from academe and industry came together with non-profit and for-profit software providers for crystallography and with experts in computational chemistry and data archiving to discuss and make recommendations on best practices, as framed by a series of questions central to structural studies of macromolecule-ligand complexes. What data concerning bound ligands should be archived in the PDB? How should the ligands be best represented? How should structural models of macromolecule-ligand complexes be validated? What supplementary information should accompany publications of structural studies of biological macromolecules? Consensus recommendations on best practices developed in response to each of these questions are provided, together with some details regarding implementation. Important issues addressed but not resolved at the workshop are also enumerated.
Assuntos
Bases de Dados de Proteínas , Proteínas/química , Cristalografia por Raios X , Curadoria de Dados , Guias como Assunto , Ligantes , Modelos Moleculares , Conformação ProteicaRESUMO
The type II transmembrane serine protease dipeptidyl peptidase IV (DPPIV), also known as CD26 or adenosine deaminase binding protein, is a major regulator of various physiological processes, including immune, inflammatory, nervous, and endocrine functions. It has been generally accepted that glycosylation of DPPIV and of other transmembrane dipeptidyl peptidases is a prerequisite for enzyme activity and correct protein folding. Crystallographic studies on DPPIV reveal clear N-linked glycosylation of nine Asn residues in DPPIV. However, the importance of each glycosylation site on physiologically relevant reactions such as dipeptide cleavage, dimer formation, and adenosine deaminase (ADA) binding remains obscure. Individual Asn-->Ala point mutants were introduced at the nine glycosylation sites in the extracellular domain of DPPIV (residues 39-766). Crystallographic and biochemical data demonstrate that N-linked glycosylation of DPPIV does not contribute significantly to its peptidase activity. The kinetic parameters of dipeptidyl peptidase cleavage of wild-type DPPIV and the N-glycosylation site mutants were determined by using Ala-Pro-AFC and Gly-Pro-pNA as substrates and varied by <50%. DPPIV is active as a homodimer. Size-exclusion chromatographic analysis showed that the glycosylation site mutants do not affect dimerization. ADA binds to the highly glycosylated beta-propeller domain of DPPIV, but the impact of glycosylation on binding had not previously been determined. Our studies indicate that glycosylation of DPPIV is not required for ADA binding. Taken together, these data indicate that in contrast to the generally accepted view, glycosylation of DPPIV is not a prerequisite for catalysis, dimerization, or ADA binding.
Assuntos
Adenosina Desaminase/metabolismo , Dipeptidil Peptidase 4/metabolismo , Alanina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Catálise , Cromatografia em Gel , Cristalografia por Raios X , Dimerização , Dipeptidil Peptidase 4/química , Dipeptidil Peptidase 4/genética , Dipeptidil Peptidase 4/isolamento & purificação , Dissulfetos , Glicosilação , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutação Puntual , Dobramento de Proteína , Estrutura Terciária de Proteína , Transporte Proteico , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Dipeptidyl peptidase IV (DPPIV) is a member of the prolyl oligopeptidase family of serine proteases. DPPIV removes dipeptides from the N terminus of substrates, including many chemokines, neuropeptides, and peptide hormones. Specific inhibition of DPPIV is being investigated in human trials for the treatment of type II diabetes. To understand better the molecular determinants that underlie enzyme catalysis and substrate specificity, we report the crystal structures of DPPIV in the free form and in complex with the first 10 residues of the physiological substrate, Neuropeptide Y (residues 1-10; tNPY). The crystal structure of the free form of the enzyme reveals two potential channels through which substrates could access the active site-a so-called propeller opening, and side opening. The crystal structure of the DPPIV/tNPY complex suggests that bioactive peptides utilize the side opening unique to DPPIV to access the active site. Other structural features in the active site such as the presence of a Glu motif, a well-defined hydrophobic S1 subsite, and minimal long-range interactions explain the substrate recognition and binding properties of DPPIV. Moreover, in the DPPIV/tNPY complex structure, the peptide is not cleaved but trapped in a tetrahedral intermediate that occurs during catalysis. Conformational changes of S630 and H740 between DPPIV in its free form and in complex with tNPY were observed and contribute to the stabilization of the tetrahedral intermediate. Our results facilitate the design of potent, selective small molecule inhibitors of DPPIV that may yield compounds for the development of novel drugs to treat type II diabetes.
Assuntos
Dipeptidil Peptidase 4/química , Dipeptidil Peptidase 4/metabolismo , Neuropeptídeo Y/química , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Animais , Sítios de Ligação , Domínio Catalítico , Células Cultivadas , Cristalização , Cristalografia por Raios X , Humanos , Insetos , Modelos Moleculares , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
With the aim of discovering a selective kinase inhibitor targeting pan-RAF kinase inhibition, we designed novel 1,3-benzothiazole derivatives based on our thiazolo[5,4-b]pyridine class RAF/VEGFR2 inhibitor 1 and developed a regioselective cyclization methodology for the C-7-substituted 1,3-benzothiazole scaffold utilizing meta-substituted anilines. Eventually, we selected 7-cyano derivative 8B (TAK-632) as a development candidate and confirmed its binding mode by cocrystal structure with BRAF. Accommodation of the 7-cyano group into the BRAF-selectivity pocket and the 3-(trifluoromethyl)phenyl acetamide moiety into the hydrophobic back pocket of BRAF in the DFG-out conformation contributed to enhanced RAF potency and selectivity vs VEGFR2. Reflecting its potent pan-RAF inhibition and slow off-rate profile, 8B demonstrated significant cellular activity against mutated BRAF or mutated NRAS cancer cell lines. Furthermore, in both A375 (BRAF(V600E)) and HMVII (NRAS(Q61K)) xenograft models in rats, 8B demonstrated regressive antitumor efficacy by twice daily, 14-day repetitive administration without significant body weight loss.
Assuntos
Benzotiazóis/síntese química , Benzotiazóis/farmacologia , Descoberta de Drogas , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Benzotiazóis/química , Barreira Hematoencefálica , Linhagem Celular Tumoral , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Humanos , Modelos Moleculares , Inibidores de Proteínas Quinases/química , Ressonância de Plasmônio de SuperfícieRESUMO
To develop RAF/VEGFR2 inhibitors that bind to the inactive DFG-out conformation, we conducted structure-based drug design using the X-ray cocrystal structures of BRAF, starting from an imidazo[1,2-b]pyridazine derivative. We designed various [5,6]-fused bicyclic scaffolds (ring A, 1-6) possessing an anilide group that forms two hydrogen bond interactions with Cys532. Stabilizing the planarity of this anilide and the nitrogen atom on the six-membered ring of the scaffold was critical for enhancing BRAF inhibition. The selected [1,3]thiazolo[5,4-b]pyridine derivative 6d showed potent inhibitory activity in both BRAF and VEGFR2. Solid dispersion formulation of 6d (6d-SD) maximized its oral absorption in rats and showed significant suppression of ERK1/2 phosphorylation in an A375 melanoma xenograft model in rats by single administration. Tumor regression (T/C = -7.0%) in twice-daily repetitive studies at a dose of 50 mg/kg in rats confirmed that 6d is a promising RAF/VEGFR2 inhibitor showing potent anticancer activity.
Assuntos
Antineoplásicos/síntese química , Benzamidas/síntese química , Compostos Bicíclicos Heterocíclicos com Pontes/síntese química , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Tiazóis/síntese química , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Administração Oral , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Benzamidas/farmacocinética , Benzamidas/farmacologia , Benzoatos/síntese química , Benzoatos/farmacocinética , Benzoatos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacocinética , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Imidazóis/síntese química , Imidazóis/farmacocinética , Imidazóis/farmacologia , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Modelos Moleculares , Estrutura Molecular , Fosforilação , Piridazinas/síntese química , Piridazinas/farmacocinética , Piridazinas/farmacologia , Ratos , Relação Estrutura-Atividade , Tiazóis/farmacocinética , Tiazóis/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Dual inhibitors of human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor (EGFR) have been investigated for breast, lung, gastric, prostate, and other cancers; one, lapatinib, is currently approved for breast cancer. To develop novel HER2/EGFR dual kinase inhibitors, we designed and synthesized pyrrolo[3,2-d]pyrimidine derivatives capable of fitting into the receptors' ATP binding site. Among the prepared compounds, 34e showed potent HER2 and EGFR (HER1) inhibitory activities as well as tumor growth inhibitory activity. The X-ray cocrystal structures of 34e with both HER2 and EGFR demonstrated that 34e interacts with the expected residues in their respective ATP pockets. Furthermore, reflecting its good oral bioavailability, 34e exhibited potent in vivo efficacy in HER2-overexpressing tumor xenograft models. On the basis of these findings, we report 34e (TAK-285) as a promising candidate for clinical development as a novel HER2/EGFR dual kinase inhibitor.