RESUMO
In the originally published version of this Letter, the authors Arthur F. Kluge, Michael A. Patane and Ce Wang were inadvertently omitted from the author list. Their affiliations are: I-to-D, Inc., PO Box 6177, Lincoln, Massachusetts 01773, USA (A.F.K.); Mitobridge, Inc. 1030 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA (M.A.P.); and China Novartis Institutes for BioMedical Research, No. 4218 Jinke Road, Zhangjiang Hi-Tech Park, Pudong District, Shanghai 201203, China (C.W.). These authors contributed to the interpretation of results and design of compounds. In addition, author 'Edward A. Kesicki' was misspelled as 'Ed Kesicki'. These errors have been corrected online.
RESUMO
The dynamic and reversible acetylation of proteins, catalysed by histone acetyltransferases (HATs) and histone deacetylases (HDACs), is a major epigenetic regulatory mechanism of gene transcription and is associated with multiple diseases. Histone deacetylase inhibitors are currently approved to treat certain cancers, but progress on the development of drug-like histone actyltransferase inhibitors has lagged behind. The histone acetyltransferase paralogues p300 and CREB-binding protein (CBP) are key transcriptional co-activators that are essential for a multitude of cellular processes, and have also been implicated in human pathological conditions (including cancer). Current inhibitors of the p300 and CBP histone acetyltransferase domains, including natural products, bi-substrate analogues and the widely used small molecule C646, lack potency or selectivity. Here, we describe A-485, a potent, selective and drug-like catalytic inhibitor of p300 and CBP. We present a high resolution (1.95 Å) co-crystal structure of a small molecule bound to the catalytic active site of p300 and demonstrate that A-485 competes with acetyl coenzyme A (acetyl-CoA). A-485 selectively inhibited proliferation in lineage-specific tumour types, including several haematological malignancies and androgen receptor-positive prostate cancer. A-485 inhibited the androgen receptor transcriptional program in both androgen-sensitive and castration-resistant prostate cancer and inhibited tumour growth in a castration-resistant xenograft model. These results demonstrate the feasibility of using small molecule inhibitors to selectively target the catalytic activity of histone acetyltransferases, which may provide effective treatments for transcriptional activator-driven malignancies and diseases.
Assuntos
Linhagem da Célula , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Compostos Heterocíclicos de 4 ou mais Anéis/uso terapêutico , Histona Acetiltransferases/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Fatores de Transcrição de p300-CBP/antagonistas & inibidores , Acetilcoenzima A/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Ligação Competitiva , Biocatálise/efeitos dos fármacos , Domínio Catalítico/efeitos dos fármacos , Linhagem Celular Tumoral , Linhagem da Célula/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Hematológicas/tratamento farmacológico , Neoplasias Hematológicas/enzimologia , Neoplasias Hematológicas/patologia , Compostos Heterocíclicos de 4 ou mais Anéis/química , Histona Acetiltransferases/química , Histona Acetiltransferases/metabolismo , Humanos , Masculino , Camundongos , Camundongos SCID , Modelos Moleculares , Neoplasias/enzimologia , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/enzimologia , Neoplasias de Próstata Resistentes à Castração/patologia , Conformação Proteica , Receptores Androgênicos/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Fatores de Transcrição de p300-CBP/química , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
p300 and CREB-binding protein (CBP) are essential for a multitude of cellular processes. Dysregulation of p300/CBP histone acetyltransferase activity is linked to a broad spectrum of human diseases including cancers. A novel drug-like spirohydantoin (21) has been discovered as a selective orally bioavailable inhibitor of p300/CBP histone acetyltransferase. Lead compound 21 is more potent than the first-in-class lead A-485 in both enzymatic and cellular assays and lacks the off-target inhibition of dopamine and serotonin transporters, that was observed with A-485.
Assuntos
Proteína de Ligação a CREB/antagonistas & inibidores , Descoberta de Drogas , Proteína p300 Associada a E1A/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Hidantoínas/farmacologia , Compostos de Espiro/farmacologia , Administração Oral , Disponibilidade Biológica , Proteína de Ligação a CREB/metabolismo , Relação Dose-Resposta a Droga , Proteína p300 Associada a E1A/metabolismo , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/metabolismo , Humanos , Hidantoínas/administração & dosagem , Hidantoínas/metabolismo , Estrutura Molecular , Compostos de Espiro/administração & dosagem , Compostos de Espiro/metabolismo , Relação Estrutura-AtividadeRESUMO
Protein lysine methyltransferases (PKMTs) regulate diverse physiological processes including transcription and the maintenance of genomic integrity. Genetic studies suggest that the PKMTs SUV420H1 and SUV420H2 facilitate proficient nonhomologous end-joining (NHEJ)-directed DNA repair by catalyzing the di- and trimethylation (me2 and me3, respectively) of lysine 20 on histone 4 (H4K20). Here we report the identification of A-196, a potent and selective inhibitor of SUV420H1 and SUV420H2. Biochemical and co-crystallization analyses demonstrate that A-196 is a substrate-competitive inhibitor of both SUV4-20 enzymes. In cells, A-196 induced a global decrease in H4K20me2 and H4K20me3 and a concomitant increase in H4K20me1. A-196 inhibited 53BP1 foci formation upon ionizing radiation and reduced NHEJ-mediated DNA-break repair but did not affect homology-directed repair. These results demonstrate the role of SUV4-20 enzymatic activity in H4K20 methylation and DNA repair. A-196 represents a first-in-class chemical probe of SUV4-20 to investigate the role of histone methyltransferases in genomic integrity.
Assuntos
Inibidores Enzimáticos/farmacologia , Epigênese Genética/efeitos dos fármacos , Instabilidade Genômica/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Linhagem Celular Tumoral , Cristalografia por Raios X , Reparo do DNA/efeitos dos fármacos , Inibidores Enzimáticos/química , Compostos Heterocíclicos de 4 ou mais Anéis/química , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Metilação/efeitos dos fármacos , Modelos Moleculares , Estrutura MolecularRESUMO
Polycomb repressive complex 2 (PRC2) is a regulator of epigenetic states required for development and homeostasis. PRC2 trimethylates histone H3 at lysine 27 (H3K27me3), which leads to gene silencing, and is dysregulated in many cancers. The embryonic ectoderm development (EED) protein is an essential subunit of PRC2 that has both a scaffolding function and an H3K27me3-binding function. Here we report the identification of A-395, a potent antagonist of the H3K27me3 binding functions of EED. Structural studies demonstrate that A-395 binds to EED in the H3K27me3-binding pocket, thereby preventing allosteric activation of the catalytic activity of PRC2. Phenotypic effects observed in vitro and in vivo are similar to those of known PRC2 enzymatic inhibitors; however, A-395 retains potent activity against cell lines resistant to the catalytic inhibitors. A-395 represents a first-in-class antagonist of PRC2 protein-protein interactions (PPI) for use as a chemical probe to investigate the roles of EED-containing protein complexes.
Assuntos
Antineoplásicos/farmacologia , Indanos/farmacologia , Complexo Repressor Polycomb 2/antagonistas & inibidores , Sulfonamidas/farmacologia , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Indanos/química , Modelos Moleculares , Estrutura Molecular , Complexo Repressor Polycomb 2/química , Complexo Repressor Polycomb 2/metabolismo , Ligação Proteica/efeitos dos fármacos , Relação Estrutura-Atividade , Sulfonamidas/química , Células Tumorais CultivadasRESUMO
Herein we disclose SAR studies of a series of dimethylamino pyrrolidines which we recently reported as novel inhibitors of the PRC2 complex through disruption of EED/H3K27me3 binding. Modification of the indole and benzyl moieties of screening hit 1 provided analogs with substantially improved binding and cellular activities. This work culminated in the identification of compound 2, our nanomolar proof-of-concept (PoC) inhibitor which provided on-target tumor growth inhibition in a mouse xenograft model. X-ray crystal structures of several inhibitors bound in the EED active-site are also discussed.
Assuntos
Complexo Repressor Polycomb 2/antagonistas & inibidores , Complexo Repressor Polycomb 2/metabolismo , Pirrolidinas/farmacologia , Sulfonamidas/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Humanos , Ligantes , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Complexo Repressor Polycomb 2/química , Ligação Proteica , Pirrolidinas/síntese química , Pirrolidinas/química , Estereoisomerismo , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Apolipoprotein E is a 299-residue lipid carrier protein produced in both the liver and the brain. The protein has three major isoforms denoted apoE2, apoE3, and apoE4 which differ at positions 112 and 158 and which occur at different frequencies in the human population. Genome-wide association studies indicate that the possession of two apoE4 alleles is a strong genetic risk factor for late-onset Alzheimer's disease (LOAD). In an attempt to identify a small molecule stabilizer of apoE4 function that may have utility as a therapy for Alzheimer's disease, we carried out an NMR-based fragment screen on the N-terminal domain of apoE4 and identified a benzyl amidine based fragment binder. In addition to NMR, binding was characterized using various other biophysical techniques, and a crystal structure of the bound core was obtained. Core elaboration ultimately yielded a compound that showed activity in an IL-6 and IL-8 cytokine release assay.
Assuntos
Apolipoproteína E4/metabolismo , Bibliotecas de Moléculas Pequenas/química , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Amidinas/química , Amidinas/metabolismo , Apolipoproteína E4/química , Apolipoproteína E4/genética , Sítios de Ligação , Cristalografia por Raios X , Descoberta de Drogas , Humanos , Lipossomos/química , Lipossomos/metabolismo , Espectroscopia de Ressonância Magnética , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Domínios Proteicos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/uso terapêutico , Relação Estrutura-Atividade , Temperatura de TransiçãoRESUMO
IDH1 plays a critical role in a number of metabolic processes and serves as a key source of cytosolic NADPH under conditions of cellular stress. However, few inhibitors of wild-type IDH1 have been reported. Here we present the discovery and biochemical characterization of two novel inhibitors of wild-type IDH1. In addition, we present the first ligand-bound crystallographic characterization of these novel small molecule IDH1 binding pockets. Importantly, the NADPH competitive α,ß-unsaturated enone 1 makes a unique covalent linkage through active site H315. As few small molecules have been shown to covalently react with histidine residues, these data support the potential utility of an underutilized strategy for reversible covalent small molecule design.
Assuntos
Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Histidina , Isocitrato Desidrogenase/antagonistas & inibidores , Isocitrato Desidrogenase/química , Linhagem Celular Tumoral , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Ligantes , Simulação de Acoplamento Molecular , Mutação , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
In a broad genomics analysis to find novel protein targets for antibiotic discovery, MurF was identified as an essential gene product for Streptococcus pneumonia that catalyzes a critical reaction in the biosynthesis of the peptidoglycan in the formation of the cell wall. Lacking close relatives in mammalian biology, MurF presents attractive characteristics as a potential drug target. Initial screening of the Abbott small-molecule compound collection identified several compounds for further validation as pharmaceutical leads. Here we report the integrated efforts of NMR and X-ray crystallography, which reveal the multidomain structure of a MurF-inhibitor complex in a compact conformation that differs dramatically from related structures. The lead molecule is bound in the substrate-binding region and induces domain closure, suggestive of the domain arrangement for the as yet unobserved transition state conformation for MurF enzymes. The results form a basis for directed optimization of the compound lead by structure-based design to explore the suitability of MurF as a pharmaceutical target.
Assuntos
Inibidores Enzimáticos/química , Peptídeo Sintases/antagonistas & inibidores , Peptídeo Sintases/química , Streptococcus pneumoniae/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Peptídeo Sintases/metabolismo , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia de Sequência , Especificidade por SubstratoRESUMO
A lack of useful small molecule tools has precluded thorough interrogation of the biological function of SMYD2, a lysine methyltransferase with known tumor-suppressor substrates. Systematic exploration of the structure-activity relationships of a previously known benzoxazinone compound led to the synthesis of A-893, a potent and selective SMYD2 inhibitor (IC50: 2.8 nM). A cocrystal structure reveals the origin of enhanced potency, and effective suppression of p53K370 methylation is observed in a lung carcinoma (A549) cell line.
RESUMO
Potent inhibitors of 7,8-dihydroneopterin aldolase (DHNA; EC 4.1.2.25) have been discovered using CrystaLEAD X-ray crystallographic high-throughput screening followed by structure-directed optimization. Screening of a 10 000 compound random library provided several low affinity leads and their corresponding X-ray crystal structures bound to the enzyme. The presence of a common structural feature in each of the leads suggested a strategy for the construction of a directed library of approximately 1000 compounds that were screened for inhibitory activity in a traditional enzyme assay. Several lead compounds with IC(50) values of about 1 microM against DHNA were identified, and crystal structures of their enzyme-bound complexes were obtained by cocrystallization. Structure-directed optimization of one of the leads thus identified afforded potent inhibitors with submicromolar IC(50) values.
Assuntos
Aldeído Liases/antagonistas & inibidores , Aldeído Liases/química , Benzoatos/química , Inibidores Enzimáticos/química , Neopterina/química , Pirimidinas/química , Triazóis/química , Benzoatos/síntese química , Sítios de Ligação , Cristalografia por Raios X , Bases de Dados Factuais , Inibidores Enzimáticos/síntese química , Guanina/análogos & derivados , Guanina/síntese química , Guanina/química , Modelos Moleculares , Estrutura Molecular , Purinas/química , Pirimidinas/síntese química , Relação Estrutura-Atividade , Triazóis/síntese químicaRESUMO
A novel series of 4-[(4-cyano-2-arylbenzyloxy)-(3-methyl-3H-imidazol-4-yl)methyl]benzonitriles have been synthesized as selective farnesyltransferase inhibitors using structure-based design. X-ray cocrystal structures of compound 20-FTase-HFP and A313326-FTase-HFP confirmed our initial design. The decreased interaction between the aryl groups and Ser 48 in GGTase-I binding site could be one possible reason to explain the improved selectivity for this new series of FTase inhibitors. Medicinal chemistry efforts led to the discovery of compound 64 with potent cellular activity (EC(50) = 3.5 nM) and outstanding pharmacokinetic profiles in dog (96% bioavailable, 18.4 h oral t(1/2), and 0.19 L/(h x kg) plasma clearance).
Assuntos
Alquil e Aril Transferases/antagonistas & inibidores , Benzamidas/síntese química , Imidazóis/síntese química , Nitrilas/síntese química , Administração Oral , Alquil e Aril Transferases/química , Alquil e Aril Transferases/metabolismo , Animais , Benzamidas/farmacocinética , Benzamidas/farmacologia , Disponibilidade Biológica , Permeabilidade da Membrana Celular , Cristalografia por Raios X , Cães , Desenho de Fármacos , Farnesiltranstransferase , Imidazóis/farmacocinética , Imidazóis/farmacologia , Modelos Moleculares , Estrutura Molecular , Nitrilas/farmacocinética , Nitrilas/farmacologia , Relação Estrutura-AtividadeRESUMO
A dual-specific, tetravalent immunoglobulin G-like molecule, termed dual variable domain immunoglobulin (DVD-Ig™), is engineered to block two targets. Flexibility modulates Fc receptor and complement binding, but could result in undesirable cross-linking of surface antigens and downstream signaling. Understanding the flexibility of parental mAbs is important for designing and retaining functionality of DVD-Ig™ molecules. The architecture and dynamics of a DVD-Ig™ molecule and its parental mAbs was examined using single particle electron microscopy. Hinge angles measured for the DVD-Ig™ molecule were similar to the inner antigen parental mAb. The outer binding domain of the DVD-Ig™ molecule was highly mobile and three-dimensional (3D) analysis showed binding of inner antigen caused the outer domain to fold out of the plane with a major morphological change. Docking high-resolution X-ray structures into the 3D electron microscopy map supports the extraordinary domain flexibility observed in the DVD-Ig™ molecule allowing antigen binding with minimal steric hindrance.
Assuntos
Anticorpos Biespecíficos/química , Anticorpos Monoclonais/química , Imunoglobulina G/química , Região Variável de Imunoglobulina/química , Imunoterapia , Anticorpos Monoclonais/uso terapêutico , Antígenos/imunologia , Cristalografia por Raios X , Humanos , Interleucina-12/química , Interleucina-12/imunologia , Interleucina-18/química , Interleucina-18/imunologia , Microscopia Eletrônica de Transmissão , Ligação Proteica , Engenharia de Proteínas/métodos , Estrutura Terciária de ProteínaRESUMO
Several bispecific antibody-based formats have been developed over the past 25 years in an effort to produce a new generation of immunotherapeutics that target two or more disease mechanisms simultaneously. One such format, the dual-variable domain immunoglobulin (DVD-Ig™), combines the target binding domains of two monoclonal antibodies via flexible naturally occurring linkers, which yields a tetravalent IgG - like molecule. We report the structure of an interleukin (IL)12-IL18 DVD-Ig™ Fab (DFab) fragment with IL18 bound to the inner variable domain (VD) that reveals the remarkable flexibility of the DVD-Ig™ molecule and how the DVD-Ig™ format can function to bind four antigens simultaneously. An understanding of how the inner variable domain retains function is of critical importance for designing DVD-Ig™ molecules, and for better understanding of the flexibility of immunoglobulin variable domains and linkers, which may aid in the design of improved bi- and multi-specific biologics in general.
Assuntos
Anticorpos Biespecíficos/química , Região Variável de Imunoglobulina/química , Imunoterapia/métodos , Interleucina-18/química , Anticorpos Biespecíficos/uso terapêutico , Anticorpos Monoclonais/química , Anticorpos Monoclonais/uso terapêutico , Especificidade de Anticorpos , Antígenos/imunologia , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Interleucina-12/imunologia , Interleucina-18/imunologia , Ligação Proteica , Engenharia de Proteínas , Estrutura Terciária de ProteínaRESUMO
The DVD-Ig (TM) protein is a dual-specific immunoglobulin. Each of the two arms of the molecule contains two variable domains, an inner variable domain and an outer variable domain linked in tandem, each with binding specificity for different targets or epitopes. One area of on-going research involves determining how the proximity of the outer variable domain affects the binding of ligands to the inner variable domain. To explore this area, we prepared a series of DVD-Ig proteins with binding specificities toward TNFα and an alternate therapeutic target. Kinetic measurements of TNFα binding to this series of DVD-Ig proteins were used to probe the effects of variable domain position and linker design on ligand on- and off-rates. We found that affinities for TNFα are generally lower when binding to the inner domain than to the outer domain and that this loss of affinity is primarily due to reduced association rate. This effect could be mitigated, to some degree, by linker design. We show several linker sequences that mitigate inner domain affinity losses in this series of DVD-Ig proteins. Moreover, we show that single chain proteolytic cleavage between the inner and outer domains, or complete outer domain removal, can largely restore inner domain TNFα affinity to that approaching the reference antibody. Taken together, these results suggest that a loss of affinity for inner variable domains in this set of DVD-Ig proteins may be largely driven by simple steric hindrance effects and can be reduced by careful linker design.
Assuntos
Anticorpos Monoclonais/química , Desenho de Fármacos , Região Variável de Imunoglobulina/química , Fator de Necrose Tumoral alfa/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Humanos , Região Variável de Imunoglobulina/metabolismo , Cinética , Ligantes , Dados de Sequência Molecular , Ligação Proteica , Engenharia de Proteínas , Estrutura Terciária de ProteínaRESUMO
Recombinant human erythropoietin (rHu-EPO) is used to treat anemia by activating the erythropoietin receptor (EPOR) in erythroid progenitor cells, leading to proliferation and differentiation into mature red blood cells. To allow less frequent dosing, a hyperglycosylated version of EPO has been developed with a longer half-life. In principle, an agonistic antibody targeting EPOR would offer an even longer half-life, support robust monthly dosing, and, unlike EPO products, reduce the risk of pure red cell aplasia. The efficiency of signaling and corresponding potency of previously reported antibody mimics are generally suboptimal compared with EPO and not suitable for clinical use. Here we describe a potent, fully human, agonistic antibody (ABT007) targeting EPOR that supports potent, more sustained, and less pulsatile elevation of hematocrit in a human EPOR-expressing transgenic mouse model compared with standard doses of rHu-EPO while requiring less frequent dosing. Resolution of the crystal structure of the EPOR extracellular domain (ECD) complexed to the ABT007 Fab fragment, determined at 0.32 nm, identifies a binding site that is consistent with a novel mechanism of receptor activation based on a unique antibody-imposed conformational change. These results demonstrate that a symmetric molecule can serve as a potent activator of the EPOR.
Assuntos
Anticorpos/imunologia , Eritropoetina/metabolismo , Mimetismo Molecular , Animais , Sítios de Ligação , Linhagem Celular , Cristalografia por Raios X , Eritropoese , Hematócrito , Humanos , Camundongos , Camundongos Knockout , Modelos Moleculares , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Receptores da Eritropoetina/química , Receptores da Eritropoetina/deficiência , Receptores da Eritropoetina/genética , Receptores da Eritropoetina/metabolismo , Homologia Estrutural de ProteínaRESUMO
The D-Ala-D-Ala adding enzyme (MurF) from Streptococcus pneumoniae catalyzes the ATP-dependent formation of the UDP-MurNAc-pentapeptide, a critical component of the bacterial cell wall. MurF is a potential target for antibacterial design because it is unique to bacteria and performs an essential non-redundant function in the bacterial cell. The recent discovery and subsequent cocrystal structure determination of MurF in complex with a new class of inhibitors served as a catalyst to begin a medicinal chemistry program aimed at improving their potency. We report here a multidisciplinary approach to this effort that allowed for rapid generation of cocrystal structures, thereby providing the crystallographic information critical for driving the inhibitor optimization process. This effort resulted in the discovery of low-nanomolar inhibitors of this bacterial enzyme.
Assuntos
Inibidores Enzimáticos/química , Peptídeo Sintases/antagonistas & inibidores , Relação Estrutura-Atividade , Cristalização , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Concentração Inibidora 50 , Ligantes , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Peptídeo Sintases/química , Peptídeo Sintases/metabolismo , Especificidade por Substrato , Sulfonamidas/química , Sulfonamidas/metabolismoRESUMO
Dipeptidyl peptidase IV (DPP-IV) belongs to a family of serine peptidases, and due to its indirect regulatory role in plasma glucose modulation, DPP-IV has become an attractive pharmaceutical target for diabetes therapy. DPP-IV inactivates the glucagon-like peptide (GLP-1) and several other naturally produced bioactive peptides that contain preferentially a proline or alanine residue in the second amino acid sequence position by cleaving the N-terminal dipeptide. To elucidate the details of the active site for structure-based drug design, we crystallized a natural source preparation of DPP-IV isolated from rat kidney and determined its three-dimensional structure using X-ray diffraction techniques. With a high degree of similarity to structures of human DPP-IV, the active site architecture provides important details for the design of inhibitory compounds, and structures of inhibitor-protein complexes offer detailed insight into three-dimensional structure-activity relationships that include a conformational change of Tyr548. Such accommodation is exemplified by the response to chemical substitution on 2-cyanopyrrolidine inhibitors at the 5 position, which conveys inhibitory selectivity for DPP-IV over closely related homologues. A similar conformational change is also observed in the complex with an unrelated synthetic inhibitor containing a xanthine core that is also selective for DPP-IV. These results suggest the conformational flexibility of Tyr548 is unique among protein family members and may be utilized in drug design to achieve peptidase selectivity.
Assuntos
Dipeptidases/antagonistas & inibidores , Dipeptidil Peptidase 4/química , Dipeptidil Peptidase 4/metabolismo , Rim/enzimologia , Animais , Sítios de Ligação , Cristalização , Dimerização , Dipeptidases/química , Dipeptidases/metabolismo , Dipeptidil Peptidase 4/genética , Dipeptidil Peptidase 4/isolamento & purificação , Peptídeo 1 Semelhante ao Glucagon/química , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Humanos , Cinética , Modelos Moleculares , Estrutura Molecular , Conformação Proteica , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Tirosina/química , Difração de Raios XRESUMO
A novel series of Akt/PKB inhibitors derived from a screening lead (1) has been prepared. The novel trans-3,4'-bispyridinylethylenes described herein are potent inhibitors of Akt/PKB with IC(50) values in the low double-digit nanomolar range against Akt1. Compound 2q shows excellent selectivity against distinct families of kinases such as tyrosine kinases and CAMK, and displays poor to modest selectivity against closely related kinases in the AGC and CMGC families. The cellular activities including inhibition of cell growth and phosphorylation of downstream target GSK3 are also described. The X-ray structure of compound 2q complexed with PKA in the ATP binding site was determined.