RESUMO
Efforts to develop an HIV-1 vaccine include those focusing on conserved structural elements as the target of broadly neutralizing monoclonal antibodies. MAb D5 binds to a highly conserved hydrophobic pocket on the gp41 N-heptad repeat (NHR) coiled coil and neutralizes through prevention of viral fusion and entry. Assessment of 17-mer and 36-mer NHR peptides presenting the D5 epitope in rodent immunogenicity studies showed that the longer peptide elicited higher titers of neutralizing antibodies, suggesting that neutralizing epitopes outside of the D5 pocket may exist. Although the magnitude and breadth of neutralization elicited by NHR-targeting antigens are lower than that observed for antibodies directed to other epitopes on the envelope glycoprotein complex, it has been shown that NHR-directed antibodies are potentiated in TZM-bl cells containing the FcγRI receptor. Herein, we report the design and evaluation of covalently stabilized trimeric 51-mer peptides encompassing the complete gp41 NHR. We demonstrate that these peptide trimers function as effective antiviral entry inhibitors and retain the ability to present the D5 epitope. We further demonstrate in rodent and nonhuman primate immunization studies that our 51-mer constructs elicit a broader repertoire of neutralizing antibody and improved cross-clade neutralization of primary HIV-1 isolates relative to 17-mer and 36-mer NHR peptides in A3R5 and FcγR1-enhanced TZM-bl assays. These results demonstrate that sensitive neutralization assays can be used for structural enhancement of moderately potent neutralizing epitopes. Finally, we present expanded trimeric peptide designs which include unique low-molecular-weight scaffolds that provide versatility in our immunogen presentation strategy.
Assuntos
Vacinas contra a AIDS , Anticorpos Neutralizantes , Anticorpos Anti-HIV , Proteína gp41 do Envelope de HIV , HIV-1 , Proteína gp41 do Envelope de HIV/imunologia , Proteína gp41 do Envelope de HIV/química , HIV-1/imunologia , Animais , Vacinas contra a AIDS/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Humanos , Camundongos , Epitopos/imunologia , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , Infecções por HIV/virologia , Peptídeos/imunologia , Peptídeos/química , Feminino , Anticorpos Monoclonais/imunologiaRESUMO
Voltage-gated potassium channels (Kv) are tetrameric membrane proteins that provide a highly selective pathway for potassium ions (K+) to diffuse across a hydrophobic cell membrane. These unique voltage-gated cation channels detect changes in membrane potential and, upon activation, help to return the depolarized cell to a resting state during the repolarization stage of each action potential. The Kv3 family of potassium channels is characterized by a high activation potential and rapid kinetics, which play a crucial role for the fast-spiking neuronal phenotype. Mutations in the Kv3.1 channel have been shown to have implications in various neurological diseases like epilepsy and Alzheimer's disease. Moreover, disruptions in neuronal circuitry involving Kv3.1 have been correlated with negative symptoms of schizophrenia. Here, we report the discovery of a novel positive modulator of Kv3.1, investigate its biophysical properties, and determine the cryo-EM structure of the compound in complex with Kv3.1. Structural analysis reveals the molecular determinants of positive modulation in Kv3.1 channels by this class of compounds and provides additional opportunities for rational drug design for the treatment of associated neurological disorders.
Assuntos
Neurônios , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Humanos , Neurônios/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Canais de Potássio/metabolismo , Potenciais de Ação/fisiologia , Proteínas de Membrana/metabolismoRESUMO
The angiotensin II receptors AT1R and AT2R serve as key components of the renin-angiotensin-aldosterone system. AT1R has a central role in the regulation of blood pressure, but the function of AT2R is unclear and it has a variety of reported effects. To identify the mechanisms that underlie the differences in function and ligand selectivity between these receptors, here we report crystal structures of human AT2R bound to an AT2R-selective ligand and to an AT1R/AT2R dual ligand, capturing the receptor in an active-like conformation. Unexpectedly, helix VIII was found in a non-canonical position, stabilizing the active-like state, but at the same time preventing the recruitment of G proteins or ß-arrestins, in agreement with the lack of signalling responses in standard cellular assays. Structure-activity relationship, docking and mutagenesis studies revealed the crucial interactions for ligand binding and selectivity. Our results thus provide insights into the structural basis of the distinct functions of the angiotensin receptors, and may guide the design of new selective ligands.
Assuntos
Modelos Moleculares , Receptor Tipo 2 de Angiotensina/química , Receptor Tipo 2 de Angiotensina/metabolismo , Bloqueadores do Receptor Tipo 2 de Angiotensina II/química , Bloqueadores do Receptor Tipo 2 de Angiotensina II/metabolismo , Sítios de Ligação/genética , Cristalografia por Raios X , Desenho de Fármacos , Proteínas Heterotriméricas de Ligação ao GTP/química , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Ligantes , Simulação de Acoplamento Molecular , Mutação , Ligação Proteica , Conformação Proteica , Receptor Tipo 1 de Angiotensina/química , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/agonistas , Receptor Tipo 2 de Angiotensina/genética , Transdução de Sinais , Relação Estrutura-Atividade , Especificidade por Substrato/genética , beta-Arrestinas/metabolismoRESUMO
Cathepsin K (Cat K) is a cysteine protease involved in bone remodeling. In addition to its role in bone biology, Cat K is upregulated in osteoclasts, chondrocytes and synoviocytes in osteoarthritic (OA) disease states making it a potential therapeutic target for disease-modifying OA. Starting from a prior preclinical compound, MK-1256, lead optimization efforts were carried out in the search for potent Cat K inhibitors with improved selectivity profiles with an emphasis on cathepsin F. Herein, we report the SAR studies which led to the discovery of the highly selective oxazole compound 23, which was subsequently shown to inhibit cathepsin K in vivo as measured by reduced levels of urinary C-telopeptide of collagen type I in dog.
Assuntos
Osteoartrite , Animais , Osso e Ossos , Catepsina K , Catepsinas , Condrócitos , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/farmacologia , Inibidores de Cisteína Proteinase/uso terapêutico , Cães , Osteoartrite/tratamento farmacológico , OsteoclastosRESUMO
TREM2 has been identified by genomic analysis as a potential and novel target for the treatment of Alzheimer's disease. To enable structure-based screening of potential small molecule therapeutics, we sought to develop a robust crystallization platform for the TREM2 Ig-like domain. A systematic set of constructs containing the structural chaperone, maltose binding protein (MBP), fused to the Ig domain of TREM2, were evaluated in parallel expression and purification, followed by crystallization studies. Using protein crystallization and high-resolution diffraction as a readout, a MBP-TREM2 Ig fusion construct was identified that generates reproducible protein crystals diffracting at 2.0 Å, which makes it suitable for soaking of potential ligands. Importantly, analysis of crystal packing interfaces indicates that most of the surface of the TREM2 Ig domain is available for small molecule binding. A proof of concept co-crystallization study with a small library of fragments validated potential utility of this system for the discovery of new TREM2 therapeutics.
Assuntos
Cristalização/métodos , Glicoproteínas de Membrana , Chaperonas Moleculares , Receptores Imunológicos , Proteínas Recombinantes de Fusão , Humanos , Proteínas Ligantes de Maltose/química , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Receptores Imunológicos/química , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
High-throughput screening methods have been used to identify two novel series of inhibitors that disrupt progranulin binding to sortilin. Exploration of structure-activity relationships (SAR) resulted in compounds with sufficient potency and physicochemical properties to enable co-crystallization with sortilin. These co-crystal structures supported observed SAR trends and provided guidance for additional avenues for designing compounds with additional interactions within the binding site.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Progranulinas/metabolismo , Bibliotecas de Moléculas Pequenas/química , Proteínas Adaptadoras de Transporte Vesicular/antagonistas & inibidores , Amidas/química , Amidas/metabolismo , Aminoácidos/química , Aminoácidos/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Ensaios de Triagem em Larga Escala , Humanos , Simulação de Dinâmica Molecular , Progranulinas/antagonistas & inibidores , Ligação Proteica , Pirazóis/química , Pirazóis/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Relação Estrutura-AtividadeRESUMO
Current therapies for chronic pain can have insufficient efficacy and lead to side effects, necessitating research of novel targets against pain. Although originally identified as an oncogene, Tropomyosin-related kinase A (TrkA) is linked to pain and elevated levels of NGF (the ligand for TrkA) are associated with chronic pain. Antibodies that block TrkA interaction with its ligand, NGF, are in clinical trials for pain relief. Here, we describe the identification of TrkA-specific inhibitors and the structural basis for their selectivity over other Trk family kinases. The X-ray structures reveal a binding site outside the kinase active site that uses residues from the kinase domain and the juxtamembrane region. Three modes of binding with the juxtamembrane region are characterized through a series of ligand-bound complexes. The structures indicate a critical pharmacophore on the compounds that leads to the distinct binding modes. The mode of interaction can allow TrkA selectivity over TrkB and TrkC or promiscuous, pan-Trk inhibition. This finding highlights the difficulty in characterizing the structure-activity relationship of a chemical series in the absence of structural information because of substantial differences in the interacting residues. These structures illustrate the flexibility of binding to sequences outside of-but adjacent to-the kinase domain of TrkA. This knowledge allows development of compounds with specificity for TrkA or the family of Trk proteins.
Assuntos
Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Receptor trkA/antagonistas & inibidores , Receptor trkA/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Humanos , Cinética , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Modelos Moleculares , Conformação Proteica , Inibidores de Proteínas Quinases/síntese química , Receptor trkA/genética , Receptor trkB/antagonistas & inibidores , Receptor trkB/química , Receptor trkB/genética , Receptor trkC/antagonistas & inibidores , Receptor trkC/química , Receptor trkC/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/genética , Relação Estrutura-Atividade , Ressonância de Plasmônio de SuperfícieRESUMO
O-GlcNAc hydrolase (OGA) catalyzes removal of ßα-linked N-acetyl-D-glucosamine from serine and threonine residues. We report crystal structures of Homo sapiens OGA catalytic domain in apo and inhibited states, revealing a flexible dimer that displays three unique conformations and is characterized by subdomain α-helix swapping. These results identify new structural features of the substrate-binding groove adjacent to the catalytic site and open new opportunities for structural, mechanistic and drug discovery activities.
Assuntos
Modelos Biológicos , beta-N-Acetil-Hexosaminidases/química , beta-N-Acetil-Hexosaminidases/metabolismo , Acetilglucosamina/metabolismo , Sítios de Ligação , Calorimetria , Domínio Catalítico , Cristalografia por Raios X , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
We have identified a novel PDE2 inhibitor series using fragment-based screening. Pyrazolopyrimidine fragment 1, while possessing weak potency (Kiâ¯=â¯22.4⯵M), exhibited good binding efficiencies (LBEâ¯=â¯0.49, LLEâ¯=â¯4.48) to serve as a start for structure-based drug design. With the assistance of molecular modeling and X-ray crystallography, this fragment was developed into a series of potent PDE2 inhibitors with good physicochemical properties. Compound 16, a PDE2 selective inhibitor, was identified that exhibited favorable rat pharmacokinetic properties.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/antagonistas & inibidores , Desenho de Fármacos , Inibidores de Fosfodiesterase/química , Animais , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/metabolismo , Meia-Vida , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Conformação Molecular , Simulação de Dinâmica Molecular , Inibidores de Fosfodiesterase/metabolismo , Inibidores de Fosfodiesterase/farmacocinética , Pirazóis/química , Pirazóis/metabolismo , Pirazóis/farmacocinética , Pirimidinas/química , Pirimidinas/metabolismo , Pirimidinas/farmacocinética , Ratos , Relação Estrutura-AtividadeRESUMO
G-protein-coupled receptor (GPCR) kinases (GRKs) bind to and phosphorylate GPCRs, initiating the process of GPCR desensitization and internalization. GRK4 is implicated in the regulation of blood pressure, and three GRK4 polymorphisms (R65L, A142V, and A486V) are associated with hypertension. Here, we describe the 2.6 Å structure of human GRK4α A486V crystallized in the presence of 5'-adenylyl ß,γ-imidodiphosphate. The structure of GRK4α is similar to other GRKs, although slight differences exist within the RGS homology (RH) bundle subdomain, substrate-binding site, and kinase C-tail. The RH bundle subdomain and kinase C-terminal lobe form a strikingly acidic surface, whereas the kinase N-terminal lobe and RH terminal subdomain surfaces are much more basic. In this respect, GRK4α is more similar to GRK2 than GRK6. A fully ordered kinase C-tail reveals interactions linking the C-tail with important determinants of kinase activity, including the αB helix, αD helix, and the P-loop. Autophosphorylation of wild-type GRK4α is required for full kinase activity, as indicated by a lag in phosphorylation of a peptide from the dopamine D1 receptor without ATP preincubation. In contrast, this lag is not observed in GRK4α A486V. Phosphopeptide mapping by mass spectrometry indicates an increased rate of autophosphorylation of a number of residues in GRK4α A486V relative to wild-type GRK4α, including Ser-485 in the kinase C-tail.
Assuntos
Quinase 4 de Receptor Acoplado a Proteína G/química , Quinase 4 de Receptor Acoplado a Proteína G/metabolismo , Hipertensão/genética , Sequência de Aminoácidos , Cristalografia por Raios X , Quinase 4 de Receptor Acoplado a Proteína G/genética , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Fosforilação , Conformação Proteica , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Novel bacterial topoisomerase inhibitors (NBTIs) represent a new class of broad-spectrum antibacterial agents targeting bacterial Gyrase A and ParC and have potential utility in combating antibiotic resistance. A series of novel oxabicyclooctane-linked NBTIs with new tricyclic-1,5-naphthyridinone left hand side moieties have been described. Compounds with a (R)-hydroxy-1,5-naphthyridinone moiety (7) showed potent antibacterial activity (e.g., Staphylococcus aureus MIC 0.25 µg/mL), acceptable Gram-positive and Gram-negative spectrum with rapidly bactericidal activity. The compound 7 showed intravenous and oral efficacy (ED50) at 3.2 and 27 mg/kg doses, respectively, in a murine model of bacteremia. Most importantly they showed significant attenuation of functional hERG activity (IC50 >170 µM). In general, lower logD attenuated hERG activity but also reduced Gram-negative activity. The co-crystal structure of a hydroxy-tricyclic NBTI bound to a DNA-gyrase complex exhibited a binding mode that show enantiomeric preference for R isomer and explains the activity and SAR. The discovery, synthesis, SAR and X-ray crystal structure of the left-hand-side tricyclic 1,5-naphthyridinone based oxabicyclooctane linked NBTIs are described.
Assuntos
Antibacterianos/farmacologia , Ciclo-Octanos/farmacologia , DNA Topoisomerases Tipo II/metabolismo , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Naftiridinas/farmacologia , Inibidores da Topoisomerase II/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Ciclo-Octanos/síntese química , Ciclo-Octanos/química , Relação Dose-Resposta a Droga , Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Positivas/enzimologia , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Naftiridinas/síntese química , Naftiridinas/química , Relação Estrutura-Atividade , Inibidores da Topoisomerase II/síntese química , Inibidores da Topoisomerase II/químicaRESUMO
The emergence of antibiotic-resistant strains of pathogenic bacteria is an increasing threat to global health that underscores an urgent need for an expanded antibacterial armamentarium. Gram-negative bacteria, such as Escherichia coli, have become increasingly important clinical pathogens with limited treatment options. This is due in part to their lipopolysaccharide (LPS) outer membrane components, which dually serve as endotoxins while also protecting Gram-negative bacteria from antibiotic entry. The LpxC enzyme catalyzes the committed step of LPS biosynthesis, making LpxC a promising target for new antibacterials. Here, we present the first structure of an LpxC enzyme in complex with the deacetylation reaction product, UDP-(3-O-(R-3-hydroxymyristoyl))-glucosamine. These studies provide valuable insight into recognition of substrates and products by LpxC and a platform for structure-guided drug discovery of broad spectrum Gram-negative antibiotics.
Assuntos
Amidoidrolases/química , Escherichia coli/enzimologia , Ácidos Mirísticos/química , Prótons , Uridina Difosfato N-Acetilglicosamina/análogos & derivados , Amidoidrolases/metabolismo , Cristalografia por Raios X , Lipopolissacarídeos/biossíntese , Lipopolissacarídeos/química , Ácidos Mirísticos/metabolismo , Estrutura Terciária de Proteína , Uridina Difosfato N-Acetilglicosamina/química , Uridina Difosfato N-Acetilglicosamina/metabolismoRESUMO
The goal of designing safer, more effective drugs has led to tremendous interest in molecular mechanisms through which ligands can precisely manipulate signaling of G-protein-coupled receptors (GPCRs), the largest class of drug targets. Decades of research have led to the widely accepted view that all agonists-ligands that trigger GPCR activation-function by causing rearrangement of the GPCR's transmembrane helices, opening an intracellular pocket for binding of transducer proteins. Here we demonstrate that certain agonists instead trigger activation of free fatty acid receptor 1 by directly rearranging an intracellular loop that interacts with transducers. We validate the predictions of our atomic-level simulations by targeted mutagenesis; specific mutations which disrupt interactions with the intracellular loop convert these agonists into inverse agonists. Further analysis suggests that allosteric ligands could regulate signaling of many other GPCRs via a similar mechanism, offering rich possibilities for precise control of pharmaceutically important targets.
RESUMO
The receptor tyrosine kinase c-Met is implicated in oncogenesis and is the target for several small molecule and biologic agents in clinical trials for the treatment of cancer. Binding of the hepatocyte growth factor to the cell surface receptor of c-Met induces activation via autophosphorylation of the kinase domain. Here we describe the structural basis of c-Met activation upon autophosphorylation and the selective small molecule inhibiton of autophosphorylated c-Met. MK-2461 is a potent c-Met inhibitor that is selective for the phosphorylated state of the enzyme. Compound 1 is an MK-2461 analog with a 20-fold enthalpy-driven preference for the autophosphorylated over unphosphorylated c-Met kinase domain. The crystal structure of the unbound kinase domain phosphorylated at Tyr-1234 and Tyr-1235 shows that activation loop phosphorylation leads to the ejection and disorder of the activation loop and rearrangement of helix αC and the G loop to generate a viable active site. Helix αC adopts a orientation different from that seen in activation loop mutants. The crystal structure of the complex formed by the autophosphorylated c-Met kinase domain and compound 1 reveals a significant induced fit conformational change of the G loop and ordering of the activation loop, explaining the selectivity of compound 1 for the autophosphorylated state. The results highlight the role of structural plasticity within the kinase domain in imparting the specificity of ligand binding and provide the framework for structure-guided design of activated c-Met inhibitors.
Assuntos
Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/química , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/química , Animais , Linhagem Celular , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Fosforilação , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Spodoptera , Relação Estrutura-Atividade , c-Mer Tirosina QuinaseRESUMO
The cooperative assembly of FtsZ, the prokaryotic homologue of tubulin, plays an essential role in cell division. FtsZ is a potential drug target, as illustrated by the small-molecule cell-cycle inhibitor and antibacterial agent PC190723 that targets FtsZ. We demonstrate that PC190723 negatively modulates Staphylococcus aureus FtsZ polymerization cooperativity as reflected in polymerization at lower concentrations without a defined critical concentration. The crystal structure of the S. aureus FtsZ-PC190723 complex shows a domain movement that would stabilize the FtsZ protofilament over the monomeric state, with the conformational change mediated from the GTP-binding site to the C-terminal domain via helix 7. Together, the results reveal the molecular mechanism of FtsZ modulation by PC190723 and a conformational switch to the high-affinity state that enables polymer assembly.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto/metabolismo , Conformação Proteica/efeitos dos fármacos , Piridinas/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Tiazóis/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Proteínas do Citoesqueleto/antagonistas & inibidores , Proteínas do Citoesqueleto/química , Humanos , Simulação de Acoplamento Molecular , Estrutura Terciária de Proteína/efeitos dos fármacos , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/química , Staphylococcus aureus/metabolismoRESUMO
Bacterial infection remains a serious threat to human lives because of emerging resistance to existing antibiotics. Although the scientific community has avidly pursued the discovery of new antibiotics that interact with new targets, these efforts have met with limited success since the early 1960s. Here we report the discovery of platensimycin, a previously unknown class of antibiotics produced by Streptomyces platensis. Platensimycin demonstrates strong, broad-spectrum Gram-positive antibacterial activity by selectively inhibiting cellular lipid biosynthesis. We show that this anti-bacterial effect is exerted through the selective targeting of beta-ketoacyl-(acyl-carrier-protein (ACP)) synthase I/II (FabF/B) in the synthetic pathway of fatty acids. Direct binding assays show that platensimycin interacts specifically with the acyl-enzyme intermediate of the target protein, and X-ray crystallographic studies reveal that a specific conformational change that occurs on acylation must take place before the inhibitor can bind. Treatment with platensimycin eradicates Staphylococcus aureus infection in mice. Because of its unique mode of action, platensimycin shows no cross-resistance to other key antibiotic-resistant strains tested, including methicillin-resistant S. aureus, vancomycin-intermediate S. aureus and vancomycin-resistant enterococci. Platensimycin is the most potent inhibitor reported for the FabF/B condensing enzymes, and is the only inhibitor of these targets that shows broad-spectrum activity, in vivo efficacy and no observed toxicity.
Assuntos
Aminoglicosídeos/farmacologia , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/antagonistas & inibidores , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/química , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Acetamidas/farmacologia , Acetamidas/toxicidade , Adamantano , Aminobenzoatos , Aminoglicosídeos/química , Aminoglicosídeos/metabolismo , Aminoglicosídeos/toxicidade , Anilidas , Animais , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/toxicidade , Apoproteínas/química , Apoproteínas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Linezolida , Lipídeos/biossíntese , Camundongos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Conformação Molecular , Oxazolidinonas/farmacologia , Oxazolidinonas/toxicidade , Streptomyces/metabolismo , Especificidade por SubstratoRESUMO
The incorporation of a carboxylic acid within in a series of 3-amido-4-aryl substituted piperidines (represented by general structure 32) led to the discovery of potent, zwitterionic, renin inhibitors with improved off-target profiles (CYP3A4 time-dependent inhibition and hERG affinity) relative to analogous non-zwitterionic inhibitors of the past (i.e., 3). Strategies to address the oral absorption of these zwitterions are also discussed within.
Assuntos
Inibidores de Proteases/síntese química , Renina/antagonistas & inibidores , Administração Oral , Animais , Domínio Catalítico , Simulação por Computador , Cães , Avaliação Pré-Clínica de Medicamentos , Humanos , Piperidinas/síntese química , Piperidinas/química , Piperidinas/farmacocinética , Inibidores de Proteases/química , Inibidores de Proteases/farmacocinética , Ratos , Ratos Sprague-Dawley , Renina/metabolismo , Relação Estrutura-AtividadeRESUMO
The farnesoid X receptor (FXR), a member of the nuclear hormone receptor family, plays important roles in the regulation of bile acid and cholesterol homeostasis, glucose metabolism, and insulin sensitivity. There is intense interest in understanding the mechanisms of FXR regulation and in developing pharmaceutically suitable synthetic FXR ligands that might be used to treat metabolic syndrome. We report here the identification of a potent FXR agonist (MFA-1) and the elucidation of the structure of this ligand in ternary complex with the human receptor and a coactivator peptide fragment using x-ray crystallography at 1.9-A resolution. The steroid ring system of MFA-1 binds with its D ring-facing helix 12 (AF-2) in a manner reminiscent of hormone binding to classical steroid hormone receptors and the reverse of the pose adopted by naturally occurring bile acids when bound to FXR. This binding mode appears to be driven by the presence of a carboxylate on MFA-1 that is situated to make a salt-bridge interaction with an arginine residue in the FXR-binding pocket that is normally used to neutralize bound bile acids. Receptor activation by MFA-1 differs from that by bile acids in that it relies on direct interactions between the ligand and residues in helices 11 and 12 and only indirectly involves a protonated histidine that is part of the activation trigger. The structure of the FXR:MFA-1 complex differs significantly from that of the complex with a structurally distinct agonist, fexaramine, highlighting the inherent plasticity of the receptor.
Assuntos
Proteínas de Ligação a DNA/agonistas , Proteínas de Ligação a DNA/química , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/química , Esteroides/química , Fatores de Transcrição/agonistas , Fatores de Transcrição/química , Sítios de Ligação , Ácidos Carboxílicos , Cristalografia por Raios X , Humanos , Ligantes , Ligação Proteica , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
All herpesviruses encode a conserved DNA polymerase that is required for viral genome replication and serves as an important therapeutic target. Currently available herpesvirus therapies include nucleoside and non-nucleoside inhibitors (NNI) that target the DNA-bound state of herpesvirus polymerase and block replication. Here we report the ternary complex crystal structure of Herpes Simplex Virus 1 DNA polymerase bound to DNA and a 4-oxo-dihydroquinoline NNI, PNU-183792 (PNU), at 3.5 Å resolution. PNU bound at the polymerase active site, displacing the template strand and inducing a conformational shift of the fingers domain into an open state. These results demonstrate that PNU inhibits replication by blocking association of dNTP and stalling the enzyme in a catalytically incompetent conformation, ultimately acting as a nucleotide competing inhibitor (NCI). Sequence conservation of the NCI binding pocket further explains broad-spectrum activity while a direct interaction between PNU and residue V823 rationalizes why mutations at this position result in loss of inhibition.
Assuntos
DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/genética , Herpesviridae/efeitos dos fármacos , Herpesviridae/enzimologia , Antivirais/farmacologia , Sítios de Ligação , DNA Polimerase Dirigida por DNA/metabolismo , Farmacorresistência Viral/efeitos dos fármacos , Exodesoxirribonucleases , Nucleotídeos , Quinolinas/farmacologia , Proteínas Virais , Replicação ViralRESUMO
The leukotriene B4 receptor 1 (BLT1) regulates the recruitment and chemotaxis of different cell types and plays a role in the pathophysiology of infectious, allergic, metabolic, and tumorigenic human diseases. Here we present a crystal structure of human BLT1 (hBLT1) in complex with a selective antagonist MK-D-046, developed for the treatment of type 2 diabetes and other inflammatory conditions. Comprehensive analysis of the structure and structure-activity relationship data, reinforced by site-directed mutagenesis and docking studies, reveals molecular determinants of ligand binding and selectivity toward different BLT receptor subtypes and across species. The structure helps to identify a putative membrane-buried ligand access channel as well as potential receptor binding modes of endogenous agonists. These structural insights of hBLT1 enrich our understanding of its ligand recognition and open up future avenues in structure-based drug design.