RESUMO
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
Starting from benzylpyrimidine 2, molecular modeling and X-ray crystallography were used to design highly potent inhibitors of Interleukin-2 inducible T-cell kinase (ITK). Sulfonylpyridine 4i showed sub-nanomolar affinity against ITK, was selective versus Lck and its activity in the Jurkat cell-based assay was greatly improved over 2.
Assuntos
Desenho de Fármacos , Inibidores de Proteínas Quinases/síntese química , Proteínas Tirosina Quinases/antagonistas & inibidores , Piridinas/química , Sítios de Ligação , Cristalografia por Raios X , Cinética , Simulação de Dinâmica Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/metabolismo , Pirazóis/química , Piridinas/síntese química , Piridinas/metabolismo , Relação Estrutura-Atividade , Sulfonas/químicaRESUMO
cGMP-dependent protein kinase I-α (PKG1α) is a target for pulmonary arterial hypertension due to its role in the regulation of smooth muscle function. While most work has focused on regulation of cGMP turnover, we recently described several small molecule tool compounds which were capable of activating PKG1α via a cGMP independent pathway. Selected molecules were crystallized in the presence of PKG1α and were found to bind to an allosteric site proximal to the low-affinity nucleotide binding domain. These molecules act to displace the switch helix and cause activation of PKG1α representing a new mechanism for the activation and control of this critical therapeutic path. The described structures are vital to understanding the function and control of this key regulatory pathway.
Assuntos
Proteína Quinase Dependente de GMP Cíclico Tipo I , Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismoRESUMO
(1,1-Dioxo-2H-[1,2,4]benzothiadiazin-3-yl) azolo[1,5-a]pyridine and azolo[1,5-a]pyrimidine derivatives have been investigated as potential anti-HCV drugs. Their synthesis, HCV NS5B polymerase inhibition, and replicon activity are discussed.
Assuntos
Antivirais/síntese química , Azóis/síntese química , Benzotiadiazinas/síntese química , Inibidores Enzimáticos/síntese química , Hepatite C/tratamento farmacológico , Piridinas/síntese química , Pirimidinas/síntese química , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Antivirais/farmacologia , Azóis/farmacologia , Benzotiadiazinas/farmacologia , Química Farmacêutica/métodos , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Hepacivirus/metabolismo , Técnicas In Vitro , Concentração Inibidora 50 , Espectroscopia de Ressonância Magnética , Modelos Químicos , Estrutura Molecular , Piridinas/farmacologia , Pirimidinas/farmacologia , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/químicaRESUMO
Fragment-based drug discovery (FBDD) represents a change in strategy from the screening of molecules with higher molecular weights and physical properties more akin to fully drug-like compounds, to the screening of smaller, less complex molecules. This is because it has been recognised that fragment hit molecules can be efficiently grown and optimised into leads, particularly after the binding mode to the target protein has been first determined by 3D structural elucidation, e.g. by NMR or X-ray crystallography. Several studies have shown that medicinal chemistry optimisation of an already drug-like hit or lead compound can result in a final compound with too high molecular weight and lipophilicity. The evolution of a lower molecular weight fragment hit therefore represents an attractive alternative approach to optimisation as it allows better control of compound properties. Computational chemistry can play an important role both prior to a fragment screen, in producing a target focussed fragment library, and post-screening in the evolution of a drug-like molecule from a fragment hit, both with and without the available fragment-target co-complex structure. We will review many of the current developments in the area and illustrate with some recent examples from successful FBDD discovery projects that we have conducted.
Assuntos
Descoberta de Drogas , Proteínas de Choque Térmico HSP90 , Proteínas Proto-Oncogênicas c-bcl-2/química , Bibliotecas de Moléculas Pequenas/química , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/química , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/química , Biologia Computacional , Inibidores Enzimáticos/química , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/química , Humanos , Ligação de Hidrogênio , Ligantes , Terapia de Alvo Molecular , Diester Fosfórico Hidrolases/química , Ligação Proteica , Conformação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/uso terapêuticoRESUMO
We have developed a standardized and efficient workflow for high-throughput (HT) protein expression in E. coli and parallel purification which can be tailored to the downstream application of the target proteins. It includes a one-step purification for the purposes of functional assays and a two-step protocol for crystallographic studies, with the option of on-column tag removal.
Assuntos
Clonagem Molecular/métodos , Escherichia coli/genética , Proteínas Recombinantes/genética , Animais , Eletroforese em Gel de Poliacrilamida/métodos , Ensaios de Triagem em Larga Escala/economia , Ensaios de Triagem em Larga Escala/métodos , Humanos , Conformação Proteica , Proteômica/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Transformação Genética , Fluxo de TrabalhoRESUMO
Bacterial resistance continues to develop and pose a significant threat, both in hospitals and, more recently, in the community. A focus on other therapeutic areas by the larger pharmaceutical companies has left a shortfall in the pipeline of novel antibacterials. Recently, many new structures have been studied by structure-genomics initiatives, delivering a wealth of targets to consider. Using the tools of structure-based design, antibacterial discovery must exploit these targets to accelerate the process of drug discovery.
Assuntos
Antibacterianos/química , Bactérias/efeitos dos fármacos , Desenho de Fármacos , Inibidores da Monoaminoxidase/farmacologia , Acetamidas/química , Acetamidas/farmacologia , Aminoacil-tRNA Sintetases/antagonistas & inibidores , Aminoacil-tRNA Sintetases/metabolismo , Animais , Antibacterianos/farmacologia , Bactérias/enzimologia , Cristalografia por Raios X , DNA Topoisomerases Tipo II/química , DNA Topoisomerases Tipo II/metabolismo , Farmacorresistência Bacteriana , Humanos , Linezolida , Modelos Moleculares , Estrutura Molecular , Inibidores da Monoaminoxidase/química , Oxazolidinonas/química , Oxazolidinonas/farmacologia , Biossíntese de Proteínas , Relação Estrutura-Atividade , Inibidores da Topoisomerase II , Transcrição GênicaRESUMO
A novel exonuclease, designated as MszExo I, was cloned from Methylocaldum szegediense, a moderately thermophilic methanotroph. It specifically digests single-stranded DNA in the 3' to 5' direction. The protein is composed of 479 amino acids, and it shares 47% sequence identity with E. coli Exo I. The crystal structure of MszExo I was determined to a resolution of 2.2 Å and it aligns well with that of E. coli Exo I. Comparative studies revealed that MszExo I and E. coli Exo I have similar metal ion binding affinity and similar activity at mesophilic temperatures (25-47°C). However, the optimum working temperature of MszExo I is 10°C higher, and the melting temperature is more than 4°C higher as evaluated by both thermal inactivation assays and DSC measurements. More importantly, two thermal transitions during unfolding of MszExo I were monitored by DSC while only one transition was found in E. coli Exo I. Further analyses showed that magnesium ions not only confer structural stability, but also affect the unfolding of MszExo I. MszExo I is the first reported enzyme in the DNA repair systems of moderately thermophilic bacteria, which are predicted to have more efficient DNA repair systems than mesophilic ones.
Assuntos
Proteínas de Bactérias/química , Exodesoxirribonucleases/química , Methylococcaceae/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Reparo do DNA/fisiologia , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Temperatura Alta , Methylococcaceae/genéticaRESUMO
The medicinal chemistry community has directed considerable efforts toward the discovery of selective inhibitors of interleukin-2 inducible T-cell kinase (ITK), given its role in T-cell signaling downstream of the T-cell receptor (TCR) and the implications of this target for inflammatory disorders such as asthma. We have previously disclosed a structure- and property-guided lead optimization effort which resulted in the discovery of a new series of tetrahydroindazole-containing selective ITK inhibitors. Herein we disclose further optimization of this series that resulted in further potency improvements, reduced off-target receptor binding liabilities, and reduced cytotoxicity. Specifically, we have identified a correlation between the basicity of solubilizing elements in the ITK inhibitors and off-target antiproliferative effects, which was exploited to reduce cytotoxicity while maintaining kinase selectivity. Optimized analogues were shown to reduce IL-2 and IL-13 production in vivo following oral or intraperitoneal dosing in mice.
Assuntos
Indazóis/química , Proteínas Tirosina Quinases/antagonistas & inibidores , Animais , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Citotoxinas/química , Citotoxinas/farmacologia , Citotoxinas/toxicidade , Feminino , Humanos , Indazóis/farmacologia , Indazóis/toxicidade , Interleucina-13/biossíntese , Interleucina-2/biossíntese , Células Jurkat , Camundongos Endogâmicos C57BL , Modelos Moleculares , Estrutura Molecular , Fosforilação , Receptores de Antígenos de Linfócitos T/metabolismo , Estereoisomerismo , Relação Estrutura-Atividade , Sulfonas/química , Sulfonas/farmacologia , Sulfonas/toxicidade , Sulfóxidos/química , Sulfóxidos/farmacologia , Sulfóxidos/toxicidadeRESUMO
Phosphopantetheine adenylyltransferase (PPAT) is an essential enzyme in Coenzyme A biosynthesis. Because bacterial PPAT and mammalian PPAT are dissimilar, this enzyme is an attractive antibacterial target. Based on the structure of the substrate, 4-phosphopantetheine, a dipeptide library was designed, synthesised and tested against Escherichia coli PPAT. The most potent inhibitor PTX040334 was co-crystallised with E. coli PPAT. With this structural information, a rational iterative medicinal chemistry program was initiated, aimed at increasing the number of inhibitor-enzyme interactions. A very potent and specific inhibitor, PTX042695, with an IC(50) of 6 nM against E.coli PPAT, but with no activity against porcine PPAT, was obtained.
Assuntos
Dipeptídeos/farmacologia , Inibidores Enzimáticos/farmacologia , Nucleotidiltransferases/antagonistas & inibidores , Coenzima A/biossíntese , Dipeptídeos/síntese química , Dipeptídeos/química , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Concentração Inibidora 50 , Modelos Químicos , Modelos Moleculares , Nucleotidiltransferases/metabolismoRESUMO
Interleukin-2 inducible T-cell kinase (ITK), a member of the Tec family of tyrosine kinases, plays a major role in T-cell signaling downstream of the T-cell receptor (TCR), and considerable efforts have been directed toward discovery of ITK-selective inhibitors as potential treatments of inflammatory disorders such as asthma. Using a previously disclosed indazole series of inhibitors as a starting point, and using X-ray crystallography and solubility forecast index (SFI) as guides, we evolved a series of tetrahydroindazole inhibitors with improved potency, selectivity, and pharmaceutical properties. Highlights include identification of a selectivity pocket above the ligand plane, and identification of appropriate lipophilic substituents to occupy this space. This effort culminated in identification of a potent and selective ITK inhibitor (GNE-9822) with good ADME properties in preclinical species.
Assuntos
Indazóis/síntese química , Inibidores de Proteínas Quinases/síntese química , Proteínas Tirosina Quinases/antagonistas & inibidores , Animais , Cristalografia por Raios X , Cães , Desenho de Fármacos , Humanos , Indazóis/farmacocinética , Indazóis/farmacologia , Células Jurkat , Cinética , Camundongos , Modelos Moleculares , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/farmacologia , Ratos , Solubilidade , Relação Estrutura-AtividadeRESUMO
An X-ray crystal structure of Kelch-like ECH-associated protein (Keap1) co-crystallised with (1S,2R)-2-[(1S)-1-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1,2,3,4-tetrahydroisoquinolin-2-carbonyl]cyclohexane-1-carboxylic acid (compound (S,R,S)-1 a) was obtained. This X-ray crystal structure provides breakthrough experimental evidence for the true binding mode of the hit compound (S,R,S)-1 a, as the ligand orientation was found to differ from that of the initial docking model, which was available at the start of the project. Crystallographic elucidation of this binding mode helped to focus and drive the drug design process more effectively and efficiently.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas do Citoesqueleto/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Isoquinolinas/farmacologia , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Ftalimidas/farmacologia , Animais , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Humanos , Isoquinolinas/síntese química , Isoquinolinas/química , Proteína 1 Associada a ECH Semelhante a Kelch , Camundongos , Modelos Moleculares , Estrutura Molecular , Ftalimidas/síntese química , Ftalimidas/química , Relação Estrutura-AtividadeRESUMO
Tissue transglutaminase 2 (TG2) is a multifunctional protein primarily known for its calcium-dependent enzymatic protein cross-linking activity via isopeptide bond formation between glutamine and lysine residues. TG2 overexpression and activity have been found to be associated with Huntington's disease (HD); specifically, TG2 is up-regulated in the brains of HD patients and in animal models of the disease. Interestingly, genetic deletion of TG2 in two different HD mouse models, R6/1 and R6/2, results in improved phenotypes including a reduction in neuronal death and prolonged survival. Starting with phenylacrylamide screening hit 7d, we describe the SAR of this series leading to potent and selective TG2 inhibitors. The suitability of the compounds as in vitro tools to elucidate the biology of TG2 was demonstrated through mode of inhibition studies, characterization of druglike properties, and inhibition profiles in a cell lysate assay.
Assuntos
Acrilamidas/síntese química , Proteínas de Ligação ao GTP/antagonistas & inibidores , Doença de Huntington/tratamento farmacológico , Sulfonamidas/síntese química , Transglutaminases/antagonistas & inibidores , Acrilamidas/química , Acrilamidas/farmacologia , Animais , Células CACO-2 , Permeabilidade da Membrana Celular , Células HEK293 , Humanos , Técnicas In Vitro , Masculino , Camundongos , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Piperazinas/síntese química , Piperazinas/química , Piperazinas/farmacologia , Proteína 2 Glutamina gama-Glutamiltransferase , Piridinas/síntese química , Piridinas/química , Piridinas/farmacologia , Pirimidinas/síntese química , Pirimidinas/química , Pirimidinas/farmacologia , Ratos , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacologiaRESUMO
Heat shock protein 90 (Hsp90) plays a key role in stress response and protection of the cell against the effects of mutation. Herein we report the identification of an Hsp90 inhibitor identified by fragment screening using a high-concentration biochemical assay, as well as its optimisation by in silico searching coupled with a structure-based drug design (SBDD) approach.