RESUMO
The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. We have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). We found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC50 < 100 nm) inhibit Jak3 activity in cell-based assays. These results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases.
Assuntos
Janus Quinase 3/antagonistas & inibidores , Janus Quinase 3/química , Janus Quinase 3/metabolismo , Inibidores de Proteínas Quinases , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/química , Trifosfato de Adenosina/farmacologia , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/enzimologia , Doenças Autoimunes/genética , Domínio Catalítico , Linhagem Celular , Humanos , Janus Quinase 3/genética , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologiaRESUMO
Although it is increasingly being recognized that drug-target interaction networks can be powerful tools for the interrogation of systems biology and the rational design of multitargeted drugs, there is no generalized, statistically validated approach to harmonizing sequence-dependent and pharmacology-dependent networks. Here we demonstrate the creation of a comprehensive kinome interaction network based not only on sequence comparisons but also on multiple pharmacology parameters derived from activity profiling data. The framework described for statistical interpretation of these network connections also enables rigorous investigation of chemotype-specific interaction networks, which is critical for multitargeted drug design.
Assuntos
Farmacogenética/métodos , Proteínas Quinases/metabolismo , Proteoma/antagonistas & inibidores , Proteoma/metabolismo , Desenho de Fármacos , Proteoma/análise , Biologia de Sistemas/métodosRESUMO
Herein we describe the identification and characterization of a class of molecules that are believed to extend into a region of p38 known as the 'switch pocket'. Although these molecules lack a canonical hinge binding motif, they show K(i) values as low as 100 nM against p38. We show that molecules that interact with this region of the protein demonstrate different binding kinetics than a canonical ATP mimetic, as well as a wide range of kinome profiles. Thus, the switch pocket presents new opportunities for kinome selectivity which could result in unique biochemical responses and offer new opportunities in the field of kinase drug discovery.
Assuntos
Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Trifosfato de Adenosina/química , Sítios de Ligação , Simulação por Computador , Cristalografia por Raios X , Transferência Ressonante de Energia de Fluorescência , Cinética , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Ligação Proteica , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
The synthesis and structure-activity relationships (SAR) of Chk1 inhibitors based on a 5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-one core are described. Specifically, an exploration of the 7 and 8 positions on this previously disclosed core afforded compounds with improved enzymatic and cellular potency.
Assuntos
Antineoplásicos/farmacologia , Benzodiazepinonas/farmacologia , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Proteínas Quinases/metabolismo , Antineoplásicos/síntese química , Benzodiazepinonas/síntese química , Linhagem Celular Tumoral/efeitos dos fármacos , Quinase 1 do Ponto de Checagem , Inibidores Enzimáticos/síntese química , Células HeLa , Humanos , Modelos Químicos , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
We describe the synthesis and antibacterial activity of a series of tetracyclic naphthyridones. The members of this series act primarily via inhibition of bacterial translation and belong to the class of novel ribosome inhibitors (NRIs). In this paper we explore the structure-activity relationships (SAR) of these compounds to measure their ability both to inhibit bacterial translation and also to inhibit the growth of bacterial cells in culture. The most active of these compounds inhibit Streptococcus pneumoniae translation at concentrations of <5 microM and have minimum inhibitory concentrations (MICs) of <8 microg/mL against clinically relevant strains of bacteria.
Assuntos
Antibacterianos/síntese química , Compostos Heterocíclicos de 4 ou mais Anéis/síntese química , Naftiridinas/síntese química , Antibacterianos/química , Antibacterianos/farmacologia , Linfócitos B/efeitos dos fármacos , Farmacorresistência Bacteriana , Compostos Heterocíclicos de 4 ou mais Anéis/química , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Testes de Sensibilidade Microbiana , Naftiridinas/química , Naftiridinas/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Estereoisomerismo , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/isolamento & purificação , Relação Estrutura-AtividadeRESUMO
Microtubules are among the most successful targets for anticancer therapies and for the development of new anticancer drugs. A-432411 is a novel small molecule that destabilizes microtubules at high concentration and disrupts normal spindle formation at low concentration. A-432411 is an indolinone that is structurally different from other known synthetic microtubule inhibitors. This compound is efficacious against a variety of human cancer cell lines including drug-resistant HCT-15 that overexpresses Pgp170. Biochemical studies show that A-432411 competes with the colchicine-binding site on tubulin and inhibits microtubule polymerization. Fluorescence-activated cell sorting analysis indicates that A-432411 causes G2-M arrest and induces apoptosis. Cells treated with A-432411 have increased level of phospho-histone H3 at Ser10 and decreased level of phospho-cdc2 at Tyr15. Concurrently, securin and cyclin B1 expression levels remain the same, indicating the activation of the spindle checkpoint. Immunocytochemistry and fluorescence microscopy experiments reveal that 1 micromol/L A-432411 destabilizes microtubules in cells. At 0.1 micromol/L, the compound disrupts normal spindle pole formation possibly through stabilization of microtubule dynamic. Both structural and cellular properties of A-432411 make it an attractive candidate for further development.
Assuntos
Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Indóis/química , Pirróis/química , Pirróis/farmacologia , Fuso Acromático/efeitos dos fármacos , Antineoplásicos/farmacologia , Sítios de Ligação , Ligação Competitiva , Western Blotting , Proteína Quinase CDC2/metabolismo , Ciclo Celular , Morte Celular , Linhagem Celular Tumoral , Separação Celular , Colchicina/farmacologia , Ciclina B/química , Ciclina B1 , Relação Dose-Resposta a Droga , Endotélio Vascular/metabolismo , Citometria de Fluxo , Células HeLa , Histonas/química , Humanos , Imuno-Histoquímica , Indóis/farmacologia , Microscopia de Fluorescência , Microtúbulos/química , Microtúbulos/metabolismo , Modelos Químicos , Proteínas de Neoplasias/química , Securina , Fatores de Tempo , Tubulina (Proteína)/química , Tirosina/químicaRESUMO
Constitutive activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) axis is fundamental to the molecular pathogenesis of a host of hematological disorders, including acute leukemias and myeloproliferative neoplasms (MPN). We demonstrate here that the major JAK2 mutation observed in these diseases (JAK2V617F) enforces Mcl-1 transcription via STAT3 signaling. Targeting this lesion with JAK inhibitor I (JAKi-I) attenuates STAT3 binding to the Mcl-1 promoter and suppresses Mcl-1 transcript and protein expression. The neutralization of Mcl-1 in JAK2V617F-harboring myelodyssplastic syndrome cell lines sensitizes them to apoptosis induced by the BH3-mimetic and Bcl-xL/Bcl-2 inhibitor, ABT-263. Moreover, simultaneously targeting JAK and Bcl-xL/-2 is synergistic in the presence of the JAK2V617F mutation. These findings suggest that JAK/Bcl-xL/-2 inhibitor combination therapy may have applicability in a range of hematological disorders characterized by activating JAK2 mutations.
Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Janus Quinase 2/antagonistas & inibidores , Leucemia Mieloide Aguda/patologia , Mutação , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteína bcl-X/antagonistas & inibidores , Compostos de Anilina/farmacologia , Linhagem Celular Tumoral , Sinergismo Farmacológico , Humanos , Janus Quinase 2/genética , Inibidores de Proteínas Quinases/farmacologia , Sulfonamidas/farmacologiaRESUMO
Aided by molecular modeling, compounds with a pyrimidine-based tricyclic scaffold were designed and confirmed to inhibit Wee1 kinase. Structure-activity studies identified key pharmacophores at the aminoaryl and halo-benzene regions responsible for binding affinity with sub-nM K i values. The potent inhibitors demonstrated sub-µM activities in both functional and mechanism-based cellular assays and also possessed desirable pharmacokinetic profiles. The lead molecule, 31, showed oral efficacy in potentiating the antiproliferative activity of irinotecan, a cytotoxic agent, in a NCI-H1299 mouse xenograft model.
RESUMO
The authors report the development of a high-throughput screen for inhibitors of Streptococcus pneumoniae transcription and translation (TT) using a luciferase reporter, and the secondary assays used to determine the biochemical spectrum of activity and bacterial specificity. More than 220,000 compounds were screened in mixtures of 10 compounds per well, with 10,000 picks selected for further study. False-positive hits from inhibition of luciferase activity were an extremely common artifact. After filtering luciferase inhibitors and several known classes of antibiotics, approximately 50 hits remained. These compounds were examined for their ability to inhibit Escherichia coli TT, uncoupled S. pneumoniae translation or transcription, rabbit reticulocyte translation, and in vitro toxicity in human and bacterial cells. One of these compounds had the desired profile of broad-spectrum biochemical activity in bacteria and selectivity versus mammalian biochemical and whole-cell assays.
Assuntos
Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana/métodos , Biossíntese de Proteínas , Streptococcus pneumoniae/efeitos dos fármacos , Transcrição Gênica , Antibacterianos/efeitos adversos , Sequência de Bases , Linhagem Celular Tumoral , DNA Bacteriano , Genes Reporter , Humanos , Luciferases/genética , Dados de Sequência Molecular , Streptococcus pneumoniae/genéticaRESUMO
5-Aminopyrazole-4-carboxamide was used as an alternative scaffold to substitute for the pyrazolopyrimidine of a known "bumped kinase inhibitor" to create selective inhibitors of calcium-dependent protein kinase-1 from both Toxoplasma gondii and Cryptosporidium parvum. Compounds with low nanomolar inhibitory potencies against the target enzymes were obtained. The most selective inhibitors also exhibited submicromolar activities in T. gondii cell proliferation assays and were shown to be non-toxic to mammalian cells.
RESUMO
To investigate the role played by the unique pre-DFG residue Val 195 of Cdc7 kinase on the potency of azaindole-chloropyridines (1), a series of novel analogues with various chloro replacements were synthesized and evaluated for their inhibitory activity against Cdc7. X-ray cocrystallization using a surrogate protein, GSK3ß, and modeling studies confirmed the azaindole motif as the hinge binder. Weaker hydrophobic interactions with Met 134 and Val 195 by certain chloro replacements (e.g., H, methyl) led to reduced Cdc7 inhibition. Meanwhile, data from other replacements (e.g., F, O) indicated that loss of such hydrophobic interaction could be compensated by enhanced hydrogen bonding to Lys 90. Our findings not only provide an in-depth understanding of the pre-DFG residue as another viable position impacting kinase inhibition, they also expand the existing knowledge of ligand-Cdc7 binding.
RESUMO
A series of 1,4-dihydroindeno[1,2-c]pyrazole compounds with a cyanopyridine moiety at the 3-position of the tricyclic pyrazole core was explored as potent CHK-1 inhibitors. The impact of substitutions at the 6 and/or 7-position of the core on pharmacokinetic properties was studied in detail. Compounds carrying a side chain with an ether linker at the 7-position and a terminal morpholino group, such as 29 and 30, exhibited much-improved oral biovailability in mice as compared to earlier generation inhibitors. These compounds also possessed desirable cellular activity in potentiating doxorubicin and will serve as valuable tool compounds for in vivo evaluation of CHK-1 inhibitors to sensitize DNA-damaging agents.
Assuntos
Hidrogênio/química , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Pirazóis/química , Pirazóis/farmacologia , Piridinas/química , Administração Oral , Animais , Quinase 1 do Ponto de Checagem , Cianetos/química , Indenos/química , Concentração Inibidora 50 , Camundongos , Estrutura Molecular , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/síntese química , Pirazóis/administração & dosagem , Pirazóis/síntese química , Ratos , Relação Estrutura-AtividadeRESUMO
A study on substitutions at the four open positions on the phenyl ring of the 1,4-dihydroindeno[1,2-c]pyrazoles as potent CHK-1 inhibitors is described. Bis-substitution at both the 6- and 7-positions led to inhibitors with IC(50) values below 0.3nM. The compound with the best overall activities (36) was able to potentiate the anti-proliferative effect of doxorubicin in HeLa cells by at least 47-fold. Physicochemical, metabolic, and pharmacokinetic properties of selected inhibitors are also disclosed.
Assuntos
Antineoplásicos/farmacocinética , Química Farmacêutica/métodos , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Quinases/química , Animais , Antineoplásicos/química , Células CACO-2 , Quinase 1 do Ponto de Checagem , Dano ao DNA , Desenho de Fármacos , Citometria de Fluxo , Humanos , Concentração Inibidora 50 , Camundongos , Microssomos Hepáticos/metabolismo , Inibidores de Proteínas Quinases/química , Proteínas Quinases/metabolismo , RatosRESUMO
As part of a fully integrated and comprehensive strategy to discover novel antibacterial agents, NMR- and mass spectrometry-based affinity selection screens were performed to identify compounds that bind to protein targets uniquely found in bacteria and encoded by genes essential for microbial viability. A biphenyl acid lead series emerged from an NMR-based screen with the Haemophilus influenzae protein HI0065, a member of a family of probable ATP-binding proteins found exclusively in eubacteria. The structure-activity relationships developed around the NMR-derived biphenyl acid lead were consistent with on-target antibacterial activity as the Staphylococcus aureus antibacterial activity of the series correlated extremely well with binding affinity to HI0065, while the correlation of binding affinity with B-cell cytotoxicity was relatively poor. Although further studies are needed to conclusively establish the mode of action of the biphenyl series, these compounds represent novel leads that can serve as the basis for the development of novel antibacterial agents that appear to work via an unprecedented mechanism of action. Overall, these results support the genomics-driven hypothesis that targeting bacterial essential gene products that are not present in eukaryotic cells can identify novel antibacterial agents.
Assuntos
Adenosina Trifosfatases/metabolismo , Antibacterianos/química , Proteínas de Bactérias/metabolismo , Química Farmacêutica/métodos , Haemophilus influenzae/metabolismo , Sequência de Aminoácidos , Animais , Linfócitos B/metabolismo , Desenho de Fármacos , Genoma Bacteriano , Genômica , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Dados de Sequência Molecular , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Restriction enzyme modulation of transformation efficiencies (REMOTE) is a method that makes use of genome restriction maps and experimentally observed differences in transformation efficiencies of genomic DNA restriction digests to discover the location of mutations in genomes. The frequency with which digested genomic DNA from a resistant strain transforms a susceptible strain to resistance is primarily determined by the size of the fragment containing the resistance mutation and the distance of the mutation to the end of the fragment. The positions of restriction enzyme cleavage sites immediately flanking the resistance mutation define these parameters. The mapping procedure involves a process of elimination in which digests that transform with high frequency indicate that the restriction enzyme cleavage sites are relatively far away from the mutation, while digests that transform with low frequency indicate that the sites are close to the mutation. The transformation data are compared computationally to the genome restriction map to identify the regions that best fit the data. Transformations with PCR amplicons encompassing candidate regions identify the resistance locus and enable identification of the mutation. REMOTE was developed using Haemophilus influenzae strains with mutations in gyrA, gyrB, and rpsE that confer resistance to ciprofloxacin, novobiocin, and spectinomycin, respectively. We applied REMOTE to identify mutations that confer resistance to two novel antibacterial compounds. The resistance mutations were found in genes that can decrease the intracellular concentration of compounds: acrB, which encodes a subunit of the AcrAB-TolC efflux pump; and fadL, which encodes a long-chain fatty acid transporter.
Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Genoma Bacteriano , Haemophilus influenzae/efeitos dos fármacos , Mapeamento por Restrição/métodos , Transformação Bacteriana/genética , Antibacterianos/química , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/genética , Enzimas de Restrição do DNA/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Transporte de Ácido Graxo , Haemophilus influenzae/genética , Proteínas de Membrana Transportadoras , Testes de Sensibilidade Microbiana/métodos , MutaçãoRESUMO
The parallel synthesis and antibacterial activity of 5-hydroxy[1,2,5] oxadiazolo[3,4-b]pyrazines is reported. The compounds were synthesized by condensing diaminofurazan with alpha-keto acids to give a variety of aryl-substituted analogues. Halogenated phenyl groups at C-6 give rise to the greatest Haemophilus influenzae antibacterial activity.
Assuntos
Antibacterianos/farmacologia , Haemophilus influenzae/efeitos dos fármacos , Oxidiazóis/farmacologia , Pirazinas/farmacologia , Antibacterianos/síntese química , Avaliação Pré-Clínica de Medicamentos/métodos , Haemophilus influenzae/crescimento & desenvolvimento , Humanos , Testes de Sensibilidade Microbiana/métodos , Oxidiazóis/síntese química , Pirazinas/síntese química , Relação Estrutura-AtividadeRESUMO
We report the discovery and characterization of a novel ribosome inhibitor (NRI) class that exhibits selective and broad-spectrum antibacterial activity. Compounds in this class inhibit growth of many gram-positive and gram-negative bacteria, including the common respiratory pathogens Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, and Moraxella catarrhalis, and are nontoxic to human cell lines. The first NRI was discovered in a high-throughput screen designed to identify inhibitors of cell-free translation in extracts from S. pneumoniae. The chemical structure of the NRI class is related to antibacterial quinolones, but, interestingly, the differences in structure are sufficient to completely alter the biochemical and intracellular mechanisms of action. Expression array studies and analysis of NRI-resistant mutants confirm this difference in intracellular mechanism and provide evidence that the NRIs inhibit bacterial protein synthesis by inhibiting ribosomes. Furthermore, compounds in the NRI series appear to inhibit bacterial ribosomes by a new mechanism, because NRI-resistant strains are not cross-resistant to other ribosome inhibitors, such as macrolides, chloramphenicol, tetracycline, aminoglycosides, or oxazolidinones. The NRIs are a promising new antibacterial class with activity against all major drug-resistant respiratory pathogens.