RESUMEN
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.
Asunto(s)
Janus Quinasa 3/antagonistas & inhibidores , Janus Quinasa 3/química , Janus Quinasa 3/metabolismo , Inhibidores de Proteínas Quinasas , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/química , Adenosina Trifosfato/farmacología , Enfermedades Autoinmunes/tratamiento farmacológico , Enfermedades Autoinmunes/enzimología , Enfermedades Autoinmunes/genética , Dominio Catalítico , Línea Celular , Humanos , Janus Quinasa 3/genética , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
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.
Asunto(s)
Farmacogenética/métodos , Proteínas Quinasas/metabolismo , Proteoma/antagonistas & inhibidores , Proteoma/metabolismo , Diseño de Fármacos , Proteoma/análisis , Biología de Sistemas/métodosRESUMEN
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.
Asunto(s)
Proteína Quinasa 14 Activada por Mitógenos/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Adenosina Trifosfato/química , Sitios de Unión , Simulación por Computador , Cristalografía por Rayos X , Transferencia Resonante de Energía de Fluorescencia , Cinética , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Unión Proteica , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Estructura Terciaria de Proteína , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Benzodiazepinonas/farmacología , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Proteínas Quinasas/metabolismo , Antineoplásicos/síntesis química , Benzodiazepinonas/síntesis química , Línea Celular Tumoral/efectos de los fármacos , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Inhibidores Enzimáticos/síntesis química , Células HeLa , Humanos , Modelos Químicos , Unión Proteica , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Antibacterianos/síntesis química , Compuestos Heterocíclicos de 4 o más Anillos/síntesis química , Naftiridinas/síntesis química , Antibacterianos/química , Antibacterianos/farmacología , Linfocitos B/efectos de los fármacos , Farmacorresistencia Bacteriana , Compuestos Heterocíclicos de 4 o más Anillos/química , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Pruebas de Sensibilidad Microbiana , Naftiridinas/química , Naftiridinas/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Estereoisomerismo , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/aislamiento & purificación , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Indoles/química , Pirroles/química , Pirroles/farmacología , Huso Acromático/efectos de los fármacos , Antineoplásicos/farmacología , Sitios de Unión , Unión Competitiva , Western Blotting , Proteína Quinasa CDC2/metabolismo , Ciclo Celular , Muerte Celular , Línea Celular Tumoral , Separación Celular , Colchicina/farmacología , Ciclina B/química , Ciclina B1 , Relación Dosis-Respuesta a Droga , Endotelio Vascular/metabolismo , Citometría de Flujo , Células HeLa , Histonas/química , Humanos , Inmunohistoquímica , Indoles/farmacología , Microscopía Fluorescente , Microtúbulos/química , Microtúbulos/metabolismo , Modelos Químicos , Proteínas de Neoplasias/química , Securina , Factores de Tiempo , Tubulina (Proteína)/química , Tirosina/químicaRESUMEN
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.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Janus Quinasa 2/antagonistas & inhibidores , Leucemia Mieloide Aguda/patología , Mutación , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteína bcl-X/antagonistas & inhibidores , Compuestos de Anilina/farmacología , Línea Celular Tumoral , Sinergismo Farmacológico , Humanos , Janus Quinasa 2/genética , Inhibidores de Proteínas Quinasas/farmacología , Sulfonamidas/farmacologíaRESUMEN
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.
RESUMEN
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.
Asunto(s)
Antibacterianos/farmacología , Pruebas de Sensibilidad Microbiana/métodos , Biosíntesis de Proteínas , Streptococcus pneumoniae/efectos de los fármacos , Transcripción Genética , Antibacterianos/efectos adversos , Secuencia de Bases , Línea Celular Tumoral , ADN Bacteriano , Genes Reporteros , Humanos , Luciferasas/genética , Datos de Secuencia Molecular , Streptococcus pneumoniae/genéticaRESUMEN
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.
RESUMEN
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.
RESUMEN
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.
Asunto(s)
Hidrógeno/química , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Pirazoles/química , Pirazoles/farmacología , Piridinas/química , Administración Oral , Animales , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Cianuros/química , Indenos/química , Concentración 50 Inhibidora , Ratones , Estructura Molecular , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/síntesis química , Pirazoles/administración & dosificación , Pirazoles/síntesis química , Ratas , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Antineoplásicos/farmacocinética , Química Farmacéutica/métodos , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores de Proteínas Quinasas/farmacocinética , Proteínas Quinasas/química , Animales , Antineoplásicos/química , Células CACO-2 , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Daño del ADN , Diseño de Fármacos , Citometría de Flujo , Humanos , Concentración 50 Inhibidora , Ratones , Microsomas Hepáticos/metabolismo , Inhibidores de Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , RatasRESUMEN
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.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Antibacterianos/química , Proteínas Bacterianas/metabolismo , Química Farmacéutica/métodos , Haemophilus influenzae/metabolismo , Secuencia de Aminoácidos , Animales , Linfocitos B/metabolismo , Diseño de Fármacos , Genoma Bacteriano , Genómica , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Datos de Secuencia Molecular , Unión Proteica , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Antibacterianos/farmacología , Farmacorresistencia Bacteriana/genética , Genoma Bacteriano , Haemophilus influenzae/efectos de los fármacos , Mapeo Restrictivo/métodos , Transformación Bacteriana/genética , Antibacterianos/química , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/genética , Enzimas de Restricción del ADN/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Transporte de Ácidos Grasos , Haemophilus influenzae/genética , Proteínas de Transporte de Membrana , Pruebas de Sensibilidad Microbiana/métodos , MutaciónRESUMEN
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.
Asunto(s)
Antibacterianos/farmacología , Haemophilus influenzae/efectos de los fármacos , Oxadiazoles/farmacología , Pirazinas/farmacología , Antibacterianos/síntesis química , Evaluación Preclínica de Medicamentos/métodos , Haemophilus influenzae/crecimiento & desarrollo , Humanos , Pruebas de Sensibilidad Microbiana/métodos , Oxadiazoles/síntesis química , Pirazinas/síntesis química , Relación Estructura-ActividadRESUMEN
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.