RESUMO
Currently, there are no established pharmaceutical strategies that effectively treat social deficits in autism spectrum disorder (ASD). Oxytocin, a neurohormone that plays a role in multiple types of social behaviors, has been proposed as a possible therapeutic against social impairment and other symptoms in ASD. However, from the standpoint of pharmacotherapy, oxytocin has several liabilities as a standard clinical treatment, including rapid metabolism, low brain penetrance, and activity at the vasopressin (antidiuretic hormone) receptors. The present studies describe findings from a preclinical screening program to evaluate oxytocin receptor (OXTR) agonists and oxytocin metabolites for potential clinical use as more optimal treatments. We first investigated two synthetic oxytocin analogs, TC-OT-39 and carbetocin, using in vitro cell-based assays for pharmacological characterization and behavioral tests in the BALB/cByJ mouse model of ASD-like social deficits. Although both TC-OT-39 and carbetocin selectively activate the OXTR, neither synthetic agonist had prosocial efficacy in the BALB/cByJ model. We next evaluated two oxytocin metabolites: OT(4-9) and OT(5-9). While OT(5-9) failed to affect social deficits, the metabolite OT(4-9) led to significant social preference in the BALB/cByJ model, in a dose-dependent manner. The increased sociability was observed at both 24â¯h and 12 days following the end of a subchronic regimen with OT(4-9) (2.0â¯mg/kg). Overall, these results suggest that the prosocial effects of oxytocin could be mediated by downstream activity of oxytocin metabolites, raising the possibility of new pathways to target for drug discovery relevant to ASD.
Assuntos
Transtorno do Espectro Autista/tratamento farmacológico , Ocitocina/análogos & derivados , Psicotrópicos/farmacologia , Receptores de Ocitocina/agonistas , Comportamento Social , Animais , Transtorno do Espectro Autista/metabolismo , Transtorno do Espectro Autista/psicologia , Comportamento Compulsivo/tratamento farmacológico , Comportamento Compulsivo/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Masculino , Camundongos Endogâmicos BALB C , Ocitocina/química , Ocitocina/metabolismo , Ocitocina/farmacologia , Receptores de Ocitocina/metabolismoRESUMO
A key challenge in the development of precision medicine is defining the phenotypic consequences of pharmacological modulation of specific target macromolecules. To address this issue, a variety of genetic, molecular and chemical tools can be used. All of these approaches can produce misleading results if the specificity of the tools is not well understood and the proper controls are not performed. In this paper we illustrate these general themes by providing detailed studies of small molecule inhibitors of the enzymatic activity of two members of the SMYD branch of the protein lysine methyltransferases, SMYD2 and SMYD3. We show that tool compounds as well as CRISPR/Cas9 fail to reproduce many of the cell proliferation findings associated with SMYD2 and SMYD3 inhibition previously obtained with RNAi based approaches and with early stage chemical probes.
Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Carcinogênese/genética , Histona-Lisina N-Metiltransferase/genética , Células A549 , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Sistemas CRISPR-Cas , Carcinogênese/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/química , Humanos , Metilação/efeitos dos fármacos , Metiltransferases/antagonistas & inibidores , Interferência de RNA , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
WHSC1 is a histone methyltransferase that is responsible for mono- and dimethylation of lysine 36 on histone H3 and has been implicated as a driver in a variety of hematological and solid tumors. Currently, there is a complete lack of validated chemical matter for this important drug discovery target. Herein we report on the first fully validated WHSC1 inhibitor, PTD2, a norleucine-containing peptide derived from the histone H4 sequence. This peptide exhibits micromolar affinity towards WHSC1 in biochemical and biophysical assays. Furthermore, a crystal structure was solved with the peptide in complex with SAM and the SET domain of WHSC1L1. This inhibitor is an important first step in creating potent, selective WHSC1 tool compounds for the purposes of understanding the complex biology in relation to human disease.
Assuntos
Inibidores Enzimáticos/química , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Peptídeos/química , Proteínas Repressoras/antagonistas & inibidores , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/genética , Histonas/química , Histonas/genética , Humanos , Lisina/química , Neoplasias/enzimologia , Norleucina/análogos & derivados , Norleucina/química , Norleucina/farmacologia , Domínios PR-SET/genética , Peptídeos/genética , Conformação Proteica/efeitos dos fármacos , Proteínas Repressoras/química , Proteínas Repressoras/genéticaRESUMO
Drug repurposing approaches have the potential advantage of facilitating rapid and cost-effective development of new therapies. Particularly, the repurposing of drugs with known safety profiles in children could bypass or streamline toxicity studies. We employed a phenotypic screening paradigm on a panel of well-characterized cell lines derived from pediatric solid tumors against a collection of â¼3,800 compounds spanning approved drugs and investigational agents. Specifically, we employed titration-based screening where compounds were tested at multiple concentrations for their effect on cell viability. Molecular and cellular target enrichment analysis indicated that numerous agents across different therapeutic categories and modes of action had an antiproliferative effect, notably antiparasitic/protozoal drugs with non-classic antineoplastic activity. Focusing on active compounds with dosing and safety information in children according to the Children's Pharmacy Collaborative database, we identified compounds with therapeutic potential through further validation using 3D tumor spheroid models. Moreover, we show that antiparasitic agents induce cell death via apoptosis induction. This study demonstrates that our screening platform enables the identification of chemical agents with cytotoxic activity in pediatric cancer cell lines of which many have known safety/toxicity profiles in children. These agents constitute attractive candidates for efficacy studies in pre-clinical models of pediatric solid tumors.
RESUMO
Mutations in chromatin-modifying proteins and transcription factors are commonly associated with a wide variety of cancers. Through gain- or loss-of-function, these mutations may result in characteristic alterations of accessible chromatin, indicative of shifts in the landscape of regulatory elements genome-wide. The identification of compounds that reverse a specific chromatin signature could lead to chemical probes or potential therapies. To explore whether chromatin accessibility could serve as a platform for small molecule screening, we adapted formaldehyde-assisted isolation of regulatory elements (FAIRE), a chemical method to enrich for nucleosome-depleted genomic regions, as a high-throughput, automated assay. After demonstrating the validity and robustness of this approach, we applied this method to screen an epigenetically targeted small molecule library by evaluating regions of aberrant nucleosome depletion mediated by EWSR1-FLI1, the chimeric transcription factor critical for the bone and soft tissue tumor Ewing sarcoma. As a class, histone deacetylase inhibitors were greatly overrepresented among active compounds. These compounds resulted in diminished accessibility at targeted sites by disrupting transcription of EWSR1-FLI1. Capitalizing on precise differences in chromatin accessibility for drug discovery efforts offers significant advantages because it does not depend on the a priori selection of a single molecular target and may detect novel biologically relevant pathways.
Assuntos
Cromatina/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Transcrição Gênica/efeitos dos fármacos , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Cromatina/ultraestrutura , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Inibidores de Histona Desacetilases/isolamento & purificação , Inibidores de Histona Desacetilases/farmacologia , Histonas/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Ácidos Hidroxâmicos/farmacologia , Indóis/farmacologia , Terapia de Alvo Molecular , Nucleossomos/ultraestrutura , Proteínas de Fusão Oncogênica/genética , Panobinostat , Fenilbutiratos/farmacologia , Sarcoma de Ewing/patologia , Bibliotecas de Moléculas Pequenas , VorinostatRESUMO
Technological advances coupled with novel collaborative strategies for compound sourcing and management are poised to transform the utility of high-throughput screening.
Assuntos
Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Indústria Farmacêutica/métodos , Parcerias Público-PrivadasRESUMO
A perfluorocarbon nanodroplet formulation is shown to be an effective cavitation enhancement agent, enabling rapid and consistent fragmentation of genomic DNA in a standard ultrasonic water bath. This nanodroplet-enhanced method produces genomic DNA libraries and next-generation sequencing results indistinguishable from DNA samples fragmented in dedicated commercial acoustic sonication equipment, and with higher throughput. This technique thus enables widespread access to fast bench-top genomic DNA fragmentation.
Assuntos
Fragmentação do DNA/efeitos da radiação , Sonicação/métodos , DNA Fúngico , Microbolhas , Sonicação/instrumentaçãoRESUMO
Improving our understanding of the role of chromatin regulators in the initiation, development, and suppression of cancer and other devastating diseases is critical, as they are integral players in regulating DNA integrity and gene expression. Developing small molecule inhibitors for this target class with cellular activity is a crucial step toward elucidating their specific functions. We specifically targeted the DNA damage response protein, 53BP1, which uses its tandem tudor domain to recognize histone H4 dimethylated on lysine 20 (H4K20me2), a modification related to double-strand DNA breaks. Through a cross-screening approach, we identified UNC2170 (1) as a micromolar ligand of 53BP1, which demonstrates at least 17-fold selectivity for 53BP1 as compared to other methyl-lysine (Kme) binding proteins tested. Structural studies revealed that the tert-butyl amine of UNC2170 anchors the compound in the methyl-lysine (Kme) binding pocket of 53BP1, making it competitive with endogenous Kme substrates. X-ray crystallography also demonstrated that UNC2170 binds at the interface of two tudor domains of a 53BP1 dimer. Importantly, this compound functions as a 53BP1 antagonist in cellular lysates and shows cellular activity by suppressing class switch recombination, a process which requires a functional 53BP1 tudor domain. These results demonstrate that UNC2170 is a functionally active, fragment-like ligand for 53BP1.
Assuntos
Benzamidas/metabolismo , Diaminas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisina/metabolismo , Animais , Linfócitos B/efeitos dos fármacos , Benzamidas/química , Benzamidas/farmacologia , Sítios de Ligação , Cromatina/metabolismo , Cristalografia por Raios X , Diaminas/química , Diaminas/farmacologia , Células HEK293 , Histonas/genética , Histonas/metabolismo , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Camundongos Endogâmicos C57BL , Estrutura Terciária de Proteína , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Relação Estrutura-Atividade , Proteína 1 de Ligação à Proteína Supressora de Tumor p53RESUMO
Phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) regulate a variety of cellular processes, including signaling through G protein-coupled receptors (GPCRs), endocytosis, exocytosis, and cell migration. These lipid kinases synthesize phosphatidylinositol 4,5-bisphosphate (PIP2) from phosphatidylinositol 4-phosphate [PI(4)P]. Because small-molecule inhibitors of these lipid kinases did not exist, molecular and genetic approaches were predominantly used to study PIP5K1 regulation of these cellular processes. Moreover, standard radioisotope-based lipid kinase assays cannot be easily adapted for high-throughput screening. Here, we report a novel, high-throughput, microfluidic mobility shift assay to identify inhibitors of PIP5K1C. This assay uses fluorescently labeled phosphatidylinositol 4-phosphate as the substrate and recombinant human PIP5K1C. Our assay exhibited high reproducibility, had a calculated adenosine triphosphate Michaelis constant (Km) of 15 µM, performed with z' values >0.7, and was used to screen a kinase-focused library of ~4700 compounds. From this screen, we identified several potent inhibitors of PIP5K1C, including UNC3230, a compound that we recently found can reduce nociceptive sensitization in animal models of chronic pain. This novel assay will allow continued drug discovery efforts for PIP5K1C and can be adapted easily to screen additional lipid kinases.
Assuntos
Descoberta de Drogas/métodos , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Relação Dose-Resposta a Droga , Humanos , Cinética , Reprodutibilidade dos Testes , Bibliotecas de Moléculas PequenasRESUMO
Ror2 is a Wnt ligand receptor that is overexpressed in a variety of tumors including clear cell renal cell carcinoma (ccRCC). Here we demonstrate that expression of wild type Ror2 results in increased tumorigenic properties in in vitro cell culture and in vivo xenograft models. In addition, Ror2 expression produced positive changes in both cell migration and invasion, which were dependent on matrix metalloprotease 2 (MMP2) activity. Mutations in key regions of the kinase domain of Ror2 resulted in the abrogation of increased tumor growth, cell migration, and cell invasion observed with expression of wild-type Ror2. Finally, we examined Ror2 expression as a prognostic biomarker for ccRCC utilizing the TCGA ccRCC dataset. High expression of Ror2 showed a significant correlation with higher clinical stage, nuclear grade, and tumor stage. Furthermore, high expression of Ror2 in ccRCC patients correlated with significant lower overall survival, cancer specific survival, and recurrence free survival. Together, these findings suggest that Ror2 plays a central role in influencing the ccRCC phenotype, and can be considered as a negative prognostic biomarker and potential therapeutic target in this cancer.
Assuntos
Carcinoma de Células Renais/patologia , Neoplasias Renais/patologia , Rim/patologia , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Animais , Carcinoma de Células Renais/diagnóstico , Carcinoma de Células Renais/genética , Linhagem Celular Tumoral , Movimento Celular , Regulação Neoplásica da Expressão Gênica , Humanos , Rim/metabolismo , Neoplasias Renais/diagnóstico , Neoplasias Renais/genética , Camundongos Nus , Mutação , Invasividade Neoplásica/diagnóstico , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Prognóstico , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/análiseRESUMO
Screening, high-throughput screening, and ultra-high-throughput screening are all really just points on a spectrum that represent differing applications of the same process: the creation of biologically relevant assays that are relevant, reproducible, reliable, and robust. Whether the discovery program is developing a pharmaceutical, an academic probe, cosmetics, pesticides, or a toxicity monitoring assay, the development of a screen focuses on generating a method that will reliably deliver reproducible results over a period of weeks, months, or years and that will generate consistent results for every test along the way. This review provides both historical perspective on how this unique scientific discipline evolved and commentary on the current state of the art technologies and techniques.
Assuntos
Descoberta de Drogas , Bibliotecas de Moléculas Pequenas/química , Polarização de Fluorescência , Transferência Ressonante de Energia de Fluorescência , Ensaios de Triagem em Larga Escala , Microfluídica , Imagem Óptica , Bibliotecas de Moléculas Pequenas/metabolismoRESUMO
Drug repurposing is the use of 'old' drugs for new indications, avoiding the need for time- and cost-intensive toxicity studies. This approach should be particularly attractive for pediatrics, but its use in this population has been limited. One obstacle has been the lack of a comprehensive database of drugs for which there already is at least one indication in children. We describe the development of The Children's Pharmacy Collaborative, which should grow over time, serve as a resource for professionals and families, and stimulate drug-repurposing efforts for a range of pediatric disorders.
Assuntos
Bases de Dados de Produtos Farmacêuticos , Reposicionamento de Medicamentos , Criança , Humanos , PediatriaRESUMO
We previously reported a potent small molecule Mer tyrosine kinase inhibitor UNC1062. However, its poor PK properties prevented further assessment in vivo. We report here the sequential modification of UNC1062 to address DMPK properties and yield a new potent and highly orally bioavailable Mer inhibitor, 11, capable of inhibiting Mer phosphorylation in vivo, following oral dosing as demonstrated by pharmaco-dynamic (PD) studies examining phospho-Mer in leukemic blasts from mouse bone marrow. Kinome profiling versus more than 300 kinases in vitro and cellular selectivity assessments demonstrate that 11 has similar subnanomolar activity against Flt3, an additional important target in acute myelogenous leukemia (AML), with pharmacologically useful selectivity versus other kinases examined.
Assuntos
Adenina/análogos & derivados , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Adenina/administração & dosagem , Adenina/farmacocinética , Adenina/farmacologia , Administração Oral , Animais , Disponibilidade Biológica , Linhagem Celular Tumoral/efeitos dos fármacos , Técnicas de Química Sintética , Humanos , Concentração Inibidora 50 , Leucemia de Células B/tratamento farmacológico , Leucemia de Células B/metabolismo , Leucemia de Células B/patologia , Camundongos SCID , Terapia de Alvo Molecular , Piperazinas/administração & dosagem , Piperazinas/farmacocinética , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de Xenoenxerto , c-Mer Tirosina Quinase , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
The lysine methyltransferase SETD8 is the only known methyltransferase that catalyzes monomethylation of histone H4 lysine 20 (H4K20). Monomethylation of H4K20 has been implicated in regulating diverse biological processes including the DNA damage response. In addition to H4K20, SETD8 monomethylates non-histone substrates including proliferating cell nuclear antigen (PCNA) and promotes carcinogenesis by deregulating PCNA expression. However, selective inhibitors of SETD8 are scarce. The only known selective inhibitor of SETD8 to date is nahuoic acid A, a marine natural product, which is competitive with the cofactor. Here, we report the discovery of the first substrate-competitive inhibitor of SETD8, UNC0379 (1). This small-molecule inhibitor is active in multiple biochemical assays. Its affinity to SETD8 was confirmed by ITC (isothermal titration calorimetry) and SPR (surface plasmon resonance) studies. Importantly, compound 1 is selective for SETD8 over 15 other methyltransferases. We also describe structure-activity relationships (SAR) of this series.
Assuntos
Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Pirrolidinas/química , Quinazolinas/química , Calorimetria , Histona-Lisina N-Metiltransferase/química , Pirrolidinas/síntese química , Quinazolinas/síntese química , Relação Estrutura-Atividade , Ressonância de Plasmônio de SuperfícieRESUMO
The tendency for mycobacteria to aggregate poses a challenge for their use in microplate based assays. Good dispersions have been difficult to achieve in high-throughput screening (HTS) assays used in the search for novel antibacterial drugs to treat tuberculosis and other related diseases. Here we describe a method using filtration to overcome the problem of variability resulting from aggregation of mycobacteria. This method consistently yielded higher reproducibility and lower variability than conventional methods, such as settling under gravity and vortexing.
Assuntos
Antituberculosos/farmacologia , Filtração/métodos , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Filtração/instrumentação , Ensaios de Triagem em Larga Escala/métodos , Filtros Microporos , Mycobacterium smegmatis/fisiologia , Mycobacterium tuberculosis/fisiologia , Reprodutibilidade dos TestesRESUMO
Numerous pain-producing (pronociceptive) receptors signal via phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis. However, it is currently unknown which lipid kinases generate PIP2 in nociceptive dorsal root ganglia (DRG) neurons and if these kinases regulate pronociceptive receptor signaling. Here, we found that phosphatidylinositol 4-phosphate 5 kinase type 1C (PIP5K1C) is expressed at higher levels than any other PIP5K and, based on experiments with Pip5k1c(+/-) mice, generates at least half of all PIP2 in DRG neurons. Additionally, Pip5k1c haploinsufficiency reduces pronociceptive receptor signaling and TRPV1 sensitization in DRG neurons as well as thermal and mechanical hypersensitivity in mouse models of chronic pain. We identified a small molecule inhibitor of PIP5K1C (UNC3230) in a high-throughput screen. UNC3230 lowered PIP2 levels in DRG neurons and attenuated hypersensitivity when administered intrathecally or into the hindpaw. Our studies reveal that PIP5K1C regulates PIP2-dependent nociceptive signaling and suggest that PIP5K1C is a therapeutic target for chronic pain.
Assuntos
Hiperalgesia/metabolismo , Limiar da Dor/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Potenciais Pós-Sinápticos Excitadores/genética , Gânglios Espinais/citologia , Hiperalgesia/induzido quimicamente , Hiperalgesia/tratamento farmacológico , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Medição da Dor/efeitos dos fármacos , Limiar da Dor/efeitos dos fármacos , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfopiruvato Hidratase/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Tempo de Reação/genética , Células Receptoras Sensoriais/fisiologia , Medula Espinal/citologiaRESUMO
Current antiretroviral treatments target multiple pathways important for human immunodeficiency virus (HIV) multiplication, including viral entry, synthesis and integration of the DNA provirus, and the processing of viral polyprotein precursors. However, HIV is becoming increasingly resistant to these "combination therapies." Recent findings show that inhibition of HIV Gag protein cleavage into its two structural proteins, matrix (MA) and capsid (CA), has a devastating effect on viral production, revealing a potential new target class for HIV treatment. Unlike the widely used HIV protease inhibitors, this new class of inhibitor would target the substrate, not the protease enzyme itself. This approach offers a distinct advantage in that inhibitors of MA/CA would only need to affect a subset of the Gag molecules to disable viral replication. To discover MA/CA-specific inhibitors, we constructed a modified MA/CA fusion peptide (MA/CAΔ) that contains the HIV protease (PR) cleavage site as well as a tetracysteine motif for fluorescent labeling. The HIV PR cleavage of MA/CAΔ can then be monitored via fluorescence polarization (FP). We have adapted this FP assay for high-throughput screening and validated it according to industry standards using a 384-well plate format. We have currently tested 24,000 compounds in this assay and here detail the screening methodology and the results of this screening campaign.
Assuntos
Fármacos Anti-HIV/farmacologia , Protease de HIV/metabolismo , Ensaios de Triagem em Larga Escala , Robótica , Produtos do Gene gag do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Motivos de Aminoácidos , Fármacos Anti-HIV/isolamento & purificação , Descoberta de Drogas/métodos , Polarização de Fluorescência , Corantes Fluorescentes/análise , Corantes Fluorescentes/química , Protease de HIV/genética , Humanos , Oligopeptídeos/antagonistas & inibidores , Oligopeptídeos/metabolismo , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Proteólise/efeitos dos fármacos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos Testes , Bibliotecas de Moléculas Pequenas , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismoRESUMO
The role of Mer kinase in regulating the second phase of platelet activation generates an opportunity to use Mer inhibitors for preventing thrombosis with diminished likelihood for bleeding as compared to current therapies. Toward this end, we have discovered a novel, Mer kinase specific substituted-pyrimidine scaffold using a structure-based drug design and a pseudo ring replacement strategy. The cocrystal structure of Mer with two compounds (7 and 22) possessing distinct activity have been determined. Subsequent SAR studies identified compound 23 (UNC2881) as a lead compound for in vivo evaluation. When applied to live cells, 23 inhibits steady-state Mer kinase phosphorylation with an IC50 value of 22 nM. Treatment with 23 is also sufficient to block EGF-mediated stimulation of a chimeric receptor containing the intracellular domain of Mer fused to the extracellular domain of EGFR. In addition, 23 potently inhibits collagen-induced platelet aggregation, suggesting that this class of inhibitors may have utility for prevention and/or treatment of pathologic thrombosis.
Assuntos
Cicloexanóis/síntese química , Fibrinolíticos/síntese química , Fibrinolíticos/uso terapêutico , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Pirimidinas/síntese química , Pirimidinas/uso terapêutico , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Trombose/tratamento farmacológico , Trombose/prevenção & controle , Cicloexanóis/uso terapêutico , Desenho de Fármacos , Humanos , Modelos Moleculares , Pirimidinas/química , Receptores Proteína Tirosina Quinases/metabolismo , Relação Estrutura-Atividade , c-Mer Tirosina QuinaseRESUMO
Abnormal activation or overexpression of Mer receptor tyrosine kinase has been implicated in survival signaling and chemoresistance in many human cancers. Consequently, Mer is a promising novel cancer therapeutic target. A structure-based drug design approach using a pseudo-ring replacement strategy was developed and validated to discover a new family of pyridinepyrimidine analogues as potent Mer inhibitors. Through SAR studies, 10 (UNC2250) was identified as the lead compound for further investigation based on high selectivity against other kinases and good pharmacokinetic properties. When applied to live cells, 10 inhibited steady-state phosphorylation of endogenous Mer with an IC50 of 9.8 nM and blocked ligand-stimulated activation of a chimeric EGFR-Mer protein. Treatment with 10 also resulted in decreased colony-forming potential in rhabdoid and NSCLC tumor cells, thereby demonstrating functional antitumor activity. The results provide a rationale for further investigation of this compound for therapeutic application in patients with cancer.
Assuntos
Antineoplásicos/síntese química , Cicloexanóis/síntese química , Inibidores de Proteínas Quinases/síntese química , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Piridinas/síntese química , Pirimidinas/síntese química , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Ciclização , Cicloexanóis/farmacologia , Desenho de Fármacos , Humanos , Ligação de Hidrogênio , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Relação Estrutura-Atividade , c-Mer Tirosina QuinaseRESUMO
Among epigenetic "writers", "readers", and "erasers", the lysine methyltransferases G9a and GLP, which catalyze mono- and dimethylation of histone H3 lysine 9 (H3K9me2) and nonhistone proteins, have been implicated in a variety of human diseases. A "toolkit" of well-characterized chemical probes will allow biological and disease hypotheses concerning these proteins to be tested in cell-based and animal models with high confidence. We previously discovered potent and selective G9a/GLP inhibitors including the cellular chemical probe UNC0638, which displays an excellent separation of functional potency and cell toxicity. However, this inhibitor is not suitable for animal studies due to its poor pharmacokinetic (PK) properties. Here, we report the discovery of the first G9a and GLP in vivo chemical probe UNC0642, which not only maintains high in vitro and cellular potency, low cell toxicity, and excellent selectivity, but also displays improved in vivo PK properties, making it suitable for animal studies.