RESUMO
The National Center for Advancing Translational Sciences (NCATS) Assay Guidance Manual (AGM) Workshop on 3D Tissue Models for Antiviral Drug Development, held virtually on 7-8 June 2022, provided comprehensive coverage of critical concepts intended to help scientists establish robust, reproducible, and scalable 3D tissue models to study viruses with pandemic potential. This workshop was organized by NCATS, the National Institute of Allergy and Infectious Diseases, and the Bill and Melinda Gates Foundation. During the workshop, scientific experts from academia, industry, and government provided an overview of 3D tissue models' utility and limitations, use of existing 3D tissue models for antiviral drug development, practical advice, best practices, and case studies about the application of available 3D tissue models to infectious disease modeling. This report includes a summary of each workshop session as well as a discussion of perspectives and challenges related to the use of 3D tissues in antiviral drug discovery.
Assuntos
Antivirais , Descoberta de Drogas , Antivirais/farmacologia , Antivirais/uso terapêutico , BioensaioRESUMO
The NIH Virtual SARS-CoV-2 Antiviral Summit, held on 6 November 2020, was organized to provide an overview on the status and challenges in developing antiviral therapeutics for coronavirus disease 2019 (COVID-19), including combinations of antivirals. Scientific experts from the public and private sectors convened virtually during a live videocast to discuss severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) targets for drug discovery as well as the preclinical tools needed to develop and evaluate effective small-molecule antivirals. The goals of the Summit were to review the current state of the science, identify unmet research needs, share insights and lessons learned from treating other infectious diseases, identify opportunities for public-private partnerships, and assist the research community in designing and developing antiviral therapeutics. This report includes an overview of therapeutic approaches, individual panel summaries, and a summary of the discussions and perspectives on the challenges ahead for antiviral development.
Assuntos
Antivirais/uso terapêutico , Tratamento Farmacológico da COVID-19 , SARS-CoV-2/efeitos dos fármacos , Antivirais/farmacologia , COVID-19/virologia , Desenvolvimento de Medicamentos , Humanos , National Institutes of Health (U.S.) , Peptídeo Hidrolases/metabolismo , Inibidores de Proteases/farmacologia , Inibidores de Proteases/uso terapêutico , Estados Unidos , Replicação Viral/efeitos dos fármacosRESUMO
Lactate dehydrogenase (LDH) is a critical enzyme in the glycolytic metabolism pathway that is used by many tumor cells. Inhibitors of LDH may be expected to inhibit the metabolic processes in cancer cells and thus selectively delay or inhibit growth in transformed versus normal cells. We have previously disclosed a pyrazole-based series of potent LDH inhibitors with long residence times on the enzyme. Here, we report the elaboration of a new subseries of LDH inhibitors based on those leads. These new compounds potently inhibit both LDHA and LDHB enzymes, and inhibit lactate production in cancer cell lines.
Assuntos
Compostos de Anilina/farmacologia , Antineoplásicos/farmacologia , Desenho de Fármacos , Éteres/farmacologia , L-Lactato Desidrogenase/antagonistas & inibidores , L-Lactato Desidrogenase/metabolismo , Compostos de Anilina/química , Antineoplásicos/química , Linhagem Celular Tumoral , Éteres/química , Humanos , L-Lactato Desidrogenase/químicaRESUMO
Multiple sclerosis involves an aberrant autoimmune response and progressive failure of remyelination in the central nervous system. Prevention of neural degeneration and subsequent disability requires remyelination through the generation of new oligodendrocytes, but current treatments exclusively target the immune system. Oligodendrocyte progenitor cells are stem cells in the central nervous system and the principal source of myelinating oligodendrocytes. These cells are abundant in demyelinated regions of patients with multiple sclerosis, yet fail to differentiate, thereby representing a cellular target for pharmacological intervention. To discover therapeutic compounds for enhancing myelination from endogenous oligodendrocyte progenitor cells, we screened a library of bioactive small molecules on mouse pluripotent epiblast stem-cell-derived oligodendrocyte progenitor cells. Here we show seven drugs function at nanomolar doses selectively to enhance the generation of mature oligodendrocytes from progenitor cells in vitro. Two drugs, miconazole and clobetasol, are effective in promoting precocious myelination in organotypic cerebellar slice cultures, and in vivo in early postnatal mouse pups. Systemic delivery of each of the two drugs significantly increases the number of new oligodendrocytes and enhances remyelination in a lysolecithin-induced mouse model of focal demyelination. Administering each of the two drugs at the peak of disease in an experimental autoimmune encephalomyelitis mouse model of chronic progressive multiple sclerosis results in striking reversal of disease severity. Immune response assays show that miconazole functions directly as a remyelinating drug with no effect on the immune system, whereas clobetasol is a potent immunosuppressant as well as a remyelinating agent. Mechanistic studies show that miconazole and clobetasol function in oligodendrocyte progenitor cells through mitogen-activated protein kinase and glucocorticoid receptor signalling, respectively. Furthermore, both drugs enhance the generation of human oligodendrocytes from human oligodendrocyte progenitor cells in vitro. Collectively, our results provide a rationale for testing miconazole and clobetasol, or structurally modified derivatives, to enhance remyelination in patients.
Assuntos
Clobetasol/farmacologia , Miconazol/farmacologia , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/metabolismo , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/metabolismo , Células-Tronco Pluripotentes/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Cerebelo/patologia , Doenças Desmielinizantes/tratamento farmacológico , Doenças Desmielinizantes/metabolismo , Doenças Desmielinizantes/patologia , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Feminino , Camadas Germinativas/efeitos dos fármacos , Camadas Germinativas/metabolismo , Camadas Germinativas/patologia , Humanos , Lisofosfatidilcolinas , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Esclerose Múltipla/patologia , Oligodendroglia/citologia , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/metabolismo , Fenótipo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Receptores de Glucocorticoides/metabolismo , Regeneração/efeitos dos fármacos , Técnicas de Cultura de TecidosRESUMO
The ATP-binding cassette transporter P-glycoprotein (P-gp) is known to limit both brain penetration and oral bioavailability of many chemotherapy drugs. Although US Food and Drug Administration guidelines require that potential interactions of investigational drugs with P-gp be explored, often this information does not enter the literature. In response, we developed a high-throughput screen to identify substrates of P-gp from a series of chemical libraries, testing a total of 10,804 compounds, most of which have known mechanisms of action. We used the CellTiter-Glo viability assay to test library compounds against parental KB-3-1 human cervical adenocarcinoma cells and the colchicine-selected subline KB-8-5-11 that overexpresses P-gp. KB-8-5-11 cells were also tested in the presence of a P-gp inhibitor (tariquidar) to assess reversibility of transporter-mediated resistance. Of the tested compounds, a total of 90 P-gp substrates were identified, including 55 newly identified compounds. Substrates were confirmed using an orthogonal killing assay against human embryonic kidney-293 cells overexpressing P-gp. We confirmed that AT7159 (cyclin-dependent kinase inhibitor), AT9283, (Janus kinase 2/3 inhibitor), ispinesib (kinesin spindle protein inhibitor), gedatolisib (PKI-587, phosphoinositide 3-kinase/mammalian target of rampamycin inhibitor), GSK-690693 (AKT inhibitor), and KW-2478 (heat-shock protein 90 inhibitor) were substrates. In addition, we assessed direct ATPase stimulation. ABCG2 was also found to confer high levels of resistance to AT9283, GSK-690693, and gedatolisib, whereas ispinesib, AT7519, and KW-2478 were weaker substrates. Combinations of P-gp substrates and inhibitors were assessed to demonstrate on-target synergistic cell killing. These data identified compounds whose oral bioavailability or brain penetration may be affected by P-gp. SIGNIFICANCE STATEMENT: The ATP-binding cassette transporter P-glycoprotein (P-gp) is known to be expressed at barrier sites, where it acts to limit oral bioavailability and brain penetration of substrates. In order to identify novel compounds that are transported by P-gp, we developed a high-throughput screen using the KB-3-1 cancer cell line and its colchicine-selected subline KB-8-5-11. We screened the Mechanism Interrogation Plate (MIPE) library, the National Center for Advancing Translational Science (NCATS) pharmaceutical collection (NPC), the NCATS Pharmacologically Active Chemical Toolbox (NPACT), and a kinase inhibitor library comprising 977 compounds, for a total of 10,804 compounds. Of the 10,804 compounds screened, a total of 90 substrates were identified of which 55 were novel. P-gp expression may adversely affect the oral bioavailability or brain penetration of these compounds.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Citotoxinas/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Proteínas de Neoplasias/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Citotoxinas/química , Citotoxinas/farmacologia , Relação Dose-Resposta a Droga , Células HEK293 , Células HeLa , Humanos , Especificidade por Substrato/efeitos dos fármacos , Especificidade por Substrato/fisiologiaRESUMO
The histone lysine methyltransferase nuclear receptor-binding SET domain protein 2 (NSD2, also known as WHSC1/MMSET) is an epigenetic modifier and is thought to play a driving role in oncogenesis. Both NSD2 overexpression and point mutations that increase its catalytic activity are associated with several human cancers. Although NSD2 is an attractive therapeutic target, no potent, selective, and bioactive small molecule inhibitors of NSD2 have been reported to date, possibly due to the challenges of developing high-throughput assays for NSD2. Here, to establish a platform for the discovery and development of selective NSD2 inhibitors, we optimized and implemented multiple assays. We performed quantitative high-throughput screening with full-length WT NSD2 and a nucleosome substrate against a diverse collection of bioactive small molecules comprising 16,251 compounds. We further interrogated 174 inhibitory compounds identified in the primary screen with orthogonal and counter assays and with activity assays based on the clinically relevant NSD2 variants E1099K and T1150A. We selected five confirmed inhibitors for follow-up, which included a radiolabeled validation assay, surface plasmon resonance studies, methyltransferase profiling, and histone methylation in cells. We found that all five NSD2 inhibitors bind the catalytic SET domain and one exhibited apparent activity in cells, validating the workflow and providing a template for identifying selective NSD2 inhibitors. In summary, we have established a robust discovery pipeline for identifying potent NSD2 inhibitors from small-molecule libraries.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Nucleossomos/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Linhagem Celular Tumoral , Inibidores Enzimáticos/química , Ensaios de Triagem em Larga Escala/métodos , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Nucleossomos/efeitos dos fármacos , Proteínas Repressoras/metabolismo , Bibliotecas de Moléculas Pequenas/químicaRESUMO
Serine is both a proteinogenic amino acid and the source of one-carbon units essential for de novo purine and deoxythymidine synthesis. In the canonical pathway of glucose-derived serine synthesis, Homo sapiens phosphoglycerate dehydrogenase (PHGDH) catalyzes the first, rate-limiting step. Genetic loss of PHGDH is toxic toward PHGDH-overexpressing breast cancer cell lines even in the presence of exogenous serine. Here, we used a quantitative high-throughput screen to identify small-molecule PHGDH inhibitors. These compounds reduce the production of glucose-derived serine in cells and suppress the growth of PHGDH-dependent cancer cells in culture and in orthotopic xenograft tumors. Surprisingly, PHGDH inhibition reduced the incorporation into nucleotides of one-carbon units from glucose-derived and exogenous serine. We conclude that glycolytic serine synthesis coordinates the use of one-carbon units from endogenous and exogenous serine in nucleotide synthesis, and we suggest that one-carbon unit wasting thus may contribute to the efficacy of PHGDH inhibitors in vitro and in vivo.
Assuntos
Carbono/metabolismo , Inibidores Enzimáticos/farmacologia , Fosfoglicerato Desidrogenase/antagonistas & inibidores , Serina/biossíntese , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Carbono/química , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Feminino , Glicólise/efeitos dos fármacos , Humanos , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/enzimologia , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Estrutura Molecular , Fosfoglicerato Desidrogenase/metabolismo , Purinas/biossíntese , Serina/química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Timidina/biossíntese , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Proliferating cells, including cancer cells, obtain serine both exogenously and via the metabolism of glucose. By catalyzing the first, rate-limiting step in the synthesis of serine from glucose, phosphoglycerate dehydrogenase (PHGDH) controls flux through the biosynthetic pathway for this important amino acid and represents a putative target in oncology. To discover inhibitors of PHGDH, a coupled biochemical assay was developed and optimized to enable high-throughput screening for inhibitors of human PHGDH. Feedback inhibition was minimized by coupling PHGDH activity to two downstream enzymes (PSAT1 and PSPH), providing a marked improvement in enzymatic turnover. Further coupling of NADH to a diaphorase/resazurin system enabled a red-shifted detection readout, minimizing interference due to compound autofluorescence. With this protocol, over 400,000 small molecules were screened for PHGDH inhibition, and following hit validation and triage work, a piperazine-1-thiourea was identified. Following rounds of medicinal chemistry and SAR exploration, two probes (NCT-502 and NCT-503) were identified. These molecules demonstrated improved target activity and encouraging ADME properties, enabling in vitro assessment of the biological importance of PHGDH, and its role in the fate of serine in PHGDH-dependent cancer cells. This manuscript reports the assay development and medicinal chemistry leading to the development of NCT-502 and -503 reported in Pacold et al. (2016).
Assuntos
Inibidores Enzimáticos/farmacologia , Fosfoglicerato Desidrogenase/antagonistas & inibidores , Piperazinas/farmacologia , Tioureia/análogos & derivados , Tioureia/farmacologia , Relação Dose-Resposta a Droga , Descoberta de Drogas , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Ensaios de Triagem em Larga Escala , Humanos , Estrutura Molecular , Fosfoglicerato Desidrogenase/genética , Fosfoglicerato Desidrogenase/metabolismo , Piperazinas/síntese química , Piperazinas/química , Relação Estrutura-Atividade , Tioureia/síntese química , Tioureia/químicaRESUMO
Hutchinson-Gilford Progeria Syndrome (HGPS) is an early onset lethal premature aging disorder caused by constitutive production of progerin, a mutant form of the nuclear architectural protein lamin A. The presence of progerin causes extensive morphological, epigenetic and DNA damage related nuclear defects that ultimately disrupt tissue and organismal functions. Hypothesis-driven approaches focused on HGPS affected pathways have been used in attempts to identify druggable targets with anti-progeroid effects. Here, we report an unbiased discovery approach to HGPS by implementation of a high-throughput, high-content imaging based screening method that enables systematic identification of small molecules that prevent the formation of multiple progerin-induced aging defects. Screening a library of 2816 FDA approved drugs, we identified retinoids as a novel class of compounds that reverses aging defects in HGPS patient skin fibroblasts. These findings establish a novel approach to anti-progeroid drug discovery.
Assuntos
Senescência Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Lamina Tipo A/antagonistas & inibidores , Imagem Molecular/métodos , Retinoides/farmacologia , Linhagem Celular Transformada , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Senescência Celular/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Epigênese Genética , Fibroblastos/metabolismo , Fibroblastos/patologia , Histonas/genética , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Lamina Tipo B/genética , Lamina Tipo B/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Cultura Primária de Células , Progéria/genética , Progéria/metabolismo , Progéria/patologia , Bibliotecas de Moléculas Pequenas/farmacologia , Transfecção , Proteína 1 de Ligação à Proteína Supressora de Tumor p53RESUMO
Multidrug resistance (MDR) is a major obstacle to the successful chemotherapy of cancer. MDR is often the result of overexpression of ATP-binding cassette transporters following chemotherapy. A common ATP-binding cassette transporter that is overexpressed in MDR cancer cells is P-glycoprotein, which actively effluxes drugs against a concentration gradient, producing an MDR phenotype. Collateral sensitivity (CS), a phenomenon of drug hypersensitivity, is defined as the ability of certain compounds to selectively target MDR cells, but not the drug-sensitive parent cells from which they were derived. The drug tiopronin has been previously shown to elicit CS. However, unlike other CS agents, the mechanism of action was not dependent on the expression of P-glycoprotein in MDR cells. We have determined that the CS activity of tiopronin is mediated by the generation of reactive oxygen species (ROS) and that CS can be reversed by a variety of ROS-scavenging compounds. Specifically, selective toxicity of tiopronin toward MDR cells is achieved by inhibition of glutathione peroxidase (GPx), and the mode of inhibition of GPx1 by tiopronin is shown in this report. Why MDR cells are particularly sensitive to ROS is discussed, as is the difficulty in exploiting this hypersensitivity to tiopronin in the clinic.
Assuntos
Inibidores Enzimáticos/farmacologia , Glutationa Peroxidase/antagonistas & inibidores , Tiopronina/farmacologia , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular Tumoral , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Glutationa Peroxidase/química , Humanos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Espécies Reativas de Oxigênio/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem , Tiomalatos/farmacologiaRESUMO
Classic Galactosemia is a rare inborn error of metabolism that is caused by deficiency of galactose-1-phosphate uridyltransferase (GALT), an enzyme within the Leloir pathway that is responsible for the conversion of galactose-1-phosphate (gal-1-p) and UDP-glucose to glucose-1-phosphate and UDP-galactose. This deficiency results in elevated intracellular concentrations of its substrate, gal-1-p, and this increased concentration is believed to be the major pathogenic mechanism in Classic Galactosemia. Galactokinase (GALK) is an upstream enzyme of GALT in the Leloir pathway and is responsible for conversion of galactose and ATP to gal-1-p and ADP. Therefore, it was hypothesized that the identification of a small-molecule inhibitor of human GALK would act to prevent the accumulation of gal-1-p and offer a novel entry therapy for this disorder. Herein we describe a quantitative high-throughput screening campaign that identified a single chemotype that was optimized and validated as a GALK inhibitor.
Assuntos
Galactoquinase/antagonistas & inibidores , Animais , Benzoxazóis/síntese química , Benzoxazóis/química , Benzoxazóis/metabolismo , Cristalografia por Raios X , Galactoquinase/genética , Galactoquinase/metabolismo , Galactosefosfatos/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Cinética , Camundongos , Microssomos Hepáticos/metabolismo , Conformação Molecular , Ligação Proteica , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Compostos de Espiro/química , Relação Estrutura-AtividadeRESUMO
Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. The interaction of PKM2 with phosphotyrosine-containing proteins inhibits enzyme activity and increases the availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small-molecule PKM2 activators inhibits the growth of xenograft tumors. Structural studies reveal that small-molecule activators bind PKM2 at the subunit interaction interface, a site that is distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. These data support the notion that small-molecule activation of PKM2 can interfere with anabolic metabolism.
Assuntos
Biopolímeros/metabolismo , Transformação Celular Neoplásica , Ativadores de Enzimas/farmacologia , Piruvato Quinase/metabolismo , Animais , Biopolímeros/química , Western Blotting , Proliferação de Células , Humanos , Camundongos , Neoplasias/enzimologia , Neoplasias/metabolismo , Neoplasias/patologia , Piruvato Quinase/químicaRESUMO
The structure-activity relationship (SAR) study of two chemotypes identified as inhibitors of the human NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (HPGD, 15-PGDH) was conducted. Top compounds from both series displayed potent inhibition (IC50 <50 nM), demonstrate excellent selectivity towards HPGD and potently induce PGE2 production in A549 lung cancer and LNCaP prostate cancer cells.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Hidroxiprostaglandina Desidrogenases/antagonistas & inibidores , NAD/antagonistas & inibidores , Linhagem Celular Tumoral , Humanos , Hidroxiprostaglandina Desidrogenases/metabolismo , NAD/metabolismo , Relação Estrutura-AtividadeRESUMO
The radiotracer [(11)C]N-desmethyl-loperamide (dLop) images the in vivo function of P-glycoprotein (P-gp), a transporter that blocks the entry of drugs that are substrates into brain. When P-gp is inhibited, [(11)C]dLop, a potent opiate agonist, enters and becomes trapped in the brain. This trapping is beneficial from an imaging perspective, because it amplifies the PET signal, essentially by accumulating radioactivity over time. As we previously demonstrated that this trapping was not caused by binding to opiate receptors, we examined whether [(11)C]dLop, a weak base, is ionically trapped in acidic lysosomes. To test this hypothesis, we measured [(3)H]dLop accumulation in human cells by using lysosomotropics. Because the in vivo trapping of dLop was seen after P-gp inhibition, we also measured [(3)H]dLop uptake in P-gp-expressing cells treated with the P-gp inhibitor tariquidar. All lysosomotropics decreased [(3)H]dLop accumulation by at least 50%. In P-gp-expressing cells, tariquidar (and another P-gp inhibitor) surprisingly decreased [(3)H]dLop uptake. Consequently, we measured [(11)C]dLop uptake before and after tariquidar preadministration in lysosome-rich organs of P-gp KO mice and humans. After tariquidar pretreatment in both species, radioactivity uptake in these organs decreased by 35% to 40%. Our results indicate that dLop is trapped in lysosomes and that tariquidar competes with dLop for lysosomal accumulation in vitro and in vivo. Although tariquidar and dLop compete for lysosomal trapping in the periphery, such competition does not occur in brain because tariquidar has negligible entry into brain. In summary, tariquidar and [(11)C]dLop can be used in combination to selectively measure the function of P-gp at the blood-brain barrier.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Barreira Hematoencefálica , Loperamida/análogos & derivados , Lisossomos/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Trítio/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Animais , Barreira Hematoencefálica/diagnóstico por imagem , Barreira Hematoencefálica/metabolismo , Linhagem Celular Tumoral , Humanos , Marcação por Isótopo/métodos , Loperamida/farmacologia , Camundongos , Quinolinas/farmacologia , RadiografiaRESUMO
Compared to normal differentiated cells, cancer cells have altered metabolic regulation to support biosynthesis and the expression of the M2 isozyme of pyruvate kinase (PKM2) plays an important role in this anabolic metabolism. While the M1 isoform is a highly active enzyme, the alternatively spliced M2 variant is considerably less active and expressed in tumors. While the exact mechanism by which decreased pyruvate kinase activity contributes to anabolic metabolism remains unclear, it is hypothesized that activation of PKM2 to levels seen with PKM1 may promote a metabolic program that is not conducive to cell proliferation. Here we report the third chemotype in a series of PKM2 activators based on the 2-oxo-N-aryl-1,2,3,4-tetrahydroquinoline-6-sulfonamide scaffold. The synthesis, structure activity relationships, selectivity and notable physiochemical properties are described.
Assuntos
Ativadores de Enzimas/farmacologia , Isoenzimas/metabolismo , Neoplasias/enzimologia , Piruvato Quinase/metabolismo , Quinolinas/farmacologia , Processamento Alternativo , Células CACO-2 , Humanos , Neoplasias/patologiaRESUMO
Neomorphic mutations in isocitrate dehydrogenase 1 (IDH1) are oncogenic for a number of malignancies, primarily low-grade gliomas and acute myeloid leukemia. We report a medicinal chemistry campaign around a 7,7-dimethyl-7,8-dihydro-2H-1λ2-quinoline-2,5(6H)-dione screening hit against the R132H and R132C mutant forms of isocitrate dehydrogenase (IDH1). Systematic SAR efforts produced a series of potent pyrid-2-one mIDH1 inhibitors, including the atropisomer (+)-119 (NCATS-SM5637, NSC 791985). In an engineered mIDH1-U87-xenograft mouse model, after a single oral dose of 30 mg/kg, 16 h post dose, between 16 and 48 h, (+)-119 showed higher tumoral concentrations that corresponded to lower 2-HG concentrations, when compared with the approved drug AG-120 (ivosidenib).
Assuntos
Inibidores Enzimáticos/química , Isocitrato Desidrogenase/antagonistas & inibidores , Piridonas/química , Animais , Encéfalo/metabolismo , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/uso terapêutico , Feminino , Glicina/análogos & derivados , Glicina/uso terapêutico , Meia-Vida , Humanos , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Camundongos , Camundongos Nus , Microssomos Hepáticos/metabolismo , Mutagênese Sítio-Dirigida , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Piridinas/uso terapêutico , Piridonas/metabolismo , Piridonas/uso terapêutico , Ratos , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
[(11)C]N-desmethyl-Loperamide ([(11)C]dLop) is used in positron emission tomography (PET) to measure the in vivo activity of efflux transporters that block the passage of drugs across the blood-brain barrier. The three most prevalent ATP-binding cassette efflux transporters at the blood-brain barrier are P-glycoprotein (P-gp), multidrug resistance protein 1 (Mrp1), and breast cancer resistance protein (BCRP). We sought to measure the selectivity of dLop among these three transporters. The selectivity of dLop at low concentrations (< or =1 nM) was measured both as the accumulation of [(3)H]dLop in human cells that overexpress each transporter and as the uptake of [(11)C]dLop in brains of mice that lack genes encoding P-gp, Mrp1, or BCRP. The selectivity of dLop at high concentrations (> or =20 microM) was measured as the inhibition of uptake of a fluorescent substrate and the change in cytotoxicity of drugs effluxed at each transporter. Accumulation of [(3)H]dLop was lowest in cells overexpressing P-gp, and the uptake of [(11)C]dLop was highest in brains of mice lacking P-gp. At high concentrations, dLop selectively inhibited P-gp function and also decreased the resistance of only the P-gp-expressing cells to cytotoxic agents. dLop is selective for P-gp among these three transporters, but its activity is dependent on concentration. At low concentrations (< or =1 nM), dLop acts only as a substrate; at high concentrations (> or =20 microM), it acts as both a substrate and an inhibitor (i.e., a competitive substrate). Because low concentrations of radiotracer are used for PET imaging, [(11)C]dLop acts selectively and only as a substrate for P-gp.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Transporte Biológico/fisiologia , Barreira Hematoencefálica/metabolismo , Glicoproteínas/metabolismo , Loperamida/análogos & derivados , Loperamida/farmacocinética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Animais , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacocinética , Encéfalo/metabolismo , Circulação Cerebrovascular , Humanos , Loperamida/química , Loperamida/farmacologia , Camundongos , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Compostos Radiofarmacêuticos/farmacocinética , Distribuição Tecidual/fisiologia , Células Tumorais CultivadasRESUMO
The development of the valves and septa of the heart depends on the formation and remodeling of endocardial cushions. Here, we report that the alternative splicing regulator muscleblind-like 1 (MBNL1) exhibits a regionally restricted pattern of expression in canal region endocardium and ventricular myocardium during endocardial cushion development in chicken. Knockdown of MBNL1 in atrioventricular explants leads to a transforming growth factor beta-dependent increase in epithelial-mesenchymal transition (EMT) of endocardial cells. This reveals a novel role for MBNL1 during embryonic development, and represents the first evidence that an alternative splicing regulator is a key player in endocardial cushion development.