RESUMO
Mitochondrial uridine insertion/deletion RNA editing, catalyzed by a multiprotein complex (editosome), is essential for gene expression in trypanosomes and Leishmania parasites. As this process is absent in the human host, a drug targeting this mechanism promises high selectivity and reduced toxicity. Here, we successfully miniaturized our FRET-based full-round RNA editing assay, which replicates the complete RNA editing process, adapting it into a 1536-well format. Leveraging this assay, we screened over 100,000 compounds against purified editosomes derived from Trypanosoma brucei, identifying seven confirmed primary hits. We sourced and evaluated various analogs to enhance the inhibitory and parasiticidal effects of these primary hits. In combination with secondary assays, our compounds marked inhibition of essential catalytic activities, including the RNA editing ligase and interactions of editosome proteins. Although the primary hits did not exhibit any growth inhibitory effect on parasites, we describe eight analog compounds capable of effectively killing T. brucei and/or Leishmania donovani parasites within a low micromolar concentration. Whether parasite killing is - at least in part - due to inhibition of RNA editing in vivo remains to be assessed. Our findings introduce novel molecular scaffolds with the potential for broad antitrypanosomal effects.
Assuntos
Trypanosoma brucei brucei , Humanos , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Ensaios de Triagem em Larga Escala , Edição de RNA , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA/metabolismoRESUMO
In Plasmodium, the first two and rate-limiting enzymes of the pentose phosphate pathway, glucose 6-phosphate dehydrogenase (G6PD) and the 6-phosphogluconolactonase, are bifunctionally fused to a unique enzyme named GluPho, differing structurally and mechanistically from the respective human orthologs. Consistent with the enzyme's essentiality for malaria parasite proliferation and propagation, human G6PD deficiency has immense impact on protection against severe malaria, making PfGluPho an attractive antimalarial drug target. Herein we report on the optimized lead compound N-(((2R,4S)-1-cyclobutyl-4-hydroxypyrrolidin-2-yl)methyl)-6-fluoro-4-methyl-11-oxo-10,11-dihydrodibenzo[b,f][1,4]thiazepine-8-carboxamide (SBI-0797750), a potent and fully selective PfGluPho inhibitor with robust nanomolar activity against recombinant PfGluPho, PvG6PD, and P. falciparum blood-stage parasites. Mode-of-action studies have confirmed that SBI-0797750 disturbs the cytosolic glutathione-dependent redox potential, as well as the cytosolic and mitochondrial H2O2 homeostasis of P. falciparum blood stages, at low nanomolar concentrations. Moreover, SBI-0797750 does not harm red blood cell (RBC) integrity and phagocytosis and thus does not promote anemia. SBI-0797750 is therefore a very promising antimalarial lead compound.
Assuntos
Antimaláricos , Deficiência de Glucosefosfato Desidrogenase , Malária Falciparum , Malária Vivax , Malária , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Hidrolases de Éster Carboxílico , Glucose/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Malária Falciparum/tratamento farmacológico , Malária Vivax/tratamento farmacológico , Fosfatos , Plasmodium falciparum/metabolismo , Plasmodium vivaxRESUMO
Obesity-associated insulin resistance plays a central role in the pathogenesis of type 2 diabetes. A promising approach to decrease insulin resistance in obesity is to inhibit the protein tyrosine phosphatases that negatively regulate insulin receptor signaling. The low-molecular-weight protein tyrosine phosphatase (LMPTP) acts as a critical promoter of insulin resistance in obesity by inhibiting phosphorylation of the liver insulin receptor activation motif. Here, we report development of a novel purine-based chemical series of LMPTP inhibitors. These compounds inhibit LMPTP with an uncompetitive mechanism and are highly selective for LMPTP over other protein tyrosine phosphatases. We also report the generation of a highly orally bioavailable purine-based analogue that reverses obesity-induced diabetes in mice.
Assuntos
Inibidores Enzimáticos/química , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Purinas/química , Administração Oral , Animais , Sítios de Ligação , Cristalografia por Raios X , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/etiologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Meia-Vida , Humanos , Resistência à Insulina , Cinética , Simulação de Dinâmica Molecular , Obesidade/complicações , Obesidade/patologia , Fosforilação/efeitos dos fármacos , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Purinas/metabolismo , Purinas/farmacologia , Purinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
PURPOSE: TNF-related apoptosis inducing ligand (TRAIL) expression by immune cells contributes to antitumor immunity. A naturally occurring splice variant of TRAIL, called TRAILshort, antagonizes TRAIL-dependent cell killing. It is unknown whether tumor cells express TRAILshort and if it impacts antitumor immunity. EXPERIMENTAL DESIGN: We used an unbiased informatics approach to identify TRAILshort expression in primary human cancers, and validated those results with IHC and ISH. TRAILshort-specific mAbs were used to determine the effect of TRAILshort on tumor cell sensitivity to TRAIL, and to immune effector cell dependent killing of autologous primary tumors. RESULTS: As many as 40% of primary human tumors express TRAILshort by both RNA sequencing and IHC analysis. By ISH, TRAILshort expression is present in tumor cells and not bystander cells. TRAILshort inhibition enhances cancer cell lines sensitivity to TRAIL-dependent killing both in vitro and in immunodeficient xenograft mouse models. Immune effector cells isolated from patients with B-cell malignancies killed more autologous tumor cells in the presence compared with the absence of TRAILshort antibody (P < 0.05). CONCLUSIONS: These results identify TRAILshort in primary human malignancies, and suggest that TRAILshort blockade can augment the effector function of autologous immune effector cells.See related commentary by de Miguel and Pardo, p. 5546.
Assuntos
Imunidade Inata/genética , Neoplasias/imunologia , Isoformas de Proteínas/genética , Ligante Indutor de Apoptose Relacionado a TNF/genética , Animais , Morte Celular/genética , Morte Celular/imunologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/genética , Xenoenxertos , Humanos , Camundongos , Neoplasias/genética , Neoplasias/patologia , RNA-Seq , Ligante Indutor de Apoptose Relacionado a TNF/imunologiaRESUMO
Neurotensin receptor 1 (NTR1) is a G protein coupled receptor that is widely expressed throughout the central nervous system where it acts as a neuromodulator. Neurotensin receptors have been implicated in a wide variety of CNS disorders, but despite extensive efforts to develop small molecule ligands there are few reports of such compounds. Herein we describe the optimization of a quinazoline based lead to give 18 (SBI-553), a potent and brain penetrant NTR1 allosteric modulator.
Assuntos
Doenças do Sistema Nervoso Central/tratamento farmacológico , Descoberta de Drogas , Quinazolinas/farmacologia , Receptores de Neurotensina/antagonistas & inibidores , beta-Arrestinas/farmacologia , Administração Oral , Regulação Alostérica/efeitos dos fármacos , Animais , Disponibilidade Biológica , Doenças do Sistema Nervoso Central/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/deficiência , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Relação Dose-Resposta a Droga , Feminino , Locomoção/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estrutura Molecular , Quinazolinas/administração & dosagem , Quinazolinas/química , Ratos , Receptores de Neurotensina/metabolismo , Relação Estrutura-Atividade , beta-Arrestinas/administração & dosagem , beta-Arrestinas/químicaRESUMO
The Office of Translation to Practice (OTP) is housed in the Center for Clinical and Translational Sciences at Mayo Clinic. Established in 2015, the office was tasked with developing and managing novel tools, mechanisms, and processes to facilitate and accelerate the translation of products, such as drugs, biological agents, and medical devices, into practice. Since its inception, the OTP is credited with creating valuable services through several strategic alliances and active scientific and project management involvement. The OTP continues to move forward to assist Mayo Clinic physicians and scientists to interact effectively with internal and external collaborators to advance translational projects that will benefit patients. Best practices, innovations, and nascent successes of the OTP are presented and discussed herein.
Assuntos
Centros Médicos Acadêmicos/organização & administração , Eficiência Organizacional , Pesquisa Translacional Biomédica , Administradores Hospitalares , Humanos , Inovação Organizacional , Garantia da Qualidade dos Cuidados de SaúdeRESUMO
Inhibition of ubiquitin ligases with small molecule remains a very challenging task, given the lack of catalytic activity of the target and the requirement of disruption of its interactions with other proteins. Siah1/2, which are E3 ubiquitin ligases, are implicated in melanoma and prostate cancer and represent high-value drug targets. We utilized three independent screening approaches in our efforts to identify small-molecule Siah1/2 inhibitors: Affinity Selection-Mass Spectrometry, a protein thermal shift-based assay and an in silico based screen. Inhibitors were assessed for their effect on viability of melanoma and prostate cancer cultures, colony formation, prolyl-hydroxylase-HIF1α signaling, expression of selected Siah2-related transcripts, and Siah2 ubiquitin ligase activity. Several analogs were further characterized, demonstrating improved efficacy. Combination of the top hits identified in the different assays demonstrated an additive effect, pointing to complementing mechanisms that underlie each of these Siah1/2 inhibitors.
Assuntos
Melanoma/tratamento farmacológico , Proteínas Nucleares/antagonistas & inibidores , Neoplasias da Próstata/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/administração & dosagem , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Simulação por Computador , Regulação para Baixo , Ensaios de Seleção de Medicamentos Antitumorais , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Espectrometria de Massas , Melanoma/genética , Camundongos , Proteínas Nucleares/genética , Neoplasias da Próstata/genética , Bibliotecas de Moléculas Pequenas/isolamento & purificação , Bibliotecas de Moléculas Pequenas/farmacologia , Ubiquitina-Proteína Ligases/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Ubiquitin-like (Ubl) post-translational modifications are potential targets for therapeutics. However, the only known mechanism for inhibiting a Ubl-activating enzyme is through targeting its ATP-binding site. Here we identify an allosteric inhibitory site in the small ubiquitin-like modifier (SUMO)-activating enzyme (E1). This site was unexpected because both it and analogous sites are deeply buried in all previously solved structures of E1s of ubiquitin-like modifiers (Ubl). The inhibitor not only suppresses SUMO E1 activity, but also enhances its degradation in vivo, presumably due to a conformational change induced by the compound. In addition, the lead compound increased the expression of miR-34b and reduced c-Myc levels in lymphoma and colorectal cancer cell lines and a colorectal cancer xenograft mouse model. Identification of this first-in-class inhibitor of SUMO E1 is a major advance in modulating Ubl modifications for therapeutic aims.
Assuntos
Sumoilação , Enzimas Ativadoras de Ubiquitina/antagonistas & inibidores , Regulação Alostérica , Sítio Alostérico , Animais , Linhagem Celular Tumoral , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Camundongos SCID , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Sumoilação/efeitos dos fármacos , Transplante Heterólogo , Ubiquitina/metabolismo , Enzimas Ativadoras de Ubiquitina/metabolismo , Ubiquitinação/efeitos dos fármacosRESUMO
The Assay Guidance Manual (AGM) is an eBook of best practices for the design, development, and implementation of robust assays for early drug discovery. Initiated by pharmaceutical company scientists, the manual provides guidance for designing a "testing funnel" of assays to identify genuine hits using high-throughput screening (HTS) and advancing them through preclinical development. Combined with a workshop/tutorial component, the overall goal of the AGM is to provide a valuable resource for training translational scientists.
Assuntos
Bioensaio/métodos , Descoberta de Drogas , Geografia , Ensaios de Triagem em Larga Escala , Humanos , Pesquisa Translacional BiomédicaRESUMO
TNF-related apoptosis-inducing ligand (TRAIL) was initially described to induce apoptosis of tumor cells and/or virally infected cells, although sparing normal cells, and has been implicated in the pathogenesis of HIV disease. We previously identified TRAILshort, a TRAIL splice variant, in HIV-infected patients and characterized it as being a dominant negative ligand to subvert TRAIL-mediated killing. Herein, using single-cell genomics we demonstrate that TRAILshort is produced by HIV-infected cells, as well as by uninfected bystander cells, and that the dominant stimulus which induces TRAILshort production are type I IFNs and TLR7, TLR8, and TLR9 agonists. TRAILshort has a short t1/2 by virtue of containing a PEST domain, which targets the protein toward the ubiquitin proteasome pathway for degradation. Further we show that TRAILshort binds preferentially to TRAIL receptors 1 and 2 with significantly reduced interaction with the decoy TRAIL receptors 3 and 4. Recombinant TRAILshort is sufficient to protect cells against TRAIL-induced killing, whereas immunodepletion of TRAILshort with a specific Ab restores TRAIL sensitivity. Importantly we show that TRAILshort is shed in microvesicles into the cellular microenvironment and therefore confers TRAIL resistance not only on the cell which produces it, but also upon neighboring bystander cells. These results establish a novel paradigm for understanding and overcoming TRAIL resistance, in particular how HIV-infected cells escape immune elimination by the TRAIL:TRAILshort receptor axis.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Microambiente Celular/imunologia , Infecções por HIV/imunologia , Isoformas de Proteínas/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/genética , Processamento Alternativo/genética , Apoptose , Efeito Espectador/imunologia , Linfócitos T CD4-Positivos/virologia , Linhagem Celular Tumoral , Membrana Celular/imunologia , Células HEK293 , Infecções por HIV/patologia , Infecções por HIV/virologia , Células HeLa , Humanos , Células Jurkat , Isoformas de Proteínas/biossíntese , Ligante Indutor de Apoptose Relacionado a TNF/biossínteseRESUMO
Hepatic cystogenesis in polycystic liver disease is associated with increased levels of cyclic adenosine monophosphate (cAMP) in cholangiocytes lining liver cysts. Takeda G protein receptor 5 (TGR5), a G protein-coupled bile acid receptor, is linked to cAMP and expressed in cholangiocytes. Therefore, we hypothesized that TGR5 might contribute to disease progression. We examined expression of TGR5 and Gα proteins in cultured cholangiocytes and in livers of animal models and humans with polycystic liver disease. In vitro, we assessed cholangiocyte proliferation, cAMP levels, and cyst growth in response to (1) TGR5 agonists (taurolithocholic acid, oleanolic acid [OA], and two synthetic compounds), (2) a novel TGR5 antagonist (m-tolyl 5-chloro-2-[ethylsulfonyl] pyrimidine-4-carboxylate [SBI-115]), and (3) a combination of SBI-115 and pasireotide, a somatostatin receptor analogue. In vivo, we examined hepatic cystogenesis in OA-treated polycystic kidney rats and after genetic elimination of TGR5 in double mutant TGR5-/- ;Pkhd1del2/del2 mice. Compared to control, expression of TGR5 and Gαs (but not Gαi and Gαq ) proteins was increased 2-fold to 3-fold in cystic cholangiocytes in vitro and in vivo. In vitro, TGR5 stimulation enhanced cAMP production, cell proliferation, and cyst growth by â¼40%; these effects were abolished after TGR5 reduction by short hairpin RNA. OA increased cystogenesis in polycystic kidney rats by 35%; in contrast, hepatic cystic areas were decreased by 45% in TGR5-deficient TGR5-/- ;Pkhd1del2/del2 mice. TGR5 expression and its colocalization with Gαs were increased â¼2-fold upon OA treatment. Levels of cAMP, cell proliferation, and cyst growth in vitro were decreased by â¼30% in cystic cholangiocytes after treatment with SBI-115 alone and by â¼50% when SBI-115 was combined with pasireotide. CONCLUSION: TGR5 contributes to hepatic cystogenesis by increasing cAMP and enhancing cholangiocyte proliferation; our data suggest that a TGR5 antagonist alone or concurrently with somatostatin receptor agonists represents a potential therapeutic approach in polycystic liver disease. (Hepatology 2017;66:1197-1218).
Assuntos
AMP Cíclico/metabolismo , Cistos/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Hepatopatias/metabolismo , Pirimidinas/uso terapêutico , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proliferação de Células/efeitos dos fármacos , Cistos/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos , Quimioterapia Combinada , Humanos , Hepatopatias/tratamento farmacológico , Camundongos , Ácido Oleanólico , Doenças Renais Policísticas/metabolismo , Cultura Primária de Células , Pirimidinas/farmacologia , Ratos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/genética , Somatostatina/análogos & derivados , Somatostatina/farmacologia , Somatostatina/uso terapêuticoRESUMO
The proteasome is a vital cellular machine that maintains protein homeostasis, which is of particular importance in multiple myeloma and possibly other cancers. Targeting of proteasome 20S peptidase activity with bortezomib and carfilzomib has been widely used to treat myeloma. However, not all patients respond to these compounds, and those who do eventually suffer relapse. Therefore, there is an urgent and unmet need to develop new drugs that target proteostasis through different mechanisms. We identified quinoline-8-thiol (8TQ) as a first-in-class inhibitor of the proteasome 19S subunit Rpn11. A derivative of 8TQ, capzimin, shows >5-fold selectivity for Rpn11 over the related JAMM proteases and >2 logs selectivity over several other metalloenzymes. Capzimin stabilized proteasome substrates, induced an unfolded protein response, and blocked proliferation of cancer cells, including those resistant to bortezomib. Proteomic analysis revealed that capzimin stabilized a subset of polyubiquitinated substrates. Identification of capzimin offers an alternative path to develop proteasome inhibitors for cancer therapy.
Assuntos
Inibidores de Proteassoma/farmacologia , Quinolinas/farmacologia , Transativadores/antagonistas & inibidores , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/química , Quinolinas/química , Relação Estrutura-Atividade , Transativadores/metabolismoRESUMO
Obesity-associated insulin resistance plays a central role in type 2 diabetes. As such, tyrosine phosphatases that dephosphorylate the insulin receptor (IR) are potential therapeutic targets. The low-molecular-weight protein tyrosine phosphatase (LMPTP) is a proposed IR phosphatase, yet its role in insulin signaling in vivo has not been defined. Here we show that global and liver-specific LMPTP deletion protects mice from high-fat diet-induced diabetes without affecting body weight. To examine the role of the catalytic activity of LMPTP, we developed a small-molecule inhibitor with a novel uncompetitive mechanism, a unique binding site at the opening of the catalytic pocket, and an exquisite selectivity over other phosphatases. This inhibitor is orally bioavailable, and it increases liver IR phosphorylation in vivo and reverses high-fat diet-induced diabetes. Our findings suggest that LMPTP is a key promoter of insulin resistance and that LMPTP inhibitors would be beneficial for treating type 2 diabetes.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Tirosina Fosfatases/genética , Bibliotecas de Moléculas Pequenas , Animais , Sítios de Ligação , Diabetes Mellitus Tipo 2/enzimologia , Diabetes Mellitus Tipo 2/genética , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática/efeitos dos fármacos , Deleção de Genes , Concentração Inibidora 50 , Camundongos , Camundongos Knockout , Camundongos Obesos , Modelos Biológicos , Estrutura Molecular , Peso Molecular , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-AtividadeRESUMO
Pharmacological treatment for methamphetamine addiction will provide important societal benefits. Neurotensin receptor NTR1 and dopamine receptor distributions coincide in brain areas regulating methamphetamine-associated reward, and neurotensin peptides produce behaviors opposing psychostimulants. Therefore, undesirable methamphetamine-associated activities should be treatable with druggable NTR1 agonists, but no such FDA-approved therapeutics exist. We address this limitation with proof-of-concept data for ML314, a small-molecule, brain penetrant, ß-arrestin biased, NTR1 agonist. ML314 attenuates amphetamine-like hyperlocomotion in dopamine transporter knockout mice, and in C57BL/6J mice it attenuates methamphetamine-induced hyperlocomotion, potentiates the psychostimulant inhibitory effects of a ghrelin antagonist, and reduces methamphetamine-associated conditioned place preference. In rats, ML314 blocks methamphetamine self-administration. ML314 acts as an allosteric enhancer of endogenous neurotensin, unmasking stoichiometric numbers of hidden NTR1 binding sites in transfected-cell membranes or mouse striatal membranes, while additionally supporting NTR1 endocytosis in cells in the absence of NT peptide. These results indicate ML314 is a viable, preclinical lead for methamphetamine abuse treatment and support an allosteric model of G protein-coupled receptor signaling.
Assuntos
Transtornos Relacionados ao Uso de Anfetaminas/metabolismo , Metanfetamina/efeitos adversos , Piperazinas/metabolismo , Quinazolinas/metabolismo , Receptores de Neurotensina/metabolismo , Regulação Alostérica , Animais , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Ligantes , Locomoção/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Cell surface p32, the target of LyP-1 homing peptide, is upregulated in tumors and atherosclerotic plaques and has been widely used as a receptor for systemic delivery of payloads. Here, we identified an improved LyP-1-mimicking peptide (TT1, CKRGARSTC). We used this peptide in a fluorescence polarization-based high-throughput screening of a 50,000-compound chemical library and identified a panel of compounds that bind p32 with low micromolar affinity. Among the hits identified in the screen, two compounds were shown to specifically bind to p32 in multiple assays. One of these compounds was chosen for an in vivo study. Nanoparticles surface-functionalized with this compound specifically adhered to surfaces coated with recombinant p32 and, when injected intravenously, homed to p32-expressing breast tumors in mice. This compound provides a lead for the development of p32-targeted affinity ligands that circumvent some of the limitations of peptide-based probes in guided drug delivery.
Assuntos
Aminopiridinas/química , Neoplasias da Mama/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Etilenodiaminas/química , Proteínas Mitocondriais/administração & dosagem , Peptídeos Cíclicos/administração & dosagem , Aminopiridinas/farmacologia , Animais , Antineoplásicos/administração & dosagem , Proteínas de Transporte , Linhagem Celular Tumoral , Etilenodiaminas/farmacologia , Feminino , Humanos , Ligantes , Camundongos , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Nanopartículas/químicaRESUMO
Persistent opening of the mitochondrial permeability transition pore (PTP), an inner membrane channel, leads to mitochondrial dysfunction and renders the PTP a therapeutic target for a host of life-threatening diseases. Herein, we report our effort toward identifying small-molecule inhibitors of this target through structure-activity relationship optimization studies, which led to the identification of several potent analogues around the N-phenylbenzamide compound series identified by high-throughput screening. In particular, compound 4 (3-(benzyloxy)-5-chloro-N-(4-(piperidin-1-ylmethyl)phenyl)benzamide) displayed noteworthy inhibitory activity in the mitochondrial swelling assay (EC50 =280â nm), poor-to-very-good physicochemical as well as in vitro pharmacokinetic properties, and conferred very high calcium retention capacity to mitochondria. From the data, we believe compoundâ 4 in this series represents a promising lead for the development of PTP inhibitors of pharmacological relevance.
Assuntos
Benzamidas/farmacologia , Proteínas de Transporte da Membrana Mitocondrial/antagonistas & inibidores , Benzamidas/síntese química , Benzamidas/química , Relação Dose-Resposta a Droga , Humanos , Poro de Transição de Permeabilidade Mitocondrial , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
The mitochondrial permeability transition pore (mtPTP) is a Ca(2+) -requiring mega-channel which, under pathological conditions, leads to the deregulated release of Ca(2+) and mitochondrial dysfunction, ultimately resulting in cell death. Although the mtPTP is a potential therapeutic target for many human pathologies, its potential as a drug target is currently unrealized. Herein we describe an optimization effort initiated around hit 1, 5-(3-hydroxyphenyl)-N-(3,4,5-trimethoxyphenyl)isoxazole-3-carboxamide, which was found to possess promising inhibitory activity against mitochondrial swelling (EC50 <0.39â µM) and showed no interference on the inner mitochondrial membrane potential (rhodamineâ 123 uptake EC50 >100â µM). This enabled the construction of a series of picomolar mtPTP inhibitors that also potently increase the calcium retention capacity of the mitochondria. Finally, the therapeutic potential and in vivo efficacy of one of the most potent analogues, N-(3-chloro-2-methylphenyl)-5-(4-fluoro-3-hydroxyphenyl)isoxazole-3-carboxamide (60), was validated in a biologically relevant zebrafish model of collagenâ VI congenital muscular dystrophies.
Assuntos
Descoberta de Drogas , Isoxazóis/farmacologia , Proteínas de Transporte da Membrana Mitocondrial/antagonistas & inibidores , Cálcio/metabolismo , Relação Dose-Resposta a Droga , Humanos , Isoxazóis/síntese química , Isoxazóis/química , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Small-molecule probes can illuminate biological processes and aid in the assessment of emerging therapeutic targets by perturbing biological systems in a manner distinct from other experimental approaches. Despite the tremendous promise of chemical tools for investigating biology and disease, small-molecule probes were unavailable for most targets and pathways as recently as a decade ago. In 2005, the NIH launched the decade-long Molecular Libraries Program with the intent of innovating in and broadening access to small-molecule science. This Perspective describes how novel small-molecule probes identified through the program are enabling the exploration of biological pathways and therapeutic hypotheses not otherwise testable. These experiences illustrate how small-molecule probes can help bridge the chasm between biological research and the development of medicines but also highlight the need to innovate the science of therapeutic discovery.
Assuntos
Descoberta de Drogas , Bibliotecas de Moléculas Pequenas , Animais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala , Humanos , National Institutes of Health (U.S.) , Estados UnidosRESUMO
Protein-protein interactions are generally challenging to target by small molecules. To address the challenge, we have used a multidisciplinary approach to identify small-molecule disruptors of protein-protein interactions that are mediated by SUMO (small ubiquitin-like modifier) proteins. SUMO modifications have emerged as a target with importance in treating cancer, neurodegenerative disorders, and viral infections. It has been shown that inhibiting SUMO-mediated protein-protein interactions can sensitize cancer cells to chemotherapy and radiation. We have developed highly sensitive assays using time-resolved fluorescence resonance energy transfer (TR-FRET) and fluorescence polarization (FP) that were used for high-throughput screening (HTS) to identify inhibitors for SUMO-dependent protein-protein interactions. Using these assays, we have identified a nonpeptidomimetic small molecule chemotype that binds to SUMO1 but not SUMO2 or 3. NMR chemical shift perturbation studies have shown that the compounds of this chemotype bind to the SUMO1 surface required for protein-protein interaction, despite the high sequence similarity of SUMO1 and SUMO2 and 3 at this surface.