RESUMO
Dysfunctional liver regeneration following surgical resection remains a major cause of postoperative mortality and has no therapeutic options. Without targeted therapies, the current treatment paradigm relies on supportive therapy until homeostasis can be achieved. Pharmacologic acceleration of regeneration represents an alternative therapeutic avenue. Therefore, we aimed to generate a small molecule inhibitor that could accelerate liver regeneration with an emphasis on diseased models, which represent a significant portion of patients who require surgical resection and are often not studied. Utilizing a clinically approved small molecule inhibitor as a parent compound, standard medicinal chemistry approaches were utilized to generate a small molecule inhibitor targeting serine/threonine kinase 4/3 (MST1/2) with reduced off-target effects. This compound, mCLC846, was then applied to preclinical models of murine partial hepatectomy, which included models of diet-induced metabolic dysfunction-associated steatohepatitis (MASH). mCLC846 demonstrated on target inhibition of MST1/2 and reduced epidermal growth factor receptor inhibition. The inhibitory effects resulted in restored pancreatic beta-cell function and survival under diabetogenic conditions. Liver-specific cell-line exposure resulted in Yes-associated protein activation. Oral delivery of mCLC846 perioperatively resulted in accelerated murine liver regeneration and improved survival in diet-induced MASH models. Bulk transcriptional analysis of regenerating liver remnants suggested that mCLC846 enhanced the normal regenerative pathways and induced them following liver resection. Overall, pharmacological acceleration of liver regeneration with mCLC846 was feasible, had an acceptable therapeutic index, and provided a survival benefit in models of diet-induced MASH.
RESUMO
The loss of functional insulin-producing ß-cells is a hallmark of diabetes. Mammalian sterile 20-like kinase 1 (MST1) is a key regulator of pancreatic ß-cell death and dysfunction; its deficiency restores functional ß-cells and normoglycemia. The identification of MST1 inhibitors represents a promising approach for a ß-cell-protective diabetes therapy. Here, we identify neratinib, an FDA-approved drug targeting HER2/EGFR dual kinases, as a potent MST1 inhibitor, which improves ß-cell survival under multiple diabetogenic conditions in human islets and INS-1E cells. In a pre-clinical study, neratinib attenuates hyperglycemia and improves ß-cell function, survival and ß-cell mass in type 1 (streptozotocin) and type 2 (obese Leprdb/db) diabetic mouse models. In summary, neratinib is a previously unrecognized inhibitor of MST1 and represents a potential ß-cell-protective drug with proof-of-concept in vitro in human islets and in vivo in rodent models of both type 1 and type 2 diabetes.
Assuntos
Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Tipo 2/fisiopatologia , Células Secretoras de Insulina/efeitos dos fármacos , Quinolinas/farmacologia , Animais , Linhagem Celular Tumoral , Células Cultivadas , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Obesos , Substâncias Protetoras/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
BACKGROUND AND PURPOSE: Beta cell apoptosis is a major feature of type 1 diabetes, and pro-inflammatory cytokines are key drivers of the deterioration of beta cell mass through induction of apoptosis. Mitochondrial stress plays a critical role in mediating apoptosis by releasing cytochrome C into the cytoplasm, directly activating caspase-9 and its downstream signalling cascade. We aimed to identify new compounds that protect beta cells from cytokine-induced activation of the intrinsic (mitochondrial) pathway of apoptosis. EXPERIMENTAL APPROACH: Diabetogenic media, composed of IL-1ß, IFN-γ and high glucose, were used to induce mitochondrial stress in rat insulin-producing INS1E cells, and a high-content image-based screen of small molecule modulators of Casp9 pathway was performed. KEY RESULTS: A novel small molecule, ATV399, was identified from a high-content image-based screen for compounds that inhibit cleaved caspase-9 activation and subsequent beta cell apoptosis induced by a combination of IL-1ß, IFN-γ and high glucose, which together mimic the pathogenic diabetic milieu. Through medicinal chemistry optimization, potency was markedly improved (6-30 fold), with reduced inhibitory effects on CYP3A4. Improved analogues, such as CAT639, improved beta cell viability and insulin secretion in cytokine-treated rat insulin-producing INS1E cells and primary dispersed islet cells. Mechanistically, CAT639 reduced the production of NO by allosterically inhibiting dimerization of inducible NOS (iNOS) without affecting its mRNA levels. CONCLUSION AND IMPLICATIONS: Taken together, these studies demonstrate a successful phenotypic screening campaign resulting in identification of an inhibitor of iNOS dimerization that protects beta cell viability and function through modulation of mitochondrial stress induced by cytokines.
Assuntos
Células Secretoras de Insulina/efeitos dos fármacos , Interferon gama/farmacologia , Interleucina-1beta/farmacologia , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Animais , Caspase 9/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citocromos c/metabolismo , Dimerização , Ativação Enzimática , Glucose/farmacologia , Células Secretoras de Insulina/citologia , Óxido Nítrico Sintase Tipo II/química , Ratos , Transdução de SinaisRESUMO
Dysregulation of protein tyrosine phosphorylation has been implicated in a number of human diseases, including cancer, diabetes, and neurodegenerative diseases. As a result of their essential role in regulating protein tyrosine phosphorylation levels, protein tyrosine phosphatases (PTPs) have emerged as important yet challenging therapeutic targets. Here we report on the development and application of a glutathione-responsive motif to facilitate the efficient intracellular delivery of a novel class of selenosulfide phosphatase inhibitors for the selective active site directed inhibition of the targeted PTP by selenosulfide exchange with the active site cysteine. The strategy leverages the large difference in extracellular and intracellular glutathione levels to deliver selenosulfide phosphatase inhibitors to cells. As an initial exploration of the prodrug platform and the corresponding selenosulfide covalent inhibitor class, potent and selective inhibitors were developed for two therapeutically relevant PTP targets: the Mycobacterium tuberculosis virulence factor mPTPA and the CNS-specific tyrosine phosphatase, striatal-enriched protein tyrosine phosphatase (STEP). The lead selenosulfide inhibitors enable potent and selective inhibition of their respective targets over a panel of human PTPs and a representative cysteine protease. Kinetic parameters of the inhibitors were characterized, including reversibility of inhibition and rapid rate of GSH exchange at intracellular GSH concentrations. Additionally, active site covalent inhibitor-labeling with an mPTPA inhibitor was rigorously confirmed by mass spectrometry, and cellular activity was demonstrated with a STEP prodrug inhibitor in cortical neurons.
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Brain-derived neurotrophic factor (BDNF) regulates synaptic strengthening and memory consolidation, and altered BDNF expression is implicated in a number of neuropsychiatric and neurodegenerative disorders. BDNF potentiates N-methyl-D-aspartate receptor function through activation of Fyn and ERK1/2. STriatal-Enriched protein tyrosine Phosphatase (STEP) is also implicated in many of the same disorders as BDNF but, in contrast to BDNF, STEP opposes the development of synaptic strengthening. STEP-mediated dephosphorylation of the NMDA receptor subunit GluN2B promotes internalization of GluN2B-containing NMDA receptors, while dephosphorylation of the kinases Fyn, Pyk2, and ERK1/2 leads to their inactivation. Thus, STEP and BDNF have opposing functions. In this study, we demonstrate that manipulation of BDNF expression has a reciprocal effect on STEP61 levels. Reduced BDNF signaling leads to elevation of STEP61 both in BDNF(+/-) mice and after acute BDNF knockdown in cortical cultures. Moreover, a newly identified STEP inhibitor reverses the biochemical and motor abnormalities in BDNF(+/-) mice. In contrast, increased BDNF signaling upon treatment with a tropomyosin receptor kinase B agonist results in degradation of STEP61 and a subsequent increase in the tyrosine phosphorylation of STEP substrates in cultured neurons and in mouse frontal cortex. These findings indicate that BDNF-tropomyosin receptor kinase B signaling leads to degradation of STEP61 , while decreased BDNF expression results in increased STEP61 activity. A better understanding of the opposing interaction between STEP and BDNF in normal cognitive functions and in neuropsychiatric disorders will hopefully lead to better therapeutic strategies. Altered expression of BDNF and STEP61 has been implicated in several neurological disorders. BDNF and STEP61 are known to regulate synaptic strengthening, but in opposite directions. Here, we report that reduced BDNF signaling leads to elevation of STEP61 both in BDNF(+/-) mice and after acute BDNF knockdown in cortical cultures. In contrast, activation of TrkB receptor results in the degradation of STEP61 and reverses hyperlocomotor activity in BDNF(+/-) mice. Moreover, inhibition of STEP61 by TC-2153 is sufficient to enhance the Tyr phosphorylation of STEP substrates and also reverses hyperlocomotion in BDNF(+/-) mice. These findings give us a better understanding of the regulation of STEP61 by BDNF in normal cognitive functions and in neuropsychiatric disorders.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Regulação para Baixo/fisiologia , Neurônios/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Animais , Benzotiepinas/farmacologia , Encéfalo/citologia , Fator Neurotrófico Derivado do Encéfalo/genética , Células Cultivadas , Inibidores de Cisteína Proteinase/farmacologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Embrião de Mamíferos , Feminino , Flavonas/farmacologia , Leupeptinas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Atividade Motora/efeitos dos fármacos , Atividade Motora/genética , Neurônios/efeitos dos fármacos , Proteínas Tirosina Fosfatases/genética , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
Brain-derived neurotrophic factor (BDNF) and STriatal-Enriched protein tyrosine Phosphatase 61 (STEP61) have opposing functions in the brain, with BDNF supporting and STEP61 opposing synaptic strengthening. BDNF and STEP61 also exhibit an inverse pattern of expression in a number of brain disorders, including schizophrenia (SZ). NMDAR antagonists such as phencyclidine (PCP) elicit SZ-like symptoms in rodent models and unaffected individuals, and exacerbate psychotic episodes in SZ. Here we characterize the regulation of BDNF expression by STEP61, utilizing PCP-treated cortical culture and PCP-treated mice. PCP-treated cortical neurons showed both an increase in STEP61 levels and a decrease in BDNF expression. The reduction in BDNF expression was prevented by STEP61 knockdown or use of the STEP inhibitor, TC-2153. The PCP-induced increase in STEP61 expression was associated with the inhibition of CREB-dependent BDNF transcription. Similarly, both genetic and pharmacologic inhibition of STEP prevented the PCP-induced reduction in BDNF expression in vivo and normalized PCP-induced hyperlocomotion and cognitive deficits. These results suggest a mechanism by which STEP61 regulates BDNF expression, with implications for cognitive functioning in CNS disorders.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Transtornos Cognitivos/tratamento farmacológico , Fenciclidina/uso terapêutico , Proteínas Tirosina Fosfatases/metabolismo , Animais , Benzotiepinas/farmacologia , Fator Neurotrófico Derivado do Encéfalo/análise , Proteína de Ligação a CREB/antagonistas & inibidores , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Células Cultivadas , Transtornos Cognitivos/metabolismo , Transtornos Cognitivos/patologia , Regulação para Baixo/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Atividade Motora/efeitos dos fármacos , Neurônios/citologia , Neurônios/metabolismo , Fenciclidina/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Tirosina Fosfatases/genética , Interferência de RNA , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/metabolismo , UbiquitinaçãoRESUMO
The information from nociceptors is processed in the dorsal horn of the spinal cord by complex circuits involving excitatory and inhibitory interneurons. It is well documented that GluN2B and ERK1/2 phosphorylation contributes to central sensitization. Striatal-enriched protein tyrosine phosphatase (STEP) dephosphorylates GluN2B and ERK1/2, promoting internalization of GluN2B and inactivation of ERK1/2. The activity of STEP was modulated by genetic (STEP knockout mice) and pharmacological (recently synthesized STEP inhibitor, TC-2153) approaches. STEP(61) protein levels in the lumbar spinal cord were determined in male and female mice of different ages. Inflammatory pain was induced by complete Freund's adjuvant injection. Behavioral tests, immunoblotting, and electrophysiology were used to analyze the effect of STEP on nociception. Our results show that both genetic deletion and pharmacological inhibition of STEP induced thermal hyperalgesia and mechanical allodynia, which were accompanied by increased pGluN2B(Tyr1472) and pERK1/2(Thr202/Tyr204)levels in the lumbar spinal cord. Striatal-enriched protein tyrosine phosphatase heterozygous and knockout mice presented a similar phenotype. Furthermore, electrophysiological experiments showed that TC-2153 increased C fiber-evoked spinal field potentials. Interestingly, we found that STEP(61) protein levels in the lumbar spinal cord inversely correlated with thermal hyperalgesia associated with age and female gender in mice. Consistently, STEP knockout mice failed to show age-related thermal hyperalgesia, although gender-related differences were preserved. Moreover, in a model of inflammatory pain, hyperalgesia was associated with increased phosphorylation-mediated STEP(61) inactivation and increased pGluN2B(Tyr1472) and pERK1/2(Thr202/Tyr204)levels in the lumbar spinal cord. Collectively, the present results underscore an important role of spinal STEP activity in the modulation of nociception.
Assuntos
Nociceptividade/fisiologia , Limiar da Dor/efeitos dos fármacos , Dor/patologia , Dor/fisiopatologia , Proteínas Tirosina Fosfatases não Receptoras/deficiência , Animais , Benzotiepinas/farmacologia , Benzotiepinas/uso terapêutico , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Potenciais Evocados/efeitos dos fármacos , Potenciais Evocados/genética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Hiperalgesia/tratamento farmacológico , Hiperalgesia/genética , Hiperalgesia/metabolismo , Hiperalgesia/patologia , Inflamação/induzido quimicamente , Inflamação/complicações , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fibras Nervosas Amielínicas/efeitos dos fármacos , Fibras Nervosas Amielínicas/fisiologia , Nociceptividade/efeitos dos fármacos , Dor/etiologia , Proteínas Tirosina Fosfatases não Receptoras/genética , Ratos , Ratos Sprague-Dawley , Transdução de SinaisRESUMO
Striatal-enriched protein tyrosine phosphatase (STEP) is a brain specific protein tyrosine phosphatase that has been implicated in many neurodegenerative diseases, such as Alzheimer's disease. We recently reported the benzopentathiepin TC-2153 as a potent inhibitor of STEP in vitro, cells and animals. Herein, we report the synthesis and evaluation of TC-2153 analogs in order to define what structural features are important for inhibition and to identify positions tolerant of substitution for further study. The trifluoromethyl substitution is beneficial for inhibitor potency, and the amine is tolerant of acylation, and thus provides a convenient handle for introducing additional functionality such as reporter groups.
Assuntos
Benzotiepinas/química , Benzotiepinas/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/enzimologia , Animais , Benzotiepinas/síntese química , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/enzimologia , Inibidores Enzimáticos/síntese química , Halogenação , Metilação , Camundongos , Proteínas Tirosina Fosfatases/metabolismo , RatosRESUMO
STEP (STriatal-Enriched protein tyrosine Phosphatase) is a neuron-specific phosphatase that regulates N-methyl-D-aspartate receptor (NMDAR) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) trafficking, as well as ERK1/2, p38, Fyn, and Pyk2 activity. STEP is overactive in several neuropsychiatric and neurodegenerative disorders, including Alzheimer's disease (AD). The increase in STEP activity likely disrupts synaptic function and contributes to the cognitive deficits in AD. AD mice lacking STEP have restored levels of glutamate receptors on synaptosomal membranes and improved cognitive function, results that suggest STEP as a novel therapeutic target for AD. Here we describe the first large-scale effort to identify and characterize small-molecule STEP inhibitors. We identified the benzopentathiepin 8-(trifluoromethyl)-1,2,3,4,5-benzopentathiepin-6-amine hydrochloride (known as TC-2153) as an inhibitor of STEP with an IC50 of 24.6 nM. TC-2153 represents a novel class of PTP inhibitors based upon a cyclic polysulfide pharmacophore that forms a reversible covalent bond with the catalytic cysteine in STEP. In cell-based secondary assays, TC-2153 increased tyrosine phosphorylation of STEP substrates ERK1/2, Pyk2, and GluN2B, and exhibited no toxicity in cortical cultures. Validation and specificity experiments performed in wild-type (WT) and STEP knockout (KO) cortical cells and in vivo in WT and STEP KO mice suggest specificity of inhibitors towards STEP compared to highly homologous tyrosine phosphatases. Furthermore, TC-2153 improved cognitive function in several cognitive tasks in 6- and 12-mo-old triple transgenic AD (3xTg-AD) mice, with no change in beta amyloid and phospho-tau levels.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/enzimologia , Transtornos Cognitivos/tratamento farmacológico , Transtornos Cognitivos/enzimologia , Inibidores Enzimáticos/uso terapêutico , Proteínas Tirosina Fosfatases não Receptoras/antagonistas & inibidores , Doença de Alzheimer/complicações , Doença de Alzheimer/patologia , Sequência de Aminoácidos , Animais , Benzotiepinas/farmacologia , Benzotiepinas/uso terapêutico , Domínio Catalítico , Morte Celular/efeitos dos fármacos , Córtex Cerebral/patologia , Transtornos Cognitivos/complicações , Transtornos Cognitivos/patologia , Cisteína/metabolismo , Modelos Animais de Doenças , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Neurônios/efeitos dos fármacos , Neurônios/patologia , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , Proteínas Tirosina Fosfatases não Receptoras/química , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Especificidade por Substrato/efeitos dos fármacosRESUMO
High levels of striatal-enriched protein tyrosine phosphatase (STEP) activity are observed in a number of neuropsychiatric disorders such as Alzheimer's disease. Overexpression of STEP results in the dephosphorylation and inactivation of many key neuronal signaling molecules, including ionotropic glutamate receptors. Moreover, genetically reducing STEP levels in AD mouse models significantly reversed cognitive deficits and decreased glutamate receptor internalization. These results support STEP as a potential target for drug discovery for the treatment of Alzheimer's disease. Herein, a substrate-based approach for the discovery and optimization of fragments called substrate activity screening (SAS) has been applied to the development of low molecular weight (<450 Da) and nonpeptidic, single-digit micromolar mechanism-based STEP inhibitors with greater than 20-fold selectivity across multiple tyrosine and dual specificity phosphatases. Significant levels of STEP inhibition in rat cortical neurons are also observed.
Assuntos
Compostos de Bifenilo/química , Proteínas Tirosina Fosfatases não Receptoras/antagonistas & inibidores , Animais , Compostos de Bifenilo/síntese química , Compostos de Bifenilo/farmacologia , Barreira Hematoencefálica/metabolismo , Ácidos Borônicos/síntese química , Ácidos Borônicos/química , Células Cultivadas , Córtex Cerebral/citologia , Humanos , Neurônios/enzimologia , Permeabilidade , Ácidos Fosforosos/síntese química , Ácidos Fosforosos/química , Ácidos Fosforosos/farmacologia , Ratos , Ratos Sprague-Dawley , Bibliotecas de Moléculas Pequenas , Estereoisomerismo , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
The MgCl(2)-enhanced addition of benzyl zinc reagents to N-tert-butanesulfinyl imines proceeds readily at room temperature to afford the N-tert-butanesulfinyl-protected amine products in good yields and diastereomeric ratios. This method is functional group tolerant in both the imine substrate and benzyl zinc coupling partner. Moreover, benzyl zinc reagent addition to the N-tert-butanesulfinyl imine 3o prepared from isopropylidene-protected glyceraldehyde proceeds in high yield and with exceptional selectivity to provide rapid entry to hydroxyethylamine-based aspartyl protease inhibitors.
RESUMO
The development of low muM inhibitors of the Mycobacterium tuberculosis phosphatase PtpA is reported. The most potent of these inhibitors (K(i)=1.4+/-0.3 microM) was found to be selective when tested against a panel of human tyrosine and dual-specificity phosphatases (11-fold vs the highly homologous HCPtpA, and >70-fold vs all others tested). Modeling the inhibitor-PtpA complexes explained the structure-activity relationships observed in vitro and revealed further possibilities for compound development.