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Striatal-enriched protein tyrosine phosphatase (STEP) is a brain-specific enzyme that regulates the signaling molecules that control synaptic plasticity and neuronal function. Dysregulation of STEP is linked to the pathophysiology of Alzheimer's disease and other neuropsychiatric disorders. Experimental results from neurological deficit disease models suggest that the modulation of STEP could be beneficial in a number of these disorders. This prompted our work to identify small-molecule modulators of STEP to provide the foundation of a drug discovery program. As a component of our testing funnel to identify small-molecule STEP inhibitors, we have developed a cellular target engagement assay that can identify compounds that interact with STEP46. We provide a comprehensive protocol to enable the use of this miniaturized assay, and we demonstrate its utility to benchmark the binding of newly discovered compounds.
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Enfermedad de Alzheimer , Proteínas Tirosina Fosfatasas no Receptoras , Humanos , Proteínas Tirosina Fosfatasas no Receptoras/genética , Enfermedad de Alzheimer/metabolismo , Encéfalo/metabolismo , Neuronas/metabolismo , Transducción de SeñalRESUMEN
Despite effective new therapies, adaptive resistance remains the main obstacle in acute myelogenous leukemia (AML) therapy. Autophagy induction is a key mechanism for adaptive resistance. Leukemic blasts at diagnosis express higher levels of the apical autophagy kinase ULK1 compared with normal hematopoietic cells. Exposure to chemotherapy and targeted agents upregulate ULK1, hence we hypothesize that developing ULK1 inhibitors may present the unique opportunity for clinical translation of autophagy inhibition. Accordingly, we demonstrate that ULK1 inhibition, by genetic and pharmacologic means, suppresses treatment-induced autophagy, overcomes adaptive drug-resistance, and synergizes with chemotherapy and emerging antileukemia agents like venetoclax (ABT-199). The study next aims at exploring the underlying mechanisms. Mechanistically, ULK1 inhibition downregulates MCL1 antiapoptotic gene, impairs mitochondrial function and downregulates components of the CD44-xCT system, resulting in impaired reactive oxygen species (ROS) mitigation, DNA damage, and apoptosis. For further validation, several mouse models of AML were generated. In these mouse models, ULK1 deficiency impaired leukemic cell homing and engraftment, delayed disease progression, and improved survival. Therefore, in the study, we validated our hypothesis and identified ULK1 as an important mediator of adaptive resistance to therapy and an ideal candidate for combination therapy in AML. Therefore, we propose ULK1 inhibition as a therapeutically relevant treatment option to overcome adaptive drug-resistance in AML. IMPLICATIONS: ULK1 drives a cell-intrinsic adaptive resistance in AML and targeting ULK1-mediated autophagy can synergize with existing and emerging AML therapies to overcome drug-resistance and induce apoptosis.
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Antineoplásicos , Leucemia Mieloide Aguda , Animales , Ratones , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Antineoplásicos/farmacología , Autofagia , Resistencia a Antineoplásicos , ApoptosisRESUMEN
Disturbance of the dynamic balance between protein tyrosine phosphorylation and dephosphorylation, modulated by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), is known to be crucial for the development of many human diseases. The discovery of agents that restore this balance has been the subject of many drug research efforts, most of which have focused on tyrosine kinase inhibitors (TKIs), resulting in the development of more than 50 FDA-approved TKIs during the past two decades. More recently, accumulating evidence has suggested that members of the PTP superfamily are also promising drug targets, and efforts to discover tyrosine phosphatase inhibitors (TPIs) have increased dramatically. Here, we provide protocols for determining the potency of TPIs in vitro. We focus on the use of fluorescence-based substrates, which exhibit a dramatic increase in fluorescence emission when dephosphorylated by the PTP, and thus allow setting up highly sensitive and miniaturized phosphatase activity assays using 384-well or 1536-well microplates and a continuous (kinetic) assay format. The protocols cover PTP specific activity assays, Michaelis-Menten kinetics, dose-response inhibition assays, and dose-response data analysis for determining IC 50 values. Potential pitfalls are also discussed. While advanced instrumentation is utilized for compound spotting and liquid dispensing, all the assays can be adapted to existing equipment in most laboratories. Assays are described for selected PTP drug targets, including SHP2 ( PTPN11 ), PTP1B ( PTPN1 ), STEP ( PTPN5 ), and VHR ( DUSP3 ). However, all protocols are applicable to members of the PTP enzyme family in general. Graphical abstract.
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BACKGROUND: Stress responses are believed to involve corticotropin releasing factor (CRF), its two cognate receptors (CRF1 and CRF2), and the CRF-binding protein (CRFBP). Whereas decades of research has focused on CRF1, the role of CRF2 in the central nervous system (CNS) has not been thoroughly investigated. We have previously reported that CRF2, interacting with a C terminal fragment of CRFBP, CRFBP(10kD), may have a role in the modulation of neuronal activity. However, the mechanism by which CRF interacts with CRFBP(10kD) and CRF2 has not been fully elucidated due to the lack of useful chemical tools to probe CRFBP. METHODS: We miniaturized a cell-based assay, where CRFBP(10kD) is fused as a chimera with CRF2, and performed a high-throughput screen (HTS) of 350,000 small molecules to find negative allosteric modulators (NAMs) of the CRFBP(10kD)-CRF2 complex. Hits were confirmed by evaluating activity toward parental HEK293 cells, toward CRF2 in the absence of CRFBP(10kD), and toward CRF1 in vitro. Hits were further characterized in ex vivo electrophysiology assays that target: 1) the CRF1+ neurons in the central nucleus of the amygdala (CeA) of CRF1:GFP mice that express GFP under the CRF1 promoter, and 2) the CRF-induced potentiation of N-methyl-D-aspartic acid receptor (NMDAR)-mediated synaptic transmission in dopamine neurons in the ventral tegmental area (VTA). RESULTS: We found that CRFBP(10kD) potentiates CRF-intracellular Ca2+ release specifically via CRF2, indicating that CRFBP may possess excitatory roles in addition to the inhibitory role established by the N-terminal fragment of CRFBP, CRFBP(27kD). We identified novel small molecule CRFBP-CRF2 NAMs that do not alter the CRF1-mediated effects of exogenous CRF but blunt CRF-induced potentiation of NMDAR-mediated synaptic transmission in dopamine neurons in the VTA, an effect mediated by CRF2 and CRFBP. CONCLUSION: These results provide the first evidence of specific roles for CRF2 and CRFBP(10kD) in the modulation of neuronal activity and suggest that CRFBP(10kD)-CRF2 NAMs can be further developed for the treatment of stress-related disorders including alcohol and substance use disorders.
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Hormona Liberadora de Corticotropina , Proyectos de Investigación , Humanos , Animales , Ratones , Células HEK293RESUMEN
Signal transducer and activator of transcription (Stat)3 is a valid anticancer therapeutic target. We have discovered a highly potent chemotype that amplifies the Stat3-inhibitory activity of lead compounds to levels previously unseen. The azetidine-based compounds, including H172 (9f) and H182, irreversibly bind to Stat3 and selectively inhibit Stat3 activity (IC50 0.38-0.98 µM) over Stat1 or Stat5 (IC50 > 15.8 µM) in vitro. Mass spectrometry detected the Stat3 cysteine peptides covalently bound to the azetidine compounds, and the key residues, Cys426 and Cys468, essential for the high potency inhibition, were confirmed by site-directed mutagenesis. In triple-negative breast cancer (TNBC) models, treatment with the azetidine compounds inhibited constitutive and ligand-induced Stat3 signaling, and induced loss of viable cells and tumor cell death, compared to no effect on the induction of Janus kinase (JAK)2, Src, epidermal growth factor receptor (EGFR), and other proteins, or weak effects on cells that do not harbor aberrantly-active Stat3. H120 (8e) and H182 as a single agent inhibited growth of TNBC xenografts, and H278 (hydrochloric acid salt of H182) in combination with radiation completely blocked mouse TNBC growth and improved survival in syngeneic models. We identify potent azetidine-based, selective, irreversible Stat3 inhibitors that inhibit TNBC growth in vivo.
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Azetidinas , Neoplasias de la Mama Triple Negativas , Animales , Apoptosis , Azetidinas/farmacología , Línea Celular Tumoral , Humanos , Ratones , Fosforilación , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genéticaRESUMEN
Serine/threonine-protein kinases 3 and 4 (STK3 and STK4, respectively) are key components of the Hippo signaling pathway, which regulates cell proliferation and death and provides a potential therapeutic target for acute myeloid leukemia (AML). Herein, we report the structure-based design of a series of pyrrolopyrimidine derivatives as STK3 and STK4 inhibitors. In an initial screen, the compounds exhibited low nanomolar potency against both STK3 and STK4. Crystallization of compound 6 with STK4 revealed two-point hinge binding in the ATP-binding pocket. Further characterization and analysis demonstrated that compound 20 (SBP-3264) specifically inhibited the Hippo signaling pathway in cultured mammalian cells and possessed favorable pharmacokinetic and pharmacodynamic properties in mice. We show that genetic knockdown and pharmacological inhibition of STK3 and STK4 suppress the proliferation of AML cells in vitro. Thus, SBP-3264 is a valuable chemical probe for understanding the roles of STK3 and STK4 in AML and is a promising candidate for further advancement as a potential therapy.
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Vía de Señalización Hippo/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Leucemia Mieloide Aguda/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Serina-Treonina Quinasa 3/antagonistas & inhibidores , Animales , Femenino , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Ratones , Ratones Endogámicos C57BL , Inhibidores de Proteínas Quinasas/químicaRESUMEN
Disturbance of the dynamic balance between tyrosine phosphorylation and dephosphorylation of signaling molecules, controlled by protein tyrosine kinases and protein tyrosine phosphatases (PTPs), is known to lead to the development of cancer. While most approved targeted cancer therapies are tyrosine kinase inhibitors, PTPs have long been stigmatized as undruggable and have only recently gained renewed attention in drug discovery. One PTP target is the Src-homology 2 domain-containing phosphatase 2 (SHP2). SHP2 is implicated in tumor initiation, progression, metastasis, and treatment resistance, primarily because of its role as a signaling nexus of the extracellular signal-regulated kinase pathway, acting upstream of the small GTPase Ras. Efforts to develop small molecules that target SHP2 are ongoing, and several SHP2 allosteric inhibitors are currently in clinical trials for the treatment of solid tumors. However, while the reported allosteric inhibitors are highly effective against cells expressing WT SHP2, none have significant activity against the most frequent oncogenic SHP2 variants that drive leukemogenesis in several juvenile and acute leukemias. Here, we report the discovery of novel furanylbenzamide molecules as inhibitors of both WT and oncogenic SHP2. Importantly, these inhibitors readily cross cell membranes, bind and inhibit SHP2 under physiological conditions, and effectively decrease the growth of cancer cells, including triple-negative breast cancer cells, acute myeloid leukemia cells expressing either WT or oncogenic SHP2, and patient-derived acute myeloid leukemia cells. These novel compounds are effective chemical probes of active SHP2 and may serve as starting points for therapeutics targeting WT or mutant SHP2 in cancer.
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Benzamidas , Inhibidores Enzimáticos , Leucemia Mieloide Aguda , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Benzamidas/farmacología , Carcinogénesis , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/enzimología , Oncogenes , Proteína Tirosina Fosfatasa no Receptora Tipo 11/antagonistas & inhibidores , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismoRESUMEN
Autophagy and apoptosis are functionally distinct mechanisms for cytoplasmic and cellular turnover. While these two pathways are distinct, they can also regulate each other, and central components of the apoptosis or autophagy pathway regulate both processes directly. Furthermore, several upstream stress-inducing signaling pathways can influence both autophagy and apoptosis. The crosstalk between autophagy and apoptosis has an integral role in pathological processes, including those related to cancer, homeostasis, and aging. Apoptosis is a form of programmed cell death, tightly regulated by various cellular and biochemical mechanisms, some of which have been the focus of drug discovery efforts targeting cancer therapeutics. Autophagy is a cellular degradation pathway whereby cells recycle macromolecules and organelles to generate energy when subjected to stress. Autophagy can act as either a prodeath or a prosurvival process and is both tissue and microenvironment specific. In this review we describe five groups of proteins that are integral to the apoptosis pathway and discuss their role in regulating autophagy. We highlight several apoptosis-inducing small molecules and biologics that have been developed and advanced into the clinic and discuss their effects on autophagy. For the most part, these apoptosis-inducing compounds appear to elevate autophagy activity. Under certain circumstances autophagy demonstrates cytoprotective functions and is overactivated in response to chemo- or radiotherapy which can lead to drug resistance, representing a clinical obstacle for successful cancer treatment. Thus, targeting the autophagy pathway in combination with apoptosis-inducing compounds may be a promising strategy for cancer therapy.
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Many human diseases are the result of abnormal expression or activation of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Not surprisingly, more than 30 tyrosine kinase inhibitors (TKIs) are currently in clinical use and provide unique treatment options for many patients. PTPs on the other hand have long been regarded as "undruggable" and only recently have gained increased attention in drug discovery. Striatal-enriched tyrosine phosphatase (STEP) is a neuron-specific PTP that is overactive in Alzheimer's disease (AD) and other neurodegenerative and neuropsychiatric disorders, including Parkinson's disease, schizophrenia, and fragile X syndrome. An emergent model suggests that the increase in STEP activity interferes with synaptic function and contributes to the characteristic cognitive and behavioral deficits present in these diseases. Prior efforts to generate STEP inhibitors with properties that warrant clinical development have largely failed. To identify novel STEP inhibitor scaffolds, we developed a biophysical, label-free high-throughput screening (HTS) platform based on the protein thermal shift (PTS) technology. In contrast to conventional HTS using STEP enzymatic assays, we found the PTS platform highly robust and capable of identifying true hits with confirmed STEP inhibitory activity and selectivity. This new platform promises to greatly advance STEP drug discovery and should be applicable to other PTP targets.
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Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Ensayos Analíticos de Alto Rendimiento/métodos , Proteínas Tirosina Fosfatasas no Receptoras/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas no Receptoras/metabolismo , Humanos , Estructura MolecularRESUMEN
Inhibition of autophagy, the major cellular recycling pathway in mammalian cells, is a promising strategy for the treatment of triple-negative breast cancer (TNBC). We previously reported SBI-0206965, a small molecule inhibitor of unc-51-like autophagy activating kinase 1 (ULK1), which is a key regulator of autophagy initiation. Herein, we describe the design, synthesis, and characterization of new dual inhibitors of ULK1 and ULK2 (ULK1/2). One inhibitor, SBP-7455 (compound 26), displayed improved binding affinity for ULK1/2 compared with SBI-0206965, potently inhibited ULK1/2 enzymatic activity in vitro and in cells, reduced the viability of TNBC cells and had oral bioavailability in mice. SBP-7455 inhibited starvation-induced autophagic flux in TNBC cells that were dependent on autophagy for survival and displayed synergistic cytotoxicity with the poly (ADP-ribose) polymerase (PARP) inhibitor olaparib against TNBC cells. These data suggest that combining ULK1/2 and PARP inhibition may have clinical utility for the treatment of TNBC.
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Antineoplásicos/farmacología , Homólogo de la Proteína 1 Relacionada con la Autofagia/antagonistas & inhibidores , Autofagia/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Ftalazinas/farmacología , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Animales , Antineoplásicos/síntesis química , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Femenino , Células HEK293 , Humanos , Ratones Endogámicos C57BL , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Proteínas Quinasas/síntesis química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pirimidinas/síntesis química , Neoplasias de la Mama Triple Negativas/tratamiento farmacológicoRESUMEN
"Shock and kill" strategies focus on purging the latent HIV-1 reservoir by treating infected individuals with therapeutics that activate the latent virus and subsequently eliminating infected cells. We have previously reported that induction of non-canonical nuclear factor κB (NF-κB) signaling through a class of small-molecule antagonists known as Smac mimetics can reverse HIV-1 latency. Here, we describe the development of Ciapavir (SBI-0953294), a molecule specifically optimized for HIV-1 latency reversal that was found to be more efficacious as a latency-reversing agent than other Smac mimetics under clinical development for cancer. Critically, this molecule induced activation of HIV-1 reservoirs in vivo in a bone marrow, liver, thymus (BLT) humanized mouse model without mediating systemic T cell activation. This study provides proof of concept for the in vivo efficacy and safety of Ciapavir and indicates that Smac mimetics can constitute a critical component of a safe and efficacious treatment strategy to eliminate the latent HIV-1 reservoir.
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Antirretrovirales/farmacología , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , FN-kappa B/metabolismo , Transducción de Señal/efectos de los fármacos , Latencia del Virus/efectos de los fármacos , Animales , Médula Ósea/efectos de los fármacos , Células Cultivadas , Infecciones por VIH/metabolismo , Seropositividad para VIH/tratamiento farmacológico , Humanos , Hígado/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Bibliotecas de Moléculas Pequeñas/farmacología , Linfocitos T/efectos de los fármacos , Timo/efectos de los fármacos , Activación Viral/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
Selective modulation of metabotropic glutamate receptor 2 (mGlu2) represents a novel therapeutic approach for treating brain disorders, including schizophrenia, depression, Parkinson's disease (PD), Alzheimer's disease (AD), drug abuse and addiction. Imaging mGlu2 using positron emission tomography (PET) would allow for in vivo quantification under physiological and pathological conditions and facilitate drug discovery by enabling target engagement studies. In this paper, we aimed to develop a novel specific radioligand derived from negative allosteric modulators (NAMs) for PET imaging of mGlu2. Methods. A focused small molecule library of mGlu2 NAMs with tetrahydro naphthyridine scaffold was synthesized for pharmacology and physicochemical evaluation. GIRK dose-response assays and CNS panel binding selectivity assays were performed to study the affinity and selectivity of mGlu2 NAMs, among which compounds 14a and 14b were selected as PET ligand candidates. Autoradiography in SD rat brain sections was used to confirm the in vitro binding specificity and selectivity of [11C]14a and [11C]14b towards mGlu2. In vivo binding specificity was then studied by PET imaging. Whole body biodistribution study and radiometabolite analysis were conducted to demonstrate the pharmacokinetic properties of [11C]14b as most promising PET mGlu2 PET ligand. Results. mGlu2 NAMs 14a-14g were synthesized in 14%-20% yields in five steps. NAMs 14a and 14b were selected to be the most promising ligands due to their high affinity in GIRK dose-response assays. [11C]14a and [11C]14b displayed similar heterogeneous distribution by autoradiography, consistent with mGlu2 expression in the brain. While PET imaging study showed good brain permeability for both tracers, compound [11C]14b demonstrated superior binding specificity compared to [11C]14a. Further radiometabolite analysis of [11C]14b showed excellent stability in the brain. Conclusions. Compound 14b exhibited high affinity and excellent subtype selectivity, which was then evaluated by in vitro autoradiography and in vivo PET imaging study after labeling with carbon-11. Ligand [11C]14b, which we named [11C]MG2-1904, demonstrated high brain uptake and excellent in vitro/in vivo specific binding towards mGlu2 with high metabolic stability in the brain. As proof-of-concept, our preliminary work demonstrated a successful example of visualizing mGlu2in vivo derived from NAMs, which represents a promising chemotype for further development and optimization aimed for clinical translation.
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Naftiridinas/administración & dosificación , Tomografía de Emisión de Positrones/métodos , Ensayo de Unión Radioligante/métodos , Radiofármacos/administración & dosificación , Receptores de Glutamato Metabotrópico/análisis , Regulación Alostérica , Animales , Autorradiografía , Encéfalo/diagnóstico por imagen , Radioisótopos de Carbono , Ligandos , Masculino , Modelos Animales , Naftiridinas/química , Naftiridinas/farmacocinética , Radiofármacos/química , Radiofármacos/farmacocinética , Ratas , Receptores de Glutamato Metabotrópico/metabolismo , Distribución TisularRESUMEN
Metabotropic glutamate receptor 2 (mGlu2) is a known target for treating several central nervous system (CNS) disorders. To develop a viable positron emission tomography (PET) ligand for mGlu2, we identified new candidates 5a-i that are potent negative allosteric modulators (NAMs) of mGlu2. Among these candidates, 4-(2-fluoro-4-methoxyphenyl)-5-((1-methyl-1H-pyrazol-3-yl)methoxy)picolinamide (5i, also named as [11C]MG2-1812) exhibited high potency, high subtype selectivity, and favorable lipophilicity. Compound 5i was labeled with positron-emitting carbon-11 (11C) to obtain [11C]5i in high radiochemical yield and high molar activity by O-[11C]methylation of the phenol precursor 12 with [11C]CH3I. In vitro autoradiography with [11C]5i showed heterogeneous radioactive accumulation in the brain tissue sections, ranked in the order: cortex > striatum > hippocampus > cerebellum â« thalamus > pons. PET study of [11C]5i indicated in vivo specific binding of mGlu2 in the rat brain. Based on the [11C]5i scaffold, further optimization for new candidates is underway to identify a more suitable ligand for imaging mGlu2.
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Imagen Molecular/métodos , Tomografía de Emisión de Positrones/métodos , Receptores de Glutamato Metabotrópico/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Animales , Autorradiografía , Encéfalo/metabolismo , Radioisótopos de Carbono , Línea Celular , Humanos , Ligandos , Masculino , Ratones , Ratones Noqueados , Estructura Molecular , Especificidad de Órganos , Ratas Sprague-Dawley , Distribución TisularRESUMEN
The Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2), encoded by the PTPN11 proto-oncogene, is a key mediator of receptor tyrosine kinase (RTK)-driven cell signaling, promoting cell survival and proliferation. In addition, SHP2 is recruited by immune check point receptors to inhibit B and T cell activation. Aberrant SHP2 function has been implicated in the development, progression, and metastasis of many cancers. Indeed, small molecule SHP2 inhibitors have recently entered clinical trials for the treatment of solid tumors with Ras/Raf/ERK pathway activation, including tumors with some oncogenic Ras mutations. However, the current class of SHP2 inhibitors is not effective against the SHP2 oncogenic variants that occur frequently in leukemias, and the development of specific small molecules that target oncogenic SHP2 is the subject of current research. A common problem with most drug discovery campaigns involving cytosolic proteins like SHP2 is that the primary assays that drive chemical discovery are often in vitro assays that do not report the cellular target engagement of candidate compounds. To provide a platform for measuring cellular target engagement, we developed both wild-type and mutant SHP2 cellular thermal shift assays. These assays reliably detect target engagement of SHP2 inhibitors in cells. Here, we provide a comprehensive protocol of this assay, which provides a valuable tool for the assessment and characterization of SHP2 inhibitors.
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Inhibidores Enzimáticos/uso terapéutico , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Inhibidores Enzimáticos/farmacología , Humanos , Proto-Oncogenes Mas , Transducción de SeñalRESUMEN
The nonreceptor protein-tyrosine phosphatase (PTP) SHP2 is encoded by the proto-oncogene PTPN11 and is a ubiquitously expressed key regulator of cell signaling, acting on a number of cellular processes and components, including the Ras/Raf/Erk, PI3K/Akt, and JAK/STAT pathways and immune checkpoint receptors. Aberrant SHP2 activity has been implicated in all phases of tumor initiation, progression, and metastasis. Gain-of-function PTPN11 mutations drive oncogenesis in several leukemias and cause developmental disorders with increased risk of malignancy such as Noonan syndrome. Until recently, small molecule-based targeting of SHP2 was hampered by the failure of orthosteric active-site inhibitors to achieve selectivity and potency within a useful therapeutic window. However, new SHP2 allosteric inhibitors with excellent potency and selectivity have sparked renewed interest in the selective targeting of SHP2 and other PTP family members. Crucially, drug discovery campaigns focusing on SHP2 would greatly benefit from the ability to validate the cellular target engagement of candidate inhibitors. Here, we report a cellular thermal shift assay that reliably detects target engagement of SHP2 inhibitors. Using this assay, based on the DiscoverX InCell Pulse enzyme complementation technology, we characterized the binding of several SHP2 allosteric inhibitors in intact cells. Moreover, we demonstrate the robustness and reliability of a 384-well miniaturized version of the assay for the screening of SHP2 inhibitors targeting either WT SHP2 or its oncogenic E76K variant. Finally, we provide an example of the assay's ability to identify and characterize novel compounds with specific cellular potency for either WT or mutant SHP2.
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Descubrimiento de Drogas/métodos , Inhibidores Enzimáticos/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 11/antagonistas & inhibidores , Animales , Carcinogénesis/genética , Línea Celular , Mutación con Ganancia de Función , Humanos , Unión Proteica , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Proto-Oncogenes MasRESUMEN
Autophagy is essential for cellular homeostasis and when deregulated this survival mechanism has been associated with disease development. Inhibition of autophagy initiation by inhibiting the kinase ULK1 (Unc-51-like autophagy activating kinase 1) has been proposed as a potential cancer therapy. While inhibitors and crystal structures of ULK1 have been reported, little is known about the other closely related kinase ULK2 (Unc-51-like autophagy activating kinase 2). Here, we present the crystal structure of ULK2 in complex with ATP competitive inhibitors. Surprisingly, the ULK2 structure revealed a dimeric assembly reminiscent of dimeric arrangements of auto-activating kinases suggesting a role for this association in ULK activation. Screening of a kinase focused library of pre-clinical and clinical compounds revealed several potent ULK1/2 inhibitors and good correlation of inhibitor-binding behavior with both ULK kinases. Aurora A was identified as a major off-target of currently used ULK1 inhibitors. Autophagic flux assays demonstrated that this off-target activity by strongly inducing autophagy in different cellular systems conferred an additional layer of complexity in the interpretation of cellular data. The data presented here provide structural models and chemical starting points for the development of ULK1/2 dual inhibitors with improved selectivity for future exploitation of autophagy inhibition.
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Muerte Celular Autofágica/efectos de los fármacos , Homólogo de la Proteína 1 Relacionada con la Autofagia , Péptidos y Proteínas de Señalización Intracelular , Modelos Moleculares , Inhibidores de Proteínas Quinasas , Proteínas Serina-Treonina Quinasas , Aurora Quinasa A/metabolismo , Homólogo de la Proteína 1 Relacionada con la Autofagia/antagonistas & inhibidores , Homólogo de la Proteína 1 Relacionada con la Autofagia/química , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Línea Celular Tumoral , Cristalografía por Rayos X , Humanos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
During times of stress, autophagy is a cellular process that enables cells to reclaim damaged components by a controlled recycling pathway. This mechanism for cellular catabolism is dysregulated in cancer, with evidence indicating that cancer cells rely on autophagy in the hypoxic and nutrient-poor microenvironment of solid tumors. Mounting evidence suggests that autophagy has a role in the resistance of tumors to standard-of-care (SOC) therapies. Therefore, there is significant interest in the discovery of small molecules that can safely modulate autophagy. In this review, we describe recent advances in the identification of new pharmacological compounds that modulate autophagy, with a focus on their mode of action, value as probe compounds, and validation as potential therapeutics.
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Antineoplásicos/farmacología , Autofagia/efectos de los fármacos , Neoplasias/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Animales , Antineoplásicos/uso terapéutico , Proteínas Relacionadas con la Autofagia/antagonistas & inhibidores , Humanos , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
The ability of novel pharmacological compounds to improve outcomes in preclinical models is often not translated into clinical efficacy. Psychiatric disorders do not have biological boundaries, and identifying mechanisms to improve the translational bottleneck between preclinical and clinical research domains is an important and challenging task. Glutamate transmission is disrupted in several neuropsychiatric disorders. Metabotropic glutamate (mGlu) receptors represent a diverse class of receptors that contribute to excitatory neurotransmission. Given the wide, yet region-specific manner of expression, developing pharmacological compounds to modulate mGlu receptor activity provides an opportunity to subtly and selectively modulate excitatory neurotransmission. This review focuses on the potential involvement of mGlu5 receptor disruption in major depressive disorder and substance and/or alcohol use disorders. We provide an overview of the justification of targeting mGlu5 receptors in the treatment of these disorders, summarize the preclinical evidence for negatively modulating mGlu5 receptors as a therapeutic target for major depressive disorders and nicotine dependence, and highlight the outcomes of recent clinical trials. While the evidence of mGlu5 receptor negative allosteric modulation has been promising in preclinical investigations, these beneficial effects have not translated into clinical efficacy. In this review, we identify key challenges that may contribute to poor clinical translation and provide suggested approaches moving forward to potentially improve the translation from preclinical to clinical domains. Such approaches may increase the success of clinical trials and may reduce the translational bottleneck that exists in drug discovery for psychiatric disorders.
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Receptor del Glutamato Metabotropico 5/antagonistas & inhibidores , Receptor del Glutamato Metabotropico 5/metabolismo , Fumar/tratamiento farmacológico , Tabaquismo/tratamiento farmacológico , Animales , Trastorno Depresivo Mayor/tratamiento farmacológico , Trastorno Depresivo Mayor/metabolismo , Descubrimiento de Drogas , Antagonistas de Aminoácidos Excitadores/administración & dosificación , Humanos , Ensayos Clínicos Controlados Aleatorios como Asunto , Receptor del Glutamato Metabotropico 5/agonistas , Fumar/metabolismo , Transmisión Sináptica/efectos de los fármacos , Resultado del TratamientoRESUMEN
Benzodiazepinones are privileged scaffolds with activity against multiple therapeutically relevant biological targets. In support of our ongoing studies around allosteric modulators of metabotropic glutamate receptors (mGlus) we required the multigram synthesis of a ß-ketoester key intermediate. We report the continuous flow synthesis of tert-butyl 3-(2-cyanopyridin-4-yl)-3-oxopropanoate and its transformation to potent mGlu2/3 negative allosteric modulators (NAMs) in batch mode.
RESUMEN
Tissue-nonspecific alkaline phosphatase (TNAP) is an ectoenzyme crucial for bone matrix mineralization via its ability to hydrolyze extracellular inorganic pyrophosphate (ePPi), a potent mineralization inhibitor, to phosphate (Pi). By the controlled hydrolysis of ePPi, TNAP maintains the correct ratio of Pi to ePPi and therefore enables normal skeletal and dental calcification. In other areas of the body low ePPi levels lead to the development of pathological soft-tissue calcification, which can progress to a number of disorders. TNAP inhibitors have been shown to prevent these processes via an increase of ePPi. Herein we describe the use of a whole blood assay to optimize a previously described series of TNAP inhibitors resulting in 5-((5-chloro-2-methoxyphenyl)sulfonamido)nicotinamide (SBI-425), a potent, selective and oral bioavailable compound that robustly inhibits TNAP in vivo.