RESUMEN
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.
Asunto(s)
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
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.
Asunto(s)
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
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.
Asunto(s)
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
The PKC-θ isoform of protein kinase C is selectively expressed in T lymphocytes and plays an important role in the T cell antigen receptor (TCR)-triggered activation of mature T cells, T cell proliferation, and the subsequent release of cytokines such as interleukin-2 (IL-2). Herein, we report the synthesis and structure-activity relationship (SAR) of a novel series of PKC-θ inhibitors. Through a combination of structure-guided design and exploratory SAR, suitable replacements for the basic C4 amine of the original lead (3) were identified. Property-guided design enabled the identification of appropriately substituted C2 groups to afford potent analogs with metabolic stability and permeability to support in vivo testing. With exquisite general kinase selectivity, cellular inhibition of T cell activation as assessed by IL-2 expression, a favorable safety profile, and demonstrated in vivo efficacy in models of acute and chronic T cell activation with oral dosing, CC-90005 (57) was selected for clinical development.
Asunto(s)
Ciclohexanoles/uso terapéutico , Enfermedad Injerto contra Huésped/tratamiento farmacológico , Factores Inmunológicos/uso terapéutico , Proteína Quinasa C-theta/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirimidinas/uso terapéutico , Animales , Células CACO-2 , Proliferación Celular/efectos de los fármacos , Ciclohexanoles/síntesis química , Ciclohexanoles/metabolismo , Humanos , Factores Inmunológicos/síntesis química , Factores Inmunológicos/metabolismo , Activación de Linfocitos/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Simulación del Acoplamiento Molecular , Estructura Molecular , Unión Proteica , Proteína Quinasa C-delta/antagonistas & inhibidores , Proteína Quinasa C-delta/metabolismo , Proteína Quinasa C-theta/metabolismo , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/metabolismo , Pirimidinas/síntesis química , Pirimidinas/metabolismo , Relación Estructura-Actividad , Linfocitos T/efectos de los fármacosRESUMEN
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.
Asunto(s)
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
"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.
Asunto(s)
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
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.
Asunto(s)
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
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.
Asunto(s)
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.
Asunto(s)
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
Autoantibodies and the immunoreceptors to which they bind can contribute to the pathogenesis of autoimmune diseases such as rheumatoid arthritis (RA). Spleen Tyrosine Kinase (Syk) is a non-receptor tyrosine kinase with a central role in immunoreceptor (FcR) signaling and immune cell functionality. Syk kinase inhibitors have activity in antibody-dependent immune cell activation assays, in preclinical models of arthritis, and have progressed into clinical trials for RA and other autoimmune diseases. Here we describe the characterization of a novel triazolopyridine-based Syk kinase inhibitor, CC-509. This compound is a potent inhibitor of purified Syk enzyme, FcR-dependent and FcR-independent signaling in primary immune cells, and basophil activation in human whole blood. CC-509 is moderately selective across the kinome and against other non-kinase enzymes or receptors. Importantly, CC-509 was optimized away from and has modest activity against cellular KDR and Jak2, kinases that when inhibited in a preclinical and clinical setting may promote hypertension and neutropenia, respectively. In addition, CC-509 is orally bioavailable and displays dose-dependent efficacy in two rodent models of immune-inflammatory disease. In passive cutaneous anaphylaxis (PCA), CC-509 significantly inhibited skin edema. Moreover, CC-509 significantly reduced paw swelling and the tissue levels of pro-inflammatory cytokines RANTES and MIP-1α in the collagen-induced arthritis (CIA) model. In summary, CC-509 is a potent, moderately selective, and efficacious inhibitor of Syk that has a differentiated profile when compared to other Syk compounds that have progressed into the clinic for RA.
Asunto(s)
Indazoles/química , Inflamación/tratamiento farmacológico , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Piridinas/química , Triazoles/química , Animales , Artritis Experimental/tratamiento farmacológico , Artritis Experimental/fisiopatología , Basófilos/citología , Línea Celular , Colágeno/química , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Edema/patología , Eosinófilos/citología , Femenino , Células HEK293 , Humanos , Hipertensión/tratamiento farmacológico , Inflamación/fisiopatología , Concentración 50 Inhibidora , Janus Quinasa 2/antagonistas & inhibidores , Masculino , Neutropenia/tratamiento farmacológico , Neutrófilos/citología , Ratas , Ratas Endogámicas Lew , Ratas Sprague-Dawley , Receptores Fc/química , Piel/patología , Quinasa Syk , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
In this Letter we describe the optimization of an aminopurine lead (1) with modest potency and poor overall kinase selectivity which led to the identification of a series of potent, selective JNK inhibitors. Improvement in kinase selectivity was enabled by introduction of an aliphatic side chain at the C-2 position. CC-359 (2) was selected as a potential clinical candidate for diseases manifested by ischemia reperfusion injury.
Asunto(s)
2-Aminopurina/química , 2-Aminopurina/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Purinas/química , Daño por Reperfusión/enzimología , Animales , Dominio Catalítico , Perros , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Haplorrinos , Concentración 50 Inhibidora , Modelos Moleculares , Estructura Molecular , Purinas/farmacología , Ratas , Daño por Reperfusión/tratamiento farmacológico , Relación Estructura-ActividadRESUMEN
In this Letter we describe the discovery of potent, selective, and orally active aminopurine JNK inhibitors. Improving the physico-chemical properties as well as increasing the potency and selectivity of a subseries with rat plasma exposure, led to the identification of four structurally diverse inhibitors. Differentiation based on PK profiles in multiple species as well as activity in a chronic efficacy model led to the identification of 1 (CC-930) as a development candidate, which is currently in Phase II clinical trial for IPF.
Asunto(s)
Ciclohexanoles/química , Ciclohexanoles/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , MAP Quinasa Quinasa 4/antagonistas & inhibidores , Purinas/química , Purinas/farmacología , Administración Oral , Animales , Dominio Catalítico , Ciclohexanoles/administración & dosificación , Perros , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/administración & dosificación , Haplorrinos , Fibrosis Pulmonar Idiopática/tratamiento farmacológico , Concentración 50 Inhibidora , Modelos Moleculares , Estructura Molecular , Purinas/administración & dosificación , Ratas , Relación Estructura-ActividadRESUMEN
The IMiDs immunomodulatory drugs are an expanding family of compounds under investigation in a broad range of diseases because they exhibit immunomodulatory and anti-tumorigenic properties. Although the molecular targets remain unidentified, the broad activity of select IMiDs immunomodulatory drugs on cell signaling pathways and transcription regulation has been partly described. One characteristic of these compounds is their ability to act as a co-stimulus of TCR ligation leading to increased IL-2, TNF-alpha and IFN-gamma expression indicative of a Th1 phenotype. Because clinical evidence for this response has been observed in thalidomide and lenalidomide treated patients, we investigated the effect of CC-4047 on T cell activation and differentiation at the molecular level. We used primary human CD4(+) T cells as a model and found that CC-4047 enhances the expression of transcription factor T-bet in both naive and pre-polarized Th2 cells. This modulation leads to upregulation of Th1 markers and cytokine production. By increasing the expression of T-bet, CC-4047 promotes the differentiation of naive T-cells to Th1 as well as effectively reverting Th2 cells into Th1-like effector cells in vitro. These findings elucidate a novel mechanism of action of CC-4047 on T cell differentiation, suggesting that certain IMiDs immunomodulatory drugs may have expanded clinical application in treating both allergic diseases and certain T cell lymphomas where a predominant Th2 phenotype is displayed.