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AXL is a member of the TAM (TYRO3, AXL, MER) subfamily of receptor tyrosine kinases. It is upregulated in a variety of cancers and its overexpression is associated with poor disease prognosis and acquired drug resistance. Utilizing a fragment-based lead discovery approach, a new indazole-based AXL inhibitor was obtained. The indazole fragment hit 11, identified through a high concentration biochemical screen, was expeditiously improved to fragment 24 by screening our in-house expanded library of fragments (ELF) collection. Subsequent fragment optimization guided by docking studies provided potent inhibitor 54 with moderate exposure levels in mice. X-ray crystal structure of analog 50 complexed with the I650M mutated kinase domain of Mer revealed the key binding interactions for the scaffold. The good potency coupled with reasonable kinase selectivity, moderate in vivo exposure levels, and availability of structural information for the series makes it a suitable starting point for further optimization efforts.
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Descubrimiento de Drogas , Indazoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Indazoles/síntesis química , Indazoles/química , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Relación Estructura-Actividad , Tirosina Quinasa del Receptor AxlRESUMEN
Current JAK2 inhibitors used for myeloproliferative neoplasms (MPN) treatment are not specific enough to selectively suppress aberrant JAK2 signalling and preserve physiological JAK2 signalling. We tested whether combining a JAK2 inhibitor with a series of serine threonine kinase inhibitors, targeting nine signalling pathways and already used in clinical trials, synergized in inhibiting growth of haematopoietic cells expressing mutant and wild-type forms of JAK2 (V617F) or thrombopoietin receptor (W515L). Out of 15 kinase inhibitors, the ZSTK474 phosphatydylinositol-3'-kinase (PI3K) inhibitor molecule showed strong synergic inhibition by Chou and Talalay analysis with JAK2 and JAK2/JAK1 inhibitors. Other pan-class I, but not gamma or delta specific PI3K inhibitors, also synergized with JAK2 inhibitors. Synergy was not observed in Bcr-Abl transformed cells. The best JAK2/JAK1 and PI3K inhibitor combination pair (ruxolitinib and GDC0941) reduces spleen weight in nude mice inoculated with Ba/F3 cells expressing TpoR and JAK2 V617F. It also exerted strong inhibitory effects on erythropoietin-independent erythroid colonies from MPN patients and JAK2 V617F knock-in mice, where at certain doses, a preferential inhibition of JAK2 V617F mutated progenitors was detected. Our data support the use of a combination of JAK2 and pan-class I PI3K inhibitors in the treatment of MPNs.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Janus Quinasa 2/antagonistas & inhibidores , Trastornos Mieloproliferativos/tratamiento farmacológico , Inhibidores de las Quinasa Fosfoinosítidos-3 , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Femenino , Técnicas de Sustitución del Gen , Neoplasias Hematológicas/tratamiento farmacológico , Neoplasias Hematológicas/enzimología , Humanos , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Ratones , Ratones Desnudos , Ratones Transgénicos , Mutación Missense , Trastornos Mieloproliferativos/enzimología , Trasplante de Neoplasias , Nitrilos , Fosfatidilinositol 3-Quinasas/metabolismo , Pirazoles/administración & dosificación , Pirimidinas , Pirrolidinas/administración & dosificación , Transducción de Señal/efectos de los fármacos , Sulfonamidas/administración & dosificación , Triazinas/administración & dosificación , Células Tumorales CultivadasRESUMEN
Crystallinity and P-glycoprotein (P-gp) mediated efflux of drugs with low aqueous solubility collaboratively contributes to erratic absorption resulting in low/variable bioavailability. Herein, the amorphous solid dispersions (SD) of lumefantrine (LUMF) containing piperine (PIP), a P-gp and CYP3A4 inhibitor, were formulated with Soluplus (Sol), Klucel (Klu) and Lutrol F68 (Lut), polymeric carriers, to improve solubility and bioavailability of LUMF following oral administration. The LUMF-PIP-SD prepared with Sol exhibited higher aqueous solubility of LUMF in concentration dependent manner and LUMF-PIP-Sol demonstrating maximum aqueous LUMF solubility were characterized by DSC, FTIR and XRD. The DSC thermogram and XRD diffractogram of LUMF-PIP-SD confirmed the loss of crystallinity of LUMF ensuing improved dissolution while possible interaction of LUMF with PIP and /or Sol was evident in FTIR spectrum. DSC and dissolution studies confirmed the stability for LUMF-PIP-Sol SD stored for 90 days under stressed conditions of humidity and temperature. An in situ single-pass intestinal perfusion study in rats indicated 2.2-fold increase in intestinal permeation of LUMF co-administered with PIP. Improved bioavailability of LUMF was evidenced by increased AUC0-∞ and Cmax for LUMF in SD compared to alone LUMF or LUMF with PIP. Peter's four-day suppressive test indicated improved antimalarial activity for LUMF-PIP-Sol SD. Overall, the data suggest that the SD of LUMF incorporated with P-gp inhibitor PIP, improves the bioavailability as well as antimalarial efficacy of LUMF.
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Antimaláricos , Parásitos , Ratas , Animales , Solubilidad , Disponibilidad Biológica , Lumefantrina , Antimaláricos/farmacología , Administración OralRESUMEN
INTRODUCTION: AOD01 is a novel, fully human immunoglobulin (Ig) G1 neutralizing monoclonal antibody that was developed as a therapeutic against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). This first-in-human study assessed safety, tolerability, pharmacokinetics (PK), and pharmacodynamics of AOD01 in healthy volunteers. METHODS: Intravenous doses of AOD01 were evaluated in escalating cohorts [four single-dose cohorts (2, 5, 10, and 20 mg/kg) and one two-dose cohort (two doses of 20 mg/kg, 24 h apart)]. RESULTS: Twenty-three subjects were randomized to receive AOD01 or a placebo in blinded fashion. A total of 34 treatment-emergent adverse events (TEAEs) were reported; all were mild in severity. Related events (headache and diarrhea) were reported in one subject each. No event of infusion reactions, serious adverse event (SAE), or discontinuation due to AE were reported. The changes in laboratory parameters, vital signs, and electrocardiograms were minimal. Dose-related exposure was seen from doses 2 to 20 mg/kg as confirmed by Cmax and AUC0-tlast. The median Tmax was 1.5-3 h. Clearance was dose independent. Study results revealed long half-lives (163-465 h). Antidrug antibodies (ADA) to AOD01 were not detected among subjects, except in one subject of the two-dose cohort on day 92. Sustained ex vivo neutralization of SARS-CoV-2 was recorded until day 29 with single doses from 2 to 20 mg/kg and until day 43 with two doses of 20 mg/kg. CONCLUSIONS: AOD01 was safe and well tolerated, demonstrated dose-related PK, non-immunogenic status, and sustained ex vivo neutralization of SARS-CoV-2 after single intravenous dose ranging from 2 to 20 mg/kg and two doses of 20 mg/kg and show good potential for treatment of SARS-CoV-2 infection. (Health Sciences Authority identifier number CTA2000119).
RESUMEN
Dysregulation of translation initiation factor 4E (eIF4E) activity occurs in various cancers. Mitogen-activated protein kinase (MAPK) interacting kinases 1 and 2 (MNK1 and MNK2) play a fundamental role in activation of eIF4E. Structure-activity relationship-driven expansion of a fragment hit led to discovery of dual MNK1 and MNK2 inhibitors based on a novel pyridine-benzamide scaffold. The compounds possess promising in vitro and in vivo pharmacokinetic profiles and show potent on target inhibition of eIF4E phosphorylation in cells.
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Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Línea Celular Tumoral , Cristalografía por Rayos X , Descubrimiento de Drogas , Factor 4E Eucariótico de Iniciación/antagonistas & inhibidores , Humanos , Modelos Moleculares , Fosforilación , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacocinética , Relación Estructura-ActividadRESUMEN
[This corrects the article DOI: 10.1021/acsmedchemlett.9b00170.].
RESUMEN
SMYD3 is a histone methyltransferase that regulates gene transcription, and its overexpression is associated with multiple human cancers. A novel class of tetrahydroacridine compounds which inhibit SMYD3 through a covalent mechanism of action is identified. Optimization of these irreversible inhibitors resulted in the discovery of 4-chloroquinolines, a new class of covalent warheads. Tool compound 29 exhibits high potency by inhibiting SMYD3's enzymatic activity and showing antiproliferative activity against HepG2 in 3D cell culture. Our findings suggest that covalent inhibition of SMYD3 may have an impact on SMYD3 biology by affecting expression levels, and this warrants further exploration.
RESUMEN
Even though many GyrB and ParE inhibitors have been reported in the literature, few possess activity against Gram-negative bacteria. This is primarily due to limited permeability across Gram-negative bacterial membrane as well as bacterial efflux mechanisms. Permeability of compounds across Gram-negative bacterial membranes depends on many factors including physicochemical properties of the inhibitors. Herein, we show the optimization of pyridylureas leading to compounds with potent activity against Gram-negative bacterial species such as P.aeruginosa, E.coli and A.baumannii.
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Antibacterianos/farmacología , Girasa de ADN/metabolismo , Topoisomerasa de ADN IV/antagonistas & inhibidores , Descubrimiento de Drogas , Escherichia coli/efectos de los fármacos , Inhibidores de Topoisomerasa/farmacología , Antibacterianos/síntesis química , Antibacterianos/química , Topoisomerasa de ADN IV/metabolismo , Relación Dosis-Respuesta a Droga , Escherichia coli/enzimología , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus pneumoniae/enzimología , Relación Estructura-Actividad , Inhibidores de Topoisomerasa/síntesis química , Inhibidores de Topoisomerasa/químicaRESUMEN
Protein kinase C iota (PKC-ι) is an atypical kinase implicated in the promotion of different cancer types. A biochemical screen of a fragment library has identified several hits from which an azaindole-based scaffold was chosen for optimization. Driven by a structure-activity relationship and supported by molecular modeling, a weakly bound fragment was systematically grown into a potent and selective inhibitor against PKC-ι.
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Carcinoma Hepatocelular/tratamiento farmacológico , Proliferación Celular/efectos de los fármacos , Isoenzimas/antagonistas & inhibidores , Neoplasias Hepáticas/tratamiento farmacológico , Proteína Quinasa C/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Carcinoma Hepatocelular/patología , Humanos , Neoplasias Hepáticas/patología , Modelos Moleculares , Estructura Molecular , Conformación Proteica , Células Tumorales CultivadasRESUMEN
Chronic myeloid leukemia (CML) is a myeloproliferative disease caused by bcr-abl1, a constitutively active tyrosine kinase fusion gene responsible for an abnormal proliferation of leukemic stem cells (LSCs). Inhibition of BCR-ABL1 kinase activity offers long-term relief to CML patients. However, for a proportion of them, BCR-ABL1 inhibition will become ineffective at treating the disease, and CML will progress to blast crisis (BC) CML with poor prognosis. BC-CML is often associated with excessive phosphorylated eukaryotic translation initiation factor 4E (eIF4E), which renders LSCs capable of proliferating via self-renewal, oblivious to BCR-ABL1 inhibition. In vivo, eIF4E is exclusively phosphorylated on Ser209 by MNK1/2. Consequently, a selective inhibitor of MNK1/2 should reduce the level of phosphorylated eIF4E and re-sensitize LSCs to BCR-ABL1 inhibition, thus hindering the proliferation of BC LSCs. We report herein the structure-activity relationships and pharmacokinetic properties of a selective MNK1/2 inhibitor clinical candidate, ETC-206, which in combination with dasatinib prevents BC-CML LSC self-renewal in vitro and enhances dasatinib antitumor activity in vivo.
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Crisis Blástica/tratamiento farmacológico , Proliferación Celular , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Animales , Crisis Blástica/patología , Femenino , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Ratones , Ratones SCID , Modelos Moleculares , Estructura Molecular , Conformación Proteica , Inhibidores de Proteínas Quinasas/química , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Over the last few decades, resveratrol has gained significance due to its impressive array of biological activities; however, its true potential as a drug has been severely constrained by its poor bioavailability. Indeed, several studies have implicated this bioavailability trait as a major road-block to resveratrol's potential clinical applications. To mitigate this pharmacokinetic issue, we envisioned a tactical bioisosteric modification of resveratrol to bicyclo[1.1.1]pentane (BCP) resveratrol. Relying on the beneficial bioisosteric potential demonstrated by the BCP-scaffold, we hypothesized that BCP-resveratrol would have an inherently better in vivo PK profile as compared to its natural counterpart. To validate such a hypothesis, it was necessary to secure a synthetic access to this novel structure. Herein we describe the first synthesis of BCP-resveratrol and disclose its PK properties.
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Porcupine is an O-acyltransferase that regulates Wnt secretion. Inhibiting porcupine may block the Wnt pathway which is often dysregulated in various cancers. Consequently porcupine inhibitors are thought to be promising oncology therapeutics. A high throughput screen against porcupine revealed several potent hits that were confirmed to be Wnt pathway inhibitors in secondary assays. We developed a pharmacophore model and used the putative bioactive conformation of a xanthine inhibitor for scaffold hopping. The resulting maleimide scaffold was optimized to subnanomolar potency while retaining good physical druglike properties. A preclinical development candidate was selected for which extensive in vitro and in vivo profiling is reported.
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Aciltransferasas/antagonistas & inhibidores , Antineoplásicos/farmacología , Maleimidas/farmacología , Proteínas de la Membrana/antagonistas & inhibidores , Piridazinas/farmacología , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/síntesis química , Antineoplásicos/farmacocinética , Línea Celular Tumoral , Inhibidores del Citocromo P-450 CYP1A2/administración & dosificación , Inhibidores del Citocromo P-450 CYP1A2/síntesis química , Inhibidores del Citocromo P-450 CYP1A2/farmacocinética , Inhibidores del Citocromo P-450 CYP1A2/farmacología , Inhibidores del Citocromo P-450 CYP2D6/administración & dosificación , Inhibidores del Citocromo P-450 CYP2D6/síntesis química , Inhibidores del Citocromo P-450 CYP2D6/farmacocinética , Inhibidores del Citocromo P-450 CYP2D6/farmacología , Inhibidores del Citocromo P-450 CYP3A/administración & dosificación , Inhibidores del Citocromo P-450 CYP3A/síntesis química , Inhibidores del Citocromo P-450 CYP3A/farmacocinética , Inhibidores del Citocromo P-450 CYP3A/farmacología , Femenino , Células HEK293 , Ensayos Analíticos de Alto Rendimiento , Humanos , Maleimidas/administración & dosificación , Maleimidas/síntesis química , Maleimidas/farmacocinética , Ratones Endogámicos BALB C , Ratones Desnudos , Microsomas Hepáticos/metabolismo , Piridazinas/administración & dosificación , Piridazinas/síntesis química , Piridazinas/farmacocinética , Ratas , Relación Estructura-Actividad , Vía de Señalización Wnt , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
p53 tumor suppressor maintains genomic stability, typically acting through cell-cycle arrest, senescence, and apoptosis. We discovered a function of p53 in preventing conflicts between transcription and replication, independent of its canonical roles. p53 deficiency sensitizes cells to Topoisomerase (Topo) II inhibitors, resulting in DNA damage arising spontaneously during replication. Topoisomerase IIα (TOP2A)-DNA complexes preferentially accumulate in isogenic p53 mutant or knockout cells, reflecting an increased recruitment of TOP2A to regulate DNA topology. We propose that p53 acts to prevent DNA topological stress originating from transcription during the S phase and, therefore, promotes normal replication fork progression. Consequently, replication fork progression is impaired in the absence of p53, which is reversed by transcription inhibition. Pharmacologic inhibition of transcription also attenuates DNA damage and decreases Topo-II-DNA complexes, restoring cell viability in p53-deficient cells. Together, our results demonstrate a function of p53 that may underlie its role in tumor suppression.
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Replicación del ADN , Inestabilidad Genómica , Transcripción Genética , Proteína p53 Supresora de Tumor/metabolismo , Antígenos de Neoplasias/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Células HCT116 , Humanos , Proteínas de Unión a Poli-ADP-Ribosa , Inhibidores de Topoisomerasa/farmacología , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Clinically used BCR-ABL1 inhibitors for the treatment of chronic myeloid leukemia do not eliminate leukemic stem cells (LSC). It has been shown that MNK1 and 2 inhibitors prevent phosphorylation of eIF4E and eliminate the self-renewal capacity of LSCs. Herein, we describe the identification of novel dual MNK1 and 2 and BCR-ABL1 inhibitors, starting from the known kinase inhibitor 2. Initial structure-activity relationship studies resulted in compound 27 with loss of BCR-ABL1 inhibition. Further modification led to orally bioavailable dual MNK1 and 2 and BCR-ABL1 inhibitors 53 and 54, which are efficacious in a mouse xenograft model and also reduce the level of phosphorylated eukaryotic translation initiation factor 4E in the tumor tissues. Kinase selectivity of these compounds is also presented.
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Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Administración Oral , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Antineoplásicos/farmacología , Disponibilidad Biológica , Técnicas de Química Sintética , Relación Dosis-Respuesta a Droga , Factor 4E Eucariótico de Iniciación/metabolismo , Femenino , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Ratones SCID , Terapia Molecular Dirigida/métodos , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/química , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
The high proliferation rate of cancer cells, together with environmental factors such as hypoxia and nutrient deprivation can cause Endoplasmic Reticulum (ER) stress. The protein kinase PERK is an essential mediator in one of the three ER stress response pathways. Genetic and pharmacological inhibition of PERK has been reported to limit tumor growth in xenograft models. Here we provide evidence that inactive PERK interacts with the nuclear pore-associated Vault complex protein and that this compromises Vault-mediated nuclear transport of PTEN. Pharmacological inhibition of PERK under ER stress results is abnormal sequestration of the Vault complex, leading to increased cytoplasmic PTEN activity and lower AKT activation. As the PI3K/PTEN/AKT pathway is crucial for many aspects of cell growth and survival, this unexpected effect of PERK inhibitors on AKT activity may have implications for their potential use as therapeutic agents.
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Fosfohidrolasa PTEN/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Partículas Ribonucleoproteicas en Bóveda/metabolismo , eIF-2 Quinasa/metabolismo , Sustitución de Aminoácidos , Línea Celular Tumoral , Estrés del Retículo Endoplásmico/efectos de los fármacos , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/metabolismo , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Proteínas Nucleares/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacos , Partículas Ribonucleoproteicas en Bóveda/química , eIF-2 Quinasa/antagonistas & inhibidores , eIF-2 Quinasa/genéticaRESUMEN
Wnt proteins regulate various cellular functions and serve distinct roles in normal development throughout life. Wnt signaling is dysregulated in various diseases including cancers. Porcupine (PORCN) is a membrane-bound O-acyltransferase that palmitoleates the Wnts and hence is essential for their secretion and function. The inhibition of PORCN could serve as a therapeutic approach for the treatment of a number of Wnt-dependent cancers. Herein, we describe the identification of a Wnt secretion inhibitor from cellular high throughput screening. Classical SAR based cellular optimization provided us with a PORCN inhibitor with nanomolar activity and excellent bioavailability that demonstrated efficacy in a Wnt-driven murine tumor model. Finally, we also discovered that enantiomeric PORCN inhibitors show very different activity in our reporter assay, suggesting that such compounds may be useful for mode of action studies on the PORCN O-acyltransferase.
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Inhibidores Enzimáticos/farmacología , Proteínas de la Membrana/antagonistas & inhibidores , Aciltransferasas , Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Modelos Moleculares , Relación Estructura-Actividad , Vía de Señalización WntRESUMEN
Inflammatory responses contribute to the morbidity and mortality of severe influenza. Current antiviral therapy offers limited success in treating severe influenza infection with both H1N1 and H5N1 viruses. We evaluated the effect of a neuraminidase inhibitor in combination with immunomodulatory drugs in vitro and in a mouse model of influenza A H1N1 infection by determining survival rate, lung inflammation markers and histopathology. Sertraline and rolipram significantly improved survival in mice infected with a lethal dose of influenza A H1N1 virus. Prophylactic treatment resulted in survival rates of 40% (rolipram), 30% (oseltamivir), 0% (sertraline), 100% (rolipram/oseltamivir) and 70% (sertraline/oseltamivir). Treatment in a therapeutic setting (24 h post-infection) resulted in 80% (rolipram/oseltamivir) and 40% (sertraline/oseltamivir) survival. Sertraline and rolipram had no effect on virus replication in vitro and in vivo, but significantly reduced lung inflammation. A significant reduction in cellular infiltration (10-fold) along with inflammatory cytokines monocyte chemotactic protein-1 (10-fold), interleukin-6 (5-fold) and regulated on activation normal T cell expressed and secreted (5-fold) was observed in the animals treated with the combination compared to oseltamivir alone. Lung histopathology of mice treated with combinations revealed significantly reduced consolidation, infiltration and alveolitis compared to oseltamivir alone. Rolipram and sertraline reduced H1N1 virus-induced lung inflammation and mortality. These data support further development of immunomodulatory agents for severe influenza.
RESUMEN
Porcupine (PORCN) is a membrane bound O-acyltransferase that is required for Wnt palmitoylation, secretion, and biologic activity. All evaluable human Wnts require PORCN for their activity, suggesting that inhibition of PORCN could be an effective treatment for cancers dependent on excess Wnt activity. In this study, we evaluated the PORCN inhibitor Wnt-C59 (C59), to determine its activity and toxicity in cultured cells and mice. C59 inhibits PORCN activity in vitro at nanomolar concentrations, as assessed by inhibition of Wnt palmitoylation, Wnt interaction with the carrier protein Wntless/WLS, Wnt secretion, and Wnt activation of ß-catenin reporter activity. In mice, C59 displayed good bioavailability, as once daily oral administration was sufficient to maintain blood concentrations well above the IC(50). C59 blocked progression of mammary tumors in MMTV-WNT1 transgenic mice while downregulating Wnt/ß-catenin target genes. Surprisingly, mice exhibit no apparent toxicity, such that at a therapeutically effective dose there were no pathologic changes in the gut or other tissues. These results offer preclinical proof-of-concept that inhibiting mammalian Wnts can be achieved by targeting PORCN with small-molecule inhibitors such as C59, and that this is a safe and feasible strategy in vivo.
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Bencenoacetamidas/farmacología , Neoplasias de la Mama/enzimología , Proteínas de la Membrana/antagonistas & inhibidores , Piridinas/farmacología , Proteínas Wnt , Aciltransferasas , Animales , Neoplasias de la Mama/patología , Línea Celular Tumoral , Humanos , Ratones , Vía de Señalización Wnt , beta Catenina/metabolismoRESUMEN
Tankyrases constitute potential drug targets for cancer and myelin-degrading diseases. We have applied a structure- and biophysics-driven fragment-based ligand design strategy to discover a novel family of potent inhibitors for human tankyrases. Biophysical screening based on a thermal shift assay identified highly efficient fragments binding in the nicotinamide-binding site, a local hot spot for fragment binding. Evolution of the fragment hit 4-methyl-1,2-dihydroquinolin-2-one (2) along its 7-vector yields dramatic affinity improvements in the first cycle of expansion. A crystal structure of 7-(2-fluorophenyl)-4-methylquinolin-2(1H)-one (11) reveals that the nonplanar compound extends with its fluorine atom into a pocket, which coincides with a region of the active site where structural differences are seen between tankyrases and other poly(ADP-ribose) polymerase (PARP) family members. A further cycle of optimization yielded compounds with affinities and IC50 values in the low nanomolar range and with good solubility, PARP selectivity, and ligand efficiency.
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Quinolinas/síntesis química , Tanquirasas/antagonistas & inhibidores , Cristalografía por Rayos X , Bases de Datos Factuales , Estabilidad de Medicamentos , Humanos , Ligandos , Modelos Moleculares , Estructura Molecular , Unión Proteica , Quinolinas/química , Solubilidad , Relación Estructura-Actividad , Tanquirasas/química , TermodinámicaRESUMEN
Glioblastoma multiforme (GBM) is the most common and prognostically unfavorable form of brain tumor. The aggressive and highly invasive phenotype of these tumors makes them among the most anatomically damaging human cancers with a median survival of less than 1 year. Although canonical Wnt pathway activation in cancers has been historically linked to the presence of mutations involving key components of the pathway (APC, ß-catenin, or Axin proteins), an increasing number of studies suggest that elevated Wnt signaling in GBM is initiated by several alternative mechanisms that are involved in different steps of the disease. Therefore, inhibition of Wnt signaling may represent a therapeutically relevant approach for GBM treatment. After the selection of a GBM cell model responsive to Wnt inhibition, we set out to develop a screening approach for the identification of compounds capable of modulating canonical Wnt signaling and associated proliferative responses in GBM cells. Here, we show that the small molecule SEN461 inhibits the canonical Wnt signaling pathway in GBM cells, with relevant effects at both molecular and phenotypic levels in vitro and in vivo. These include SEN461-induced Axin stabilization, increased ß-catenin phosphorylation/degradation, and inhibition of anchorage-independent growth of human GBM cell lines and patient-derived primary tumor cells in vitro. Moreover, in vivo administration of SEN461 antagonized Wnt signaling in Xenopus embryos and reduced tumor growth in a GBM xenograft model. These data represent the first demonstration that small-molecule-mediated inhibition of Wnt signaling may be a potential approach for GBM therapeutics.