RESUMEN
ALKBH4 is a versatile demethylase capable of catalyzing the demethylation of monomethylated lysine-84 on actin and N6 -methyladenine in DNA. In this study, we conducted a quantitative proteomic experiment to reveal the altered expression of proteins in HEK293T cells upon genetic ablation of ALKBH4. Our results showed markedly diminished levels of GSTP1 and HSPB1 proteins in ALKBH4-depleted cells, which emanate from an augmented expression level of DNA (cytosine-5)-methyltransferase 1 (DNMT1) and the ensuing elevated cytosine methylation in the promoter regions of GSTP1 and HSPB1 genes. Together, our results revealed a role of ALKBH4 in modulating DNA cytosine methylation through regulating the expression level of DNMT1 protein.
Asunto(s)
Homólogo 4 de AlkB Lisina Desmetilasa , Metilación de ADN , Actinas/metabolismo , Homólogo 4 de AlkB Lisina Desmetilasa/genética , Homólogo 4 de AlkB Lisina Desmetilasa/metabolismo , ADN/metabolismo , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Células HEK293 , Humanos , ProteómicaRESUMEN
N6-Methyladenosine (m6A) and its reader, writer, and eraser (RWE) proteins assume crucial roles in regulating the splicing, stability, and translation of mRNA. Aside from m6A, RNA is known to carry many other types of chemical modifications; no systematic investigations, however, have been conducted about the crosstalk between m6A and other modified nucleosides in RNA. Here, we modified our recently established liquid chromatography-parallel-reaction monitoring (LC-PRM) method by incorporating stable isotope-labeled (SIL) peptides as internal or surrogate standards for profiling epitranscriptomic RWE proteins. We were able to detect reproducibly a total of 114 RWE proteins in HEK293T cells with the genes encoding m6A eraser proteins (i.e., ALKBH5, FTO) and the catalytic subunit of the major m6A writer complex (i.e., METTL3) being individually ablated. Notably, eight proteins, including writer proteins for 5-methylcytidine and pseudouridine, were altered by more than 1.5-fold in the opposite directions in HEK293T cells depleted of METTL3 and ALKBH5. Analysis of previously published m6A mapping results revealed the presence of m6A in the corresponding mRNAs for four of these proteins. Together, we integrated SIL peptides into our LC-PRM method for quantifying epitranscriptomic RWE proteins, and our work revealed potential crosstalks between m6A and other epitranscriptomic modifications. Our modified LC-PRM method with the use of SIL peptides should be applicable for high-throughput profiling of epitranscriptomic RWE proteins in other cell types and in tissues.
Asunto(s)
Adenosina , Seudouridina , Adenosina/metabolismo , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismo , Células HEK293 , Humanos , Isótopos , Metiltransferasas/metabolismo , Péptidos , Proteínas , ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Understanding the occurrence, repair, and biological consequences of DNA damage is important in environmental toxicology and risk assessment. The most common way to assess DNA damage elicited by exogenous sources in a laboratory setting is to expose cells or experimental animals with chemicals that modify DNA. Owing to the lack of reaction specificities of DNA damaging agents, the approach frequently does not allow for induction of a specific DNA lesion. Herein, we employed metabolic labeling to selectively incorporate N2-methyl-dG (N2-MedG) and N2-n-butyl-dG (N2-nBudG) into genomic DNA of cultured mammalian cells, and investigated how the levels of the two lesions in cellular DNA are modulated by different DNA repair factors. Our results revealed that nucleotide excision repair (NER) exert moderate effects on the removal of N2-MedG and N2-nBudG from genomic DNA. We also observed that DNA polymerases κ and η contribute to the incorporation of N2-MedG into genomic DNA and modulate its repair in human cells. In addition, loss of ALKBH3 resulted in higher frequencies of N2-MedG and N2-nBuG incorporation into genomic DNA, suggesting a role of oxidative dealkylation in the reversal of these lesions. Together, our study provided new insights into the repair of minor-groove N2-alkyl-dG lesions in mammalian cells.
Asunto(s)
Desoxiguanosina , Espectrometría de Masas en Tándem , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/genética , Animales , Cromatografía Liquida , ADN , Daño del ADN , Reparación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Desoxiguanosina/metabolismo , Genómica , Humanos , Mamíferos/genética , Mamíferos/metabolismoRESUMEN
Protein α-N-methylation is an evolutionarily conserved type of post-translational modification; however, little is known about the regulatory mechanisms for this modification. Methylation at the N6 position of adenosine in mRNAs is dynamic and modulates their stability, splicing, and translational efficiency. Here, we found that the expression of N-terminal methyltransferase 1 (NTMT1) protein is altered by depletion of those genes encoding the reader/writer/eraser proteins of N6-methyladenosine (m6A). We also observed that MRG15 is N-terminally methylated by NTMT1, and this methylation could also be modulated by reader/writer/eraser proteins of m6A. Together, these results revealed a novel m6A-based epitranscriptomic mechanism in regulating protein N-terminal methylation.
Asunto(s)
Adenosina/análogos & derivados , Epigénesis Genética , Metiltransferasas/genética , Metiltransferasas/metabolismo , Transcriptoma , Adenosina/metabolismo , Células HEK293 , Humanos , Metilación , Metiltransferasas/biosíntesis , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
N6-Methyladenosine (m6A) in messenger RNA (mRNA) regulates its stability, splicing, and translation efficiency. Here, we explored how the expression levels of small GTPase proteins are regulated by m6A modulators. We employed a high-throughput scheduled multiple-reaction monitoring (MRM)-based targeted proteomic approach to quantify systemically the changes in expression of small GTPase proteins in cells upon genetic ablation of METTL3 (the catalytic subunit of the major m6A methyltransferase complex), m6A demethylases (ALKBH5 and FTO), or m6A reader proteins (YTHDF1, YTHDF2, and YTHDF3). Depletions of METTL3 and ALKBH5 resulted in substantially diminished and augmented expression, respectively, of a subset of small GTPase proteins, including RHOB and RHOC. Our results also revealed that the stability of RHOB mRNA is significantly increased in cells depleted of METTL3, suggesting an m6A-elicited destabilization of this mRNA. Those small GTPases that are targeted by METTL3 and/or ALKBH5 also displayed higher discrepancies between protein and mRNA expression in paired primary/metastatic melanoma or colorectal cancer cells than those that are not. Together, this is the first comprehensive analysis of the alterations in small GTPase proteome regulated by epitranscriptomic modulators of m6A, and our study suggests the potential of an alternative therapeutic approach to target the currently "undruggable" small GTPases.
Asunto(s)
Adenosina/análogos & derivados , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteoma/metabolismo , Adenosina/metabolismo , Células Cultivadas , Cromatografía Liquida , Células HEK293 , Humanos , Proteínas de Unión al GTP Monoméricas/análisis , Proteínas de Unión al GTP Monoméricas/genética , Proteoma/análisis , Proteoma/genética , Espectrometría de Masas en TándemRESUMEN
Isoprenoid pyrophosphates are involved in protein prenylation and assume regulatory roles in cells; however, little is known about the cellular proteins that can interact with isoprenoid pyrophosphates. Here, we devised a chemical proteomic strategy, capitalizing on the use of a desthiobiotin-geranyl pyrophosphate (GPP) acyl phosphate probe for the enrichment and subsequent identification of GPP-binding proteins using liquid chromatography-tandem mass spectrometry (LC-MS/MS). By combining stable isotope labeling by amino acids in cell culture (SILAC) and competitive labeling with low vs high concentrations of GPP probe, with ATP vs GPP acyl phosphate probes, or with the GPP probe in the presence of different concentrations of free GPP, we uncovered a number of candidate GPP-binding proteins. We also discovered, for the first time, histone deacetylase 1 (HDAC1) as a GPP-binding protein. Furthermore, we found that the enzymatic activity of HDAC1 could be modulated by isoprenoid pyrophosphates. Together, we developed a novel chemical proteomic method for the proteome-wide discovery of GPP-binding proteins, which sets the stage for a better understanding about the biological functions of isoprenoids.
Asunto(s)
Biotina/análogos & derivados , Histona Desacetilasa 1/química , Fosfatos de Poliisoprenilo/química , Proteómica , Biotina/química , Histona Desacetilasa 1/metabolismo , Humanos , Estructura MolecularRESUMEN
Acute myeloid leukemia (AML) is reported to be vulnerable to transcription disruption due to transcriptional addiction. Cyclin-dependent kinase 9 (CDK9), which regulates transcriptional elongation, has attracted extensive attention as a drug target. Although several inhibitors, such as alvocidib and dinaciclib, have shown potent therapeutic effects in clinical trials on AML, the lack of high selectivity for CDK9 and other CDKs has limited their optimal clinical efficacy. Therefore, developing highly selective CDK9 inhibitors is still imperative for the efficacy and safety profile in treating AML. Here, we report a novel highly selective CDK9 inhibitor, JSH-009, which exhibited high potency against CDK9 and displayed great selectivity over 468 kinases/mutants. It also demonstrates impressive in vitro and in vivo antileukemic efficacy in preclinical models of AML, which makes JSH-009 a useful pharmacological tool for elucidating CDK9-mediated transcription and a novel therapeutic candidate for AML.
Asunto(s)
Antineoplásicos/uso terapéutico , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Leucemia Mieloide Aguda/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Animales , Antineoplásicos/farmacología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Humanos , Leucemia Mieloide Aguda/patología , Ratones Endogámicos NOD , Ratones SCID , Inhibidores de Proteínas Quinasas/farmacología , Resultado del Tratamiento , Carga Tumoral/efectos de los fármacosRESUMEN
BACKGROUND An increasing number of studies have demonstrated that Streptococcus bovis and its concomitant inflammatory factors concentrate in the intestine in colorectal cancer (CRC). However, the molecular mechanism of S. bovis on colorectal tumorigenesis remains unclear. This study aimed to explore the role of S. bovis in carcinogenesis and its potential mechanism in CRC of mice orally pretreated with S. bovis. MATERIAL AND METHODS The colons of experimental mice were collected and evaluated for the extent of neoplasm. In addition, comparative feces DNA sequencing was adopted to verify the abundance change of S. bovis during the progression of CRC in patients. RESULTS The results of this study found that S. bovis is more likely to be present at higher levels in patients with progressive colorectal carcinoma compared to those adenoma patients and healthy volunteers (P<0.05). Pretreatment with S. bovis aggravated tumor formation in mice, resulting in more substantial and a higher number of tumor nodes (P<0.05). A cytokine expression pattern with increased levels of IL-6, Scyb1, Ptgs2, IL-1ß, TNF, and Ccl2 was detected in S. bovis pretreated CRC mice (all P<0.05). Furthermore, S. bovis recruited myeloid cells, especially CD11bâºTLR-4⺠cells, which could promote pro-tumor immunity in the tumor microenvironment (P<0.05). CONCLUSIONS Collectively, our study indicates that S. bovis may induce a suppressive immunity that is conducive to CRC by recruiting tumor-infiltrating CD11bâºTLR-4⺠cells. In conclusion, S. bovis contributes to colorectal tumorigenesis via recruiting CD11bâºTLR-4⺠cells.
Asunto(s)
Adenoma/microbiología , Carcinogénesis/inmunología , Neoplasias del Colon/microbiología , Neoplasias Colorrectales/microbiología , Regulación Neoplásica de la Expresión Génica , Streptococcus bovis/patogenicidad , Adenoma/genética , Adenoma/inmunología , Adenoma/patología , Anciano , Animales , Carga Bacteriana , Antígeno CD11b/genética , Antígeno CD11b/inmunología , Carcinogénesis/genética , Carcinogénesis/patología , Estudios de Casos y Controles , Quimiocina CCL2/genética , Quimiocina CCL2/inmunología , Quimiocina CXCL1/genética , Quimiocina CXCL1/inmunología , Neoplasias del Colon/genética , Neoplasias del Colon/inmunología , Neoplasias del Colon/patología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/patología , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/inmunología , Heces/microbiología , Femenino , Humanos , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Interleucina-6/genética , Interleucina-6/inmunología , Metástasis Linfática , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Células Mieloides/inmunología , Células Mieloides/microbiología , Streptococcus bovis/crecimiento & desarrollo , Streptococcus bovis/inmunología , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunología , Microambiente Tumoral/genética , Microambiente Tumoral/inmunología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
BACKGROUND: For colorectal liver metastasis (CRLM) patients, hepatic resection is currently the sole cure offering the chance of long-term survival. Tumor shrinkage and planned liver remnant hypertrophy are the two key strategies for conversion of initially unresectable CRLM. First conducted in 2012, associated liver partition and portal vein ligation for staged hepatectomy (ALPPS) allows rapid liver growth. As a means to induce hypertrophy, portal vein embolization (PVE) has been widely applied before extending hepatectomy. Recently, Peng et al. present a new approach of terminal branches portal vein embolization (TBPVE), offering an efficient way to amplify FLR and making chances for surgery in 2 weeks. CASE PRESENTATION: We reported a 61-year-old woman with synchronous hepatic metastasized carcinoma of the colon sigmoideum underwent TBPVE after 6 cycles of neoadjuvant therapy in order to perform a planned right trisectionectomy. Rapid liver remnant hypertrophy and remarkable tumor shrinkage were achieved, and laparoscopic sigmoidectomy and right trisectionectomy were successfully performed. The postsurgical course was uneventful and 7 months of recurrence-free survival have been witnessed. CONCLUSIONS: The dual tactics of tumor shrinkage and planned rapid liver remnant hypertrophy will make concerted efforts to further increase the clinical candidacy for curative resection, which are valuable for further investigation.
Asunto(s)
Carcinoma/terapia , Neoplasias Colorrectales/terapia , Hepatectomía/métodos , Neoplasias Hepáticas/terapia , Regeneración Hepática , Hígado/fisiología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Camptotecina/análogos & derivados , Camptotecina/uso terapéutico , Antígeno Carcinoembrionario/sangre , Carcinoma/sangre , Carcinoma/diagnóstico por imagen , Carcinoma/secundario , Colon Sigmoide/diagnóstico por imagen , Colon Sigmoide/patología , Colon Sigmoide/cirugía , Colonoscopía , Neoplasias Colorrectales/diagnóstico por imagen , Neoplasias Colorrectales/patología , Embolización Terapéutica , Femenino , Fluorouracilo/uso terapéutico , Humanos , Hipertrofia , Laparoscopía , Leucovorina/uso terapéutico , Ligadura , Hígado/irrigación sanguínea , Hígado/diagnóstico por imagen , Hígado/cirugía , Neoplasias Hepáticas/sangre , Neoplasias Hepáticas/diagnóstico por imagen , Neoplasias Hepáticas/secundario , Imagen por Resonancia Magnética , Persona de Mediana Edad , Terapia Neoadyuvante/métodos , Vena Porta , Pronóstico , Resultado del Tratamiento , Procedimientos Quirúrgicos Vasculares/métodosRESUMEN
Drug resistance remains a major challenge in the clinical treatment of gastrointestinal stromal tumours (GISTs). While acquired on-target mutations of mast/stem cell growth factor receptor (KIT) kinase is the major resistance mechanism, activation of alternative signalling pathways may also play a role. Although several second- and third-generation KIT kinase inhibitors have been developed that could overcome some of the KIT mutations conferring resistance, the low clinical responses and narrow safety window have limited their broad application. The present study revealed that nintedanib not only overcame resistance induced by a panel of KIT primary and secondary mutations, but also overcame ERK-reactivation-mediated resistance caused by the upregulation of fibroblast growth factor (FGF) activity. In preclinical models of GISTs, nintedanib significantly inhibited the proliferation of imatinib-resistant cells, including GIST-5R, GIST-T1/T670I and GIST patient-derived primary cells. In addition, it also exhibited dose-dependent inhibition of ERK phosphorylation upon FGF ligand stimulation. In vivo antitumour activity was also observed in several xenograft GIST models. Considering the well-documented safety and pharmacokinetic profiles of nintedanib, this finding provides evidence for the repurposing of nintedanib as a new therapy for the treatment of GIST patients with de novo or acquired resistance to imatinib.
Asunto(s)
Antineoplásicos , Tumores del Estroma Gastrointestinal , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Resistencia a Antineoplásicos/genética , Tumores del Estroma Gastrointestinal/tratamiento farmacológico , Tumores del Estroma Gastrointestinal/genética , Tumores del Estroma Gastrointestinal/patología , Humanos , Mesilato de Imatinib/farmacología , Mesilato de Imatinib/uso terapéutico , Indoles , Mutación/genética , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , Pirimidinas/farmacología , Regulación hacia Arriba/genéticaRESUMEN
BACKGROUND: Obstructed defecation syndrome (ODS) is a condition that is frequently caused by rectocele and rectal intussusception. This study aimed to evaluate the effectiveness of a modified Bresler procedure for the treatment of ODS. The outcomes of this modified procedure were compared with the stapled transanal rectal resection (STARR) procedure. METHODS: We performed a retrospective analysis of the clinical data from 76 female patients who presented with ODS between June 2014 and June 2016. The patients were divided into two treatment groups, namely Modified and STARR. Patients in the Modified group (n = 36) underwent the modified Bresler procedure, which involved posterior rectal-wall resection using a circular tubular stapler with multilevel purse-string sutures. Patients in the STARR group (n = 40) underwent the standard STARR procedure. We analysed post-operative complications, Wexner constipation scores (WCS), rectocele depths, and four-point post-operative satisfaction scales. RESULTS: Patients in the Modified group exhibited shorter operative times and fewer post-operative complications (both P < 0.05). At 12 months post-operatively, both the Modified and STARR groups displayed a significant improvement in the Wexner constipation score and the depth of rectocele. The post-operative WCS for the Modified group were significantly improved compared to those for the STARR group (P < 0.05), while there was no significant difference in the rectocele depth between the two groups (P > 0.05). Post-operative interviews at post-operative 12 months showed that patients in the Modified group had a better satisfaction (P = 0.05). CONCLUSIONS: Our modified procedure may be an effective treatment strategy for patients experiencing ODS caused by rectocele and rectal intussusception, with fewer complications and effective relief of symptoms.
RESUMEN
Angiogenesis is an essential process in tumor growth, invasion and metastasis. VEGF receptor 2 (VEGFR2) inhibitors targeting tumor angiogenic pathway have been widely used in the clinical cancer treatment. However, most of currently used VEGFR2 kinase inhibitors are multi-target inhibitors which might result in target-associated side effects and therefore limited clinical toleration. Highly selective VEGFR inhibitors are still highly demanded from both basic research and clinical application point of view. Here we report the discovery and characterization of a novel VEGFR2 inhibitor (CHMFL-VEGFR2-002), which exhibited high selectivity among structurally closed kinases including PDGFRs, FGFRs, CSF1R, etc. CHMFL-VEGFR2-002 displayed potent inhibitory activity against VEGFR2 kinase in the biochemical assay (IC50 = 66 nmol/L) and VEGFR2 autophosphorylation in cells (EC50s â¼100 nmol/L) as well as potent anti-proliferation effect against VEGFR2 transformed BaF3 cells (GI50 = 150 nmol/L). In addition, CHMFL-VEGFR2-002 also displayed good anti-angiogenesis efficacy in vitro and exhibited good in vivo PK (pharmacokinetics) profile with bioavailability over 49% and anti-angiogenesis efficacy in both zebrafish and mouse models without apparent toxicity. These results suggest that CHMFL-VEGFR2-002 might be a useful research tool for dissecting new functions of VEGFR2 kinase as well as a potential anti-angiogenetic agent for the cancer therapy.
RESUMEN
All-trans retinoic acid (ATRA) is known to be a potent inhibitor of FLT3-ITD acute myeloid leukemia (AML) cells, although the exact mechanism remains unclear. In this work, we report that ATRA causes fatal mitotic catastrophe in FLT3-ITD AML cells by degrading Chk1 kinase, and therefore preventing DNA damage repair. In order to explore a further enhancement in the inhibitory effect of ATRA on FLT3-ITD AML cells, we investigated the suitability of a combination of ATRA and DNA damage drug SN38. In vitro experiments showed that this combinatorial approach effectively inhibited the proliferation of FLT3-ITD cells and induced cell apoptosis in AML. In vivo experiments confirmed that the combination could substantially improve the anti-tumor effect of SN38. Taken together, our results indicate that ATRA down-regulates Chk1 in FLT3-ITD AML cells, and the combination of ATRA and SN38 significantly improves the anti-tumor effect of either ATRA or SN38 when used alone.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Leucemia Mieloide Aguda/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Tretinoina/farmacología , Adulto , Anciano , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Apoptosis/efectos de los fármacos , Apoptosis/genética , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Preescolar , Daño del ADN/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Sinergismo Farmacológico , Humanos , Irinotecán/farmacología , Irinotecán/uso terapéutico , Leucemia Mieloide Aguda/sangre , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Masculino , Ratones , Persona de Mediana Edad , Mitosis/efectos de los fármacos , Cultivo Primario de Células , Inhibidores de Proteínas Quinasas/uso terapéutico , Secuencias Repetidas en Tándem/genética , Inhibidores de Topoisomerasa I/farmacología , Inhibidores de Topoisomerasa I/uso terapéutico , Tretinoina/uso terapéutico , Ensayos Antitumor por Modelo de Xenoinjerto , Tirosina Quinasa 3 Similar a fms/genéticaRESUMEN
Gain-of-function mutations of c-KIT kinase play crucial pathological roles for the gastrointestinal stromal tumors (GISTs). Despite the success of imatinib as the first-line treatment of GISTs, dozens of drug-acquired resistant mutations emerge, and c-KIT T670I is one of the most common mutants among them. Although several kinase inhibitors are capable of overcoming the T670I mutant, none of them can achieve the selectivity over the c-KIT wild-type (wt), which also plays important roles in a variety of physiological functions such as hematopoiesis. Starting from axitinib, through fragment hybrid type II kinase inhibitor design approach, we have discovered a novel inhibitor 24, which not only exhibits potent activity to c-KIT T670I mutant but also achieves 12-fold selectivity over c-KIT wt. Compound 24 displays good antiproliferative effects against c-KIT T670I mutant-driven GIST cell lines (GIST-T1/T670I and GIST-5R) and also exhibits suitable in vivo pharmacokinetic profiles as well as dose-dependent antitumor efficacy. This study provides a proof of concept for developing a c-KIT mutant selective inhibitor that theoretically can render a better therapeutic window.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Neoplasias Gastrointestinales/tratamiento farmacológico , Tumores del Estroma Gastrointestinal/tratamiento farmacológico , Indazoles/farmacología , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-kit/genética , Animales , Antineoplásicos/farmacocinética , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Diseño de Fármacos , Resistencia a Antineoplásicos , Femenino , Humanos , Indazoles/síntesis química , Indazoles/farmacocinética , Ratones , Modelos Moleculares , Simulación del Acoplamiento Molecular , Mutación , Inhibidores de Proteínas Quinasas/farmacocinética , Ratas , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
Most of the current FMS-like tyrosine kinase 3 (FLT3) inhibitors lack selectivity between FLT3 kinase and cKIT kinase as well as the FLT3 wt and internal tandem duplication (ITD) mutants. We report a new compound 27, which displays GI50 values of 30-80 nM against different ITD mutants and achieves selectivity over both FLT3 wt (8-fold) and cKIT kinase in the transformed BaF3 cells (>300-fold). 27 potently inhibits the proliferation of the FLT3-ITD-positive acute myeloid leukemia cancer lines through suppression of the phosphorylation of FLT3 kinase and downstream signaling pathways, induction of apoptosis, and arresting the cell cycle into the G0/G1 phase. 27 also displays potent antiproliferative effect against FLT3-ITD-positive patient primary cells, whereas it does not apparently affect FLT3 wt primary cells. In addition, it also exhibits a good therapeutic window to PBMC compared to PKC412. In the in vivo studies, 27 demonstrates favorable PK profiles and suppresses the tumor growth in the MV4-11 cell inoculated mouse xenograft model.
Asunto(s)
Acetamidas/química , Inhibidores de Proteínas Quinasas/química , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Acetamidas/farmacología , Acetamidas/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Sitios de Unión , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Femenino , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Ratones , Ratones Desnudos , Simulación de Dinámica Molecular , Mutagénesis , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Estructura Terciaria de Proteína , Transducción de Señal/efectos de los fármacos , Relación Estructura-Actividad , Tirosina Quinasa 3 Similar a fms/genética , Tirosina Quinasa 3 Similar a fms/metabolismoRESUMEN
BACKGROUND: cKIT kinase overexpression and gain-of-function mutations are the critical pathogenesis of gastrointestinal stromal tumors (GISTs). Although the multiple kinase inhibitors such as imatinib, sunitinib, and regorafenib have been approved for GISTs, the acquisition of polyclonal secondary resistance mutations in KIT is still a limitation for GIST treatment. Here we explored the KIT inhibitory activity of axitinib in preclinical models and describe initial characterization of its activity in GIST patient-derived primary cells. METHODS: The activities of axitinib against mutant KIT were evaluated using protein-based assay and a panel of engineered and GIST-derived cell lines. The binding modes of axitinib-KIT/KIT mutants were analyzed. Four primary cells derived from GIST patients were also used to assess the drug response of axitinib. RESULTS: Axitinib exhibited potent activities against a variety of cKIT associated primary and secondary mutations. It displayed better activity against cKIT wild-type, cKIT V559D/A/G, and L576P primary gain-of-function mutations than imatinib, sunitinib, and regorafenib. In addition, it could inhibit imatinib resistant cKIT T670I and V654A mutants in vitro and in vivo GIST preclinical models. CONCLUSION: Our results provide the basis for extending the application of axitinib to GISTs patients who are unresponsive or intolerant to the current therapies.
RESUMEN
PI3Kδ, which is mainly expressed in leukocytes, plays a critical role in B-cell receptor mediated signaling pathway and has been extensively studied as a drug discovery target for B cell malignances such as AML, CLL etc. In this manuscript, we report the discovery, SAR optimization and pharmacological evaluation of a novel series of aminothiazole-pyridine containing PI3Kδ inhibitors. Among them compound 15i (CHMFL-PI3KD-317) displays an IC50 of 6â¯nM against PI3Kδ in the ADP-Glo biochemical assays. It also exhibits over 10-1500 fold selectivity over other class I, II and III PIKK family isoforms. In addition, in the cellular context, 15i can selectively and potently inhibit PI3Kδ mediated phosphorylation of Akt T308 but not PI3Kα, ß, γ mediated Akt phosphorylation. 15i also exhibits an excellent selectivity profile in the protein kinases including 468 kinases/mutants at the concentration of 1⯵M. 15i has acceptable pharmacokinetic properties and can dose-dependently inhibit the tumor growth of AML cell line MOLM14 inoculated xenograft mouse model. The high selectivity and potency makes 15i a potential valuable addition to the current PI3Kδ armory.
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Antineoplásicos/farmacología , Fosfatidilinositol 3-Quinasa Clase I/antagonistas & inhibidores , Leucemia Mieloide Aguda/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Tiazoles/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Descubrimiento de Drogas , Femenino , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Masculino , Ratones Desnudos , Simulación del Acoplamiento Molecular , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/uso terapéutico , Piridinas/química , Piridinas/farmacocinética , Piridinas/uso terapéutico , Ratas Sprague-Dawley , Tiazoles/química , Tiazoles/farmacocinética , Tiazoles/uso terapéuticoRESUMEN
Currently there are several irreversible BTK inhibitors targeting Cys481 residue under preclinical or clinical development. However, most of these inhibitors also targeted other kinases such as BMX, JAK3, and EGFR that bear the highly similar active cysteine residues. Through a structure-based drug design approach, we discovered a highly potent (IC50: 7 nM) irreversible BTK inhibitor compound 9 (CHMFL-BTK-01), which displayed a high selectivity profile in KINOMEscan (S score (35) = 0.00) among 468 kinases/mutants at the concentration of 1 µM. Compound 9 completely abolished BMX, JAK3 and EGFR's activity. Both X-ray crystal structure and cysteine-serine mutation mediated rescue experiment confirmed 9's irreversible binding mode. 9 also potently inhibited BTK Y223 auto-phosphorylation (EC50: <30 nM), arrested cell cycle in G0/G1 phase and induced apoptosis in U2932 and Pfeiffer cells. We believe these features would make 9 a good pharmacological tool to study the BTK related pathology.
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Benzamidas/farmacología , Descubrimiento de Drogas , Morfolinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Agammaglobulinemia Tirosina Quinasa , Benzamidas/síntesis química , Benzamidas/química , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Morfolinas/síntesis química , Morfolinas/química , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Proteínas Tirosina Quinasas/metabolismo , Relación Estructura-ActividadRESUMEN
The discovery of a novel potent type II ABL/c-KIT dual kinase inhibitor compound 34 (CHMFL-ABL/KIT-155), which utilized a hydrogen bond formed by NH on the kinase backbone and carbonyl oxygen of 34 as a unique hinge binding, is described. 34 potently inhibited purified ABL (IC50: 46 nM) and c-KIT kinase (IC50: 75 nM) in the biochemical assays and displayed high selectivity (S Score (1) = 0.03) at the concentration of 1 µM among 468 kinases/mutants in KINOMEscan assay. It exhibited strong antiproliferative activities against BCR-ABL/c-KIT driven CML/GISTs cancer cell lines through blockage of the BCR-ABL/c-KIT mediated signaling pathways, arresting cell cycle progression and induction of apoptosis. 34 possessed a good oral PK property and effectively suppressed the tumor progression in the K562 (CML) and GIST-T1 (GISTs) cells mediated xenograft mouse model. The distinct hinge-binding mode of 34 provided a novel pharmacophore for expanding the chemical structure diversity for the type II kinase inhibitors discovery.