RESUMO
The kinase domain is highly conserved among protein kinases 'in terms of both sequence and structure. Conformational rearrangements of the kinase domain are affected by the phosphorylation of residues and the binding of kinase inhibitors. Interestingly, the conformational rearrangement of the active pocket plays an important role in kinase activity and can be used to design novel kinase inhibitors. We characterized the conformational plasticity of the active pocket when bosutinib was bound to salt-inducible kinase 2 (SIK2) using homology modeling and molecular dynamics simulations. Ten different initial complex models were constructed using the Morph server, ranging from open to closed conformations of SIK2 binding with bosutinib. Our simulation showed that bosutinib binds SIK2 with up or down conformations of the P-loop and with all the conformations of the activation loop. In addition, the αC-helix conformation was induced by the conformation of the activation loop, and the salt bridge formed only with its open conformation. The binding affinity of the models was also determined using the molecular mechanics generalized Born surface area method. Bosutinib was found to form a strong binding model with SIK2 and hydrophobic interactions were the dominant factor. This discovery may help guide the design of novel SIK2 inhibitors.
RESUMO
Janus kinase 2 (JAK2) hyperactivation by JAK2V617F mutation leads to myeloproliferative neoplasms (MPNs) and targeting JAK2 could serve as a promising therapeutic strategy for MPNs. Here, we report that Flonoltinib Maleate (FM), a selective JAK2/FLT3 inhibitor, shows high selectivity for JAK2 over the JAK family. Surface plasmon resonance assays verified that FM had a stronger affinity for the pseudokinase domain JH2 than JH1 of JAK2 and had an inhibitory effect on JAK2 JH2V617F. The cocrystal structure confirmed that FM could stably bind to JAK2 JH2, and FM suppressed endogenous colony formation of primary erythroid progenitor cells from patients with MPNs. In several JAK2V617F-induced MPN murine models, FM could dose-dependently reduce hepatosplenomegaly and prolong survival. Similar results were observed in JAK2V617F bone marrow transplantation mice. FM exhibited strong inhibitory effects on fibrosis of the spleen and bone marrow. Long-term FM treatment showed good pharmacokinetic/pharmacodynamic characteristics with high drug exposure in tumor-bearing tissues and low toxicity. Currently, FM has been approved by the National Medical Products Administration of China (CXHL2000628), and this study will guide clinical trials for patients with MPNs.
Assuntos
Transtornos Mieloproliferativos , Neoplasias , Animais , Humanos , Janus Quinase 2 , Maleatos/uso terapêutico , Camundongos , Mutação , Transtornos Mieloproliferativos/tratamento farmacológico , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/metabolismo , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
FMS-like tyrosine kinase-3 (FLT3) and cyclin-dependent kinase 4/6 (CDK4/6) inhibitors have been proven to play a significant role in tumor therapy. Herein, based on the previously reported JAK2/FLT3 inhibitor 18e, we described the synthesis, structure-activity relationship and biological evaluation of a series of unique 6-(pyrimidin-4-yl)-1H-pyrazolo[4,3-b]pyridine derivatives that inhibited FLT3 and CDK4 kinases. The optimized compound 23k exhibited low nanomolar range activities with IC50 values of 11 and 7 nM for FLT3 and CDK4, respectively. In the MV4-11 xenograft tumor model, the tumor growth inhibition rate of 23k dosed at 200 mg/kg was 67%, suggesting that 23k possessed an antitumor therapeutic effect.
Assuntos
Antineoplásicos , Leucemia Mieloide Aguda , Linhagem Celular Tumoral , Proliferação de Células , Quinase 4 Dependente de Ciclina , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Inibidores de Proteínas Quinases , Piridinas , Relação Estrutura-Atividade , Tirosina Quinase 3 Semelhante a fmsRESUMO
Given the maturation of small-interfering RNA (siRNA) techniques with nanotechnology, and because overexpression of human programmed death-ligand 1 (PD-L1) is crucial for T cell inactivation and immunosuppression of the tumor microenvironment, application of siRNA-PD-L1 has demonstrated positive progress in preclinical studies; however, the limited penetration of this compound into solid tumors remains a challenge. To decrease PD-L1 expression and increase the penetration efficacy of solid tumors, we synthesized a novel tumor-microenvironment-sensitive delivery polymer by conjugating hyaluronic acid (HA) to polyethyleneimine (PEI), with a matrix metalloproteinase-2 (MMP-2)-sensitive peptide acting as the linker (HA-P-PEI), for use in delivery of PD-L1-siRNA. Concurrent synthesis of a linker-less HA-PEI compound allowed confirmation that negatively charged siRNA can be complexed onto the positively charged HA-PEI and HA-P-PEI compounds to form nanoparticles with the same particle size and uniform distribution with serum stability. We found that the size of the HA-P-PEI/siRNA nanoparticles decreased to <10 nm upon addition of MMP-2, and that H1975 cells overexpressing CD44, PD-L1, and MMP-2 aided confirmation of the delivery efficacy of the HA-P-PEI/siRNA nanocomplexes. Additionally, the use of HA-P-PEI caused less cytotoxicity than PEI alone, demonstrating its high cellular uptake. Moreover, pretreatment with MMP-2 increased nanocomplex tumor permeability, and western blot showed that HA-P-PEI/PD-L1-siRNA efficiently downregulated the PD-L1 expression in H1975 cells. These results demonstrated a novel approach for siRNA delivery and tumor penetration for future clinical applications in cancer treatment.
Assuntos
Antígeno B7-H1/antagonistas & inibidores , Pulmão/efeitos dos fármacos , Metaloproteinase 2 da Matriz/metabolismo , Nanopartículas/química , RNA Interferente Pequeno/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular , Química Farmacêutica , Relação Dose-Resposta a Droga , Portadores de Fármacos , Estabilidade de Medicamentos , Inativação Gênica , Humanos , Receptores de Hialuronatos/biossíntese , Ácido Hialurônico/química , Micelas , Tamanho da Partícula , Polietilenoimina/química , RNA Interferente Pequeno/administração & dosagem , Propriedades de Superfície , Microambiente Tumoral/efeitos dos fármacosRESUMO
In this study, we described a series of N-(pyrimidin-2-yl)-1,2,3,4-tetrahydroisoquinolin-6-amine derivatives as selective JAK2 (Janus kinase 2) inhibitors. Systematic exploration of the structure-activity relationship though cyclization modification based on previously reported compound 18e led to the discovery of the superior derivative 13ac. Compound 13ac showed excellent potency on JAK2 kinase, SET-2, and Ba/F3V617F cells (high expression of JAK2V617F mutation) with IC50 values of 3, 11.7, and 41 nM, respectively. Further mechanistic studies demonstrated that compound 13ac could downregulate the phosphorylation of downstream proteins of JAK2 kinase in cells. Compound 13ac also showed good selectivity in kinase scanning and potent in vivo antitumor efficacy with 82.3% tumor growth inhibition in the SET-2 xenograft model. Moreover, 13ac significantly ameliorated the disease symptoms in a Ba/F3-JAK2V617F allograft model, with 77.1% normalization of spleen weight, which was more potent than Ruxolitinib.
Assuntos
Antineoplásicos/uso terapêutico , Isoquinolinas/uso terapêutico , Janus Quinase 2/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/uso terapêutico , Animais , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Feminino , Humanos , Isoquinolinas/síntese química , Isoquinolinas/metabolismo , Isoquinolinas/farmacocinética , Janus Quinase 2/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCID , Simulação de Acoplamento Molecular , Estrutura Molecular , Transtornos Mieloproliferativos/tratamento farmacológico , Ligação Proteica , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacocinética , Pirimidinas/síntese química , Pirimidinas/metabolismo , Pirimidinas/farmacocinética , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Herein, we describe the design, synthesis, and structure-activity relationships of a series of unique 4-(1H-pyrazol-4-yl)-pyrimidin-2-amine derivatives that selectively inhibit Janus kinase 2 (JAK2) and FLT3 kinases. These screening cascades revealed that 18e was a preferred compound, with IC50 values of 0.7 and 4 nM for JAK2 and FLT3, respectively. Moreover, 18e was a potent JAK2 inhibitor with 37-fold and 56-fold selectivity over JAK1 and JAK3, respectively, and possessed an excellent selectivity profile over the other 100 representative kinases. In a series of cytokine-stimulated cell-based assays, 18e exhibited a higher JAK2 selectivity over other JAK isoforms. The oral administration of 60 mg/kg of 18e could significantly inhibit tumor growth, with a tumor growth inhibition rate of 93 and 85% in MV4-11 and SET-2 xenograft models, respectively. Additionally, 18e showed an excellent bioavailability (F = 58%), a suitable half-life time (T1/2 = 4.1 h), a satisfactory metabolic stability, and a weak CYP3A4 inhibitory activity, suggesting that 18e might be a potential drug candidate for JAK2-driven myeloproliferative neoplasms and FLT3-internal tandem duplication-driven acute myelogenous leukemia.
Assuntos
Antineoplásicos/farmacologia , Janus Quinase 2/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Antineoplásicos/farmacocinética , Disponibilidade Biológica , Cães , Relação Dose-Resposta a Droga , Feminino , Meia-Vida , Humanos , Janus Quinase 2/química , Janus Quinase 2/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Leucócitos Mononucleares/efeitos dos fármacos , Masculino , Camundongos SCID , Simulação de Acoplamento Molecular , Transtornos Mieloproliferativos/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacocinética , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de Xenoenxerto , Tirosina Quinase 3 Semelhante a fms/química , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
In this study, a series of novel HDAC inhibitors, using 1,2,4-oxadiazole-containing as the cap group, were synthesized and evaluated in vitro. Compound 14b, N-hydroxy-2-(methyl((3-(1-(4-methylbenzyl)piperidin-4-yl)-1,2,4-oxadiazol-5-yl)methyl)amino)pyrimidine-5-carboxamide, displayed the most potent histone deacetylase (HDAC) inhibition, especially against HDAC1, 2, and 3 with IC50 values of 1.8, 3.6 and 3.0â¯nM, respectively. In vitro antiproliferative studies confirmed that 14b was more potent than SAHA, with IC50 values against 12 types of cancer cell lines ranging from 9.8 to 44.9â¯nM. The results of Western blot assays showed that compound 14b can significantly up-regulate the acetylation of the biomarker his-H3 and molecular docking analyses revealed the mode of action of compound 14b against HDAC1. The results of flow-cytometry analysis suggested that compound 14b induces cell cycle arrest at the G1 phase and has apoptotic effects. Further investigation of the activity of 14b on the primary cells of three patients, showed IC50 values of 21.3, 61.1, and 77.4â¯nM. More importantly, an oral bioavailability of up to 53.52% was observed for 14b. An in vivo pharmacodynamic evaluation demonstrated that compound 14b can significantly inhibit tumor growth in a Daudi Burkitt's lymphoma xenograft model, with tumor inhibition rates of 53.8 and 46.1% observed at 20 and 10â¯mg/kg when administered p.o. and i.v., respectively. These results indicate that compound 14b may be a suitable lead for further evaluation and development as an HDAC inhibitor and a potent anticancer agent.
Assuntos
Antineoplásicos/uso terapêutico , Linfoma de Burkitt/tratamento farmacológico , Inibidores de Histona Desacetilases/uso terapêutico , Ácidos Hidroxâmicos/uso terapêutico , Oxidiazóis/uso terapêutico , Acetilação/efeitos dos fármacos , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacocinética , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Descoberta de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Histona Desacetilase 1/química , Histona Desacetilase 1/metabolismo , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/farmacocinética , Histonas/metabolismo , Humanos , Ácidos Hidroxâmicos/síntese química , Ácidos Hidroxâmicos/farmacocinética , Camundongos Endogâmicos NOD , Camundongos SCID , Simulação de Acoplamento Molecular , Estrutura Molecular , Oxidiazóis/síntese química , Oxidiazóis/farmacocinética , Ligação Proteica , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Tubulina (Proteína)/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Twenty-four novel ß-carboline/acylhydrazone hybrids were synthesized and evaluated for their in vitro antiproliferative activity. Among them, 12r exhibited the most potent activity, with IC50 values of 1-2⯵M against panel of cancer cell lines and retained significant activity in multidrug resistant cancer cells. Treated cells were not arrested in any phase of cell cycle but resulted in late cellular apoptosis on both MCF-7 and MCF-7/ADR cancer cells. Importantly, 12r showed certain antitumor effect on inhibiting the tumor growth with low toxic and side effects and without significant body weight loss.
Assuntos
Antineoplásicos/farmacologia , Carbolinas/farmacologia , Descoberta de Drogas , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Hidrazonas/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Carbolinas/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Hidrazonas/química , Neoplasias Mamárias Experimentais , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Relação Estrutura-AtividadeRESUMO
Inhibitors that bind to the paclitaxel- or vinblastine-binding sites of tubulin have been part of the pharmacopoeia of anticancer therapy for decades. However, tubulin inhibitors that bind to the colchicine-binding site are not used in clinical cancer therapy, because of their low therapeutic index. To address multidrug resistance to many conventional tubulin-binding agents, numerous efforts have attempted to clinically develop inhibitors that bind the colchicine-binding site. Previously, we have found that millepachine (MIL), a natural chalcone-type small molecule extracted from the plant Millettia pachycarpa, and its two derivatives (MDs) SKLB028 and SKLB050 have potential antitumor activities both in vitro and in vivo However, their cellular targets and mechanisms are unclear. Here, biochemical and cellular experiments revealed that the MDs directly and irreversibly bind ß-tubulin. X-ray crystallography of the tubulin-MD structures disclosed that the MDs bind at the tubulin intradimer interface and to the same site as colchicine and that their binding mode is similar to that of colchicine. Of note, MDs inhibited tubulin polymerization and caused G2/M cell-cycle arrest. Comprehensive analysis further revealed that free MIL exhibits an s-cis conformation, whereas MIL in the colchicine-binding site in tubulin adopts an s-trans conformation. Moreover, introducing an α-methyl to MDs to increase the proportion of s-trans conformations augmented MDs' tubulin inhibition activity. Our study uncovers a new class of chalcone-type tubulin inhibitors that bind the colchicine-binding site in ß-tubulin and suggests that the s-trans conformation of these compounds may make them more active anticancer agents.