RESUMO
The calcium release activated calcium (CRAC) channel is highly expressed in T lymphocytes and plays a critical role in regulating T cell proliferation and functions including activation of the transcription factor nuclear factor of activated T cells (NFAT), cytokine production and cytotoxicity. The CRAC channel consists of the Orai pore subunit and STIM (stromal interacting molecule) endoplasmic reticulum calcium sensor. Loss of CRAC channel mediated calcium signaling has been identified as an underlying cause of severe combined immunodeficiency (SCID), leading to drastically weakened immunity against infections. Gain-of-function mutations in Orai and STIM have been associated with tubular aggregated myopathy (TAM), a skeletal muscle disease. While a number of small molecules have shown activity in inhibiting the CRAC signaling pathway, the usefulness of those tool compounds is limited by their off-target activity against TRPM4 and TRPM7 ion channels, high lipophilicity, and a lack of understanding of their mechanism of action. We report structure-activity relationship (SAR) studies that resulted in the characterization of compound 4k [1-(cyclopropylmethyl)-N-(3-fluoropyridin-4-yl)-1H-indazole-3-carboxamie] as a fast onset, reversible, and selective CRAC channel blocker. 4k fully blocked the CRAC current (IC50: 4.9 µM) and the nuclear translocation of NFAT at 30 and 10 µM, respectively, without affecting the electrophysiological function of TRPM4 and TRPM7 channels. Computational modeling appears to support its direction binding to Orai proteins that form the transmembrane CRACchannel.
Assuntos
Bloqueadores dos Canais de Cálcio , Indazóis , Pirazóis , Humanos , Bloqueadores dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/química , Bloqueadores dos Canais de Cálcio/síntese química , Relação Estrutura-Atividade , Indazóis/farmacologia , Indazóis/química , Indazóis/síntese química , Pirazóis/farmacologia , Pirazóis/química , Pirazóis/síntese química , Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Canais de Cálcio Ativados pela Liberação de Cálcio/antagonistas & inibidores , Estrutura Molecular , Descoberta de Drogas , Relação Dose-Resposta a Droga , Proteína ORAI1/metabolismo , Proteína ORAI1/antagonistas & inibidoresRESUMO
BACKGROUND: Fibrosis cataract occurs in patients receiving cataract extraction. Still, no medication that can cure the disease exists in clinical. This study aims to investigate the effects and mechanisms of Entrectinib on fibrotic cataract in vitro and in vivo. METHODS: The human lens cells line SRA 01/04 and C57BL/6J mice were applied in the study. Entrectinib was used in animals and cells. Cataract severity was assessed by slit lamp and Hematoxylin and Eosin staining. Expression of alpha-smooth muscle actin, fibronectin, and collagen I were examined by real-time quantitative PCR, western blotting, and immunofluorescence. Cell proliferation was evaluated by Cell Counting Kit-8. Cell migration was measured by wound healing and transwell assays. Molecular docking, Drug Affinity Responsive Target Stability, and Cellular Thermal Shift Assay were applied to seek and certify the target of Entrectinib treating fibrosis cataract. RESULTS: Entrectinib can ameliorate fibrotic cataract in vitro and in vivo. At the RNA and the protein levels, the expression of alpha-smooth muscle actin, collagen I, and fibronectin can be downgraded by Entrectinib, while E-cadherin can be upregulated. The migration and proliferation of cells were inhibited by Entrectinib. Mechanistically, Entrectinib obstructs TGFß2/Smad and TGFß2/non-Smad signaling pathways to hinder the fibrosis cataract by targeting PYK2 protein. CONCLUSIONS: Targeting with PYK2, Entrectinib can block TGF-ß2/Smad and TGF-ß2/non-Smad signaling pathways, lessen the activation of EMT, and alleviate fibrosis cataract. Entrectinib may be a potential treatment for fibrosis cataract in clinic.
Assuntos
Catarata , Quinase 2 de Adesão Focal , Transdução de Sinais , Fator de Crescimento Transformador beta2 , Animais , Camundongos , Transdução de Sinais/efeitos dos fármacos , Catarata/etiologia , Catarata/tratamento farmacológico , Catarata/metabolismo , Catarata/patologia , Humanos , Fator de Crescimento Transformador beta2/metabolismo , Quinase 2 de Adesão Focal/metabolismo , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Movimento Celular/efeitos dos fármacos , Linhagem Celular , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Indazóis/farmacologia , Indazóis/uso terapêutico , Masculino , Quinase 1 de Adesão FocalRESUMO
Inhibition of leucine-rich repeat kinase 2 is a genetically supported mechanism for the treatment of Parkinson's disease. We previously disclosed the discovery of an indazole series lead that demonstrated both safety and translational risks. The safety risks were hypothesized to be of unknown origin, so structural diversity in subsequent chemical matter was prioritized. The translational risks were identified due to a low brain Kpu,u in nonhuman primate studies, which raised concern over the use of an established peripheral biomarker as a surrogate for central target engagement. Given these challenges, the team sought to leverage structure- and property-based drug design and expanded efflux transporter profiling to identify structurally distinct leads with enhanced CNS drug-likeness. Herein, we describe the discovery of a "reinvented" indazole series with improved physicochemical properties and efflux transporter profiles while maintaining excellent potency and off-target kinase selectivity, which resulted in advanced lead, compound 23.
Assuntos
Indazóis , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Inibidores de Proteínas Quinases , Indazóis/farmacologia , Indazóis/química , Indazóis/síntese química , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/antagonistas & inibidores , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Humanos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Animais , Relação Estrutura-Atividade , Descoberta de Drogas , Ratos , Estrutura MolecularRESUMO
The inhibitory-kappaB kinases (IKKs) IKKα and IKKß play central roles in regulating the non-canonical and canonical NF-κB signalling pathways. Whilst the proteins that transduce the signals of each pathway have been extensively characterised, the clear dissection of the functional roles of IKKα-mediated non-canonical NF-κB signalling versus IKKß-driven canonical signalling remains to be fully elucidated. Progress has relied upon complementary molecular and pharmacological tools; however, the lack of highly potent and selective IKKα inhibitors has limited advances. Herein, we report the development of an aminoindazole-pyrrolo[2,3-b]pyridine scaffold into a novel series of IKKα inhibitors. We demonstrate high potency and selectivity against IKKα over IKKß in vitro and explain the structure-activity relationships using structure-based molecular modelling. We show selective target engagement with IKKα in the non-canonical NF-κB pathway for both U2OS osteosarcoma and PC-3M prostate cancer cells by employing isoform-related pharmacodynamic markers from both pathways. Two compounds (SU1261 [IKKα Ki = 10 nM; IKKß Ki = 680 nM] and SU1349 [IKKα Ki = 16 nM; IKKß Ki = 3352 nM]) represent the first selective and potent pharmacological tools that can be used to interrogate the different signalling functions of IKKα and IKKß in cells. Our understanding of the regulatory role of IKKα in various inflammatory-based conditions will be advanced using these pharmacological agents.
Assuntos
Desenho de Fármacos , Quinase I-kappa B , NF-kappa B , Inibidores de Proteínas Quinases , Transdução de Sinais , Quinase I-kappa B/metabolismo , Quinase I-kappa B/antagonistas & inibidores , Humanos , NF-kappa B/metabolismo , NF-kappa B/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Linhagem Celular Tumoral , Piridinas/farmacologia , Piridinas/química , Piridinas/síntese química , Indazóis/farmacologia , Indazóis/química , Indazóis/síntese química , Modelos MolecularesRESUMO
Obesity has shown a global epidemic trend. The high-lipid state caused by obesity can maintain the heart in a prolonged low-grade inflammatory state and cause ventricular remodeling, leading to a series of pathologies, such as hypertrophy, fibrosis, and apoptosis, which eventually develop into obese cardiomyopathy. Therefore, prolonged low-grade inflammation plays a crucial role in the progression of obese cardiomyopathy, making inflammation regulation an essential strategy for treating this disease. Cyy-272, an indazole derivative, is an anti-inflammatory compound independently synthesized by our laboratory. Our previous studies revealed that Cyy-272 can exert anti-inflammatory effects by inhibiting the phosphorylation and activation of C-Jun N-terminal kinase (JNK), thereby alleviating lipopolysaccharide (LPS)-induced acute lung injury (ALI). The current study aimed to evaluate the potential of Cyy-272 to mitigate the occurrence and progression of obese cardiomyopathy through the inhibition of the JNK signaling pathway. Our results indicate that the compound Cyy-272 has encouraging therapeutic effects on obesity-induced cardiac injury. It significantly inhibits inflammation in cardiomyocytes and heart tissues induced by high lipid concentrations, further alleviating the resulting hypertrophy, fibrosis, and apoptosis. Mechanistically, the protective effect of Cyy-272 on obese cardiomyopathy can be attributed to its direct inhibition of JNK protein phosphorylation. In conclusion, we identified a novel compound, Cyy-272, capable of alleviating obese cardiomyopathy and confirmed that its effect is achieved through direct inhibition of JNK.
Assuntos
Cardiomiopatias , Indazóis , Proteínas Quinases JNK Ativadas por Mitógeno , Obesidade , Animais , Obesidade/tratamento farmacológico , Obesidade/complicações , Cardiomiopatias/tratamento farmacológico , Indazóis/farmacologia , Indazóis/uso terapêutico , Indazóis/química , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Masculino , Apoptose/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Miócitos Cardíacos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos , Fibrose , Anti-Inflamatórios/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Inibidores de Proteínas Quinases/química , Lipopolissacarídeos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacosRESUMO
Inflammatory bowel disease (IBD) is a chronic and progressive condition with a significant global burden. Currently, available treatments primarily provide symptomatic relief and retard disease progression, yet they do not offer a cure and are frequently associated with adverse effects. Therefore, the discovery of new targets and therapeutic drugs for IBD is crucial. Phosphodiesterase 4 (PDE4) inhibitors have emerged as promising candidates in the search for effective IBD treatments, although dose-dependent side effects hamper their clinical utility. In this study, building upon heterocyclic biaryl derivatives (TPA16), we designed and synthesized a series of N2-substituted indazole-based PDE4D inhibitors, emphasizing improving safety profiles. An enzyme activity screening discovered an optimized compound, LZ-14 (Z21115), which exhibited high PDE4D7 (IC50 = 10.5 nM) inhibitory activity and good selectivity. More interestingly, LZ-14 has demonstrated promising effects in treating IBD in mouse models by improving the inflammatory response and colon injury. Furthermore, LZ-14 displayed low emetogenic potential in ketamine/xylazine anesthesia mice alternative models.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Descoberta de Drogas , Indazóis , Doenças Inflamatórias Intestinais , Inibidores da Fosfodiesterase 4 , Animais , Doenças Inflamatórias Intestinais/tratamento farmacológico , Relação Estrutura-Atividade , Inibidores da Fosfodiesterase 4/farmacologia , Inibidores da Fosfodiesterase 4/química , Inibidores da Fosfodiesterase 4/síntese química , Inibidores da Fosfodiesterase 4/uso terapêutico , Camundongos , Indazóis/farmacologia , Indazóis/química , Indazóis/síntese química , Humanos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Estrutura Molecular , Relação Dose-Resposta a Droga , Masculino , Camundongos Endogâmicos C57BLRESUMO
The DNA-encoded library (DEL) is a robust tool for chemical biology and drug discovery. In this study, we developed a DNA-compatible light-promoted reaction that is highly efficient and plate-compatible for DEL construction based on the formation of the indazolone scaffold. Employing this high-efficiency approach, we constructed a DEL featuring an indazolone core, which enabled the identification of a novel series of ligands specifically targeting E1A-binding protein (p300) after DEL selection. Taken together, our findings underscore the feasibility of light-promoted reactions in DEL synthesis and unveil promising avenues for developing p300-targeting inhibitors.
Assuntos
DNA , Descoberta de Drogas , Proteína p300 Associada a E1A , Indazóis , Bibliotecas de Moléculas Pequenas , DNA/química , Indazóis/química , Indazóis/farmacologia , Proteína p300 Associada a E1A/antagonistas & inibidores , Proteína p300 Associada a E1A/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Descoberta de Drogas/métodos , Humanos , Biblioteca Gênica , LigantesRESUMO
Although the primary elimination pathway for most tyrosine kinase inhibitors (TKI) involves CYP3A4-mediated metabolism, the mechanism by which these agents are brought into hepatocytes remains unclear. In this study, we optimized and validated a competitive counterflow (CCF) assay to examine TKIs as substrates of the hepatic uptake transporter OATP1B1. The CCF method was based on the stimulated efflux of radiolabeled estradiol-17ß-glucuronide under steady-state conditions in HEK293 cells engineered to overexpress OATP1B1. Of the 62 approved TKIs examined, 13 agents were identified as putative substrates of OATP1B1, and pazopanib was selected as a representative hit for further validation studies. The transport of pazopanib by OATP1B1 was confirmed by decreased activity of its target VEGFR2 in OATP1B1-overexpressing cells, but not cells lacking OATP1B1, consistent with molecular docking analyses indicating an overlapping binding orientation on OATP1B1 with the known substrate estrone-3-sulfate. In addition, the liver-to-plasma ratio of pazopanib in vivo was decreased in mice with a deficiency of the orthologous transporters, and this was accompanied by diminished pazopanib-induced hepatotoxicity, as determined by changes in the levels of liver transaminases. Our study supports the utility of CCF assays to assess substrate affinity for OATP1B1 within a large set of agents in the class of TKIs and sheds light on the mechanism by which these agents are taken up into hepatocytes in advance of metabolism. SIGNIFICANCE: Despite the established exposure-pharmacodynamic relationships for many TKIs, the mechanisms underlying the agents' unpredictable pharmacokinetic profiles remain poorly understood. We report here that the disposition of many TKIs depends on hepatic transport by OATP1B1, a process that has toxicologic ramifications for agents that are associated with hepatotoxicity.
Assuntos
Indazóis , Transportador 1 de Ânion Orgânico Específico do Fígado , Inibidores de Proteínas Quinases , Sulfonamidas , Transportador 1 de Ânion Orgânico Específico do Fígado/metabolismo , Transportador 1 de Ânion Orgânico Específico do Fígado/antagonistas & inibidores , Transportador 1 de Ânion Orgânico Específico do Fígado/genética , Humanos , Animais , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacocinética , Células HEK293 , Camundongos , Sulfonamidas/farmacologia , Sulfonamidas/metabolismo , Indazóis/farmacologia , Pirimidinas/farmacologia , Hepatócitos/metabolismo , Hepatócitos/efeitos dos fármacos , Estradiol/metabolismo , Estradiol/análogos & derivados , Estradiol/farmacologia , Fígado/metabolismo , Fígado/efeitos dos fármacos , Estrona/análogos & derivados , Estrona/metabolismo , Simulação de Acoplamento Molecular , Camundongos Knockout , Transporte Biológico , MasculinoRESUMO
NTRK gene fusion leads to the activation of downstream signaling pathways, which is a oncogenic driver in various cancers. NTRK fusion-positive cancers can be treated with the first-generation TRK inhibitors, larotrectinib and entrectinib. Unfortunately, the patients eventually face the dilemma of no drugs available as the emergence of certain resistance mutations. The development of efficient and broad-spectrum second-generation TRK inhibitors is still of great significance. Here, we analyzed the binding modes of compounds 6, 10 with TRKA protein, respectively, a series of novel indazole TRK inhibitors were designed and synthesized using molecular hybridization strategy. Among them, the optimal compound B31 showed strong antiproliferative activities against Km-12, Ba/F3-TRKAG595R, and Ba/F3-TRKAG667C cell lines with IC50 values of 0.3, 4.7, and 9.9 nM, respectively. And the inhibitory effect against TRKAG667C (IC50 = 9.9 nM) was better than that of selitrectinib (IC50 = 113.1 nM). Further, compound B31 exhibited moderate kinase selectivity and excellent plasma stability (t1/2 > 480 min). In vivo pharmacokinetic studies in Sprague-Dawley rats showed that B31 had acceptable pharmacokinetic properties.
Assuntos
Antineoplásicos , Proliferação de Células , Descoberta de Drogas , Indazóis , Inibidores de Proteínas Quinases , Ratos Sprague-Dawley , Receptor trkA , Indazóis/farmacologia , Indazóis/química , Indazóis/síntese química , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/síntese química , Humanos , Animais , Relação Estrutura-Atividade , Receptor trkA/antagonistas & inibidores , Receptor trkA/metabolismo , Proliferação de Células/efeitos dos fármacos , Ratos , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Estrutura Molecular , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Linhagem Celular Tumoral , MasculinoRESUMO
Alzheimer's disease is characterized by a progressive deterioration of cognitive function and memory loss, and it is closely associated with the dysregulation of cholinergic neurotransmission. Since acetylcholinesterase (AChE) is a critical enzyme in the nervous system, responsible for breaking down the neurotransmitter acetylcholine, its inhibition holds a significant interest in the treatment of various neurological disorders. Therefore, it is crucial to develop efficient AChE inhibitors capable of increasing acetylcholine levels, ultimately leading to improved cholinergic neurotransmission. The results reported here represent a step forward in the development of novel thiazoloindazole-based compounds that have the potential to serve as effective AChE inhibitors. Molecular docking studies revealed that certain of the evaluated nitroindazole-based compounds outperformed donepezil, a well-known AChE inhibitor used in Alzheimer's disease treatment. Sustained by these findings, two series of compounds were synthesized. One series included a triazole moiety (Tl45a-c), while the other incorporated a carbazole moiety (Tl58a-c). These compounds were isolated in yields ranging from 66 to 87% through nucleophilic substitution and Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) reactions. Among the synthesized compounds, the thiazoloindazole-based 6b core derivatives emerged as selective AChE inhibitors, exhibiting remarkable IC50 values of less than 1.0 µM. Notably, derivative Tl45b displays superior performance as an AChE inhibitor, boasting the lowest IC50 (0.071 ± 0.014 µM). Structure-activity relationship (SAR) analysis indicated that derivatives containing the bis(trifluoromethyl)phenyl-triazolyl group demonstrated the most promising activity against AChE, when compared to more rigid substituents such as carbazolyl moiety. The combination of molecular docking and experimental synthesis provides a suitable and promising strategy for the development of new efficient thiazoloindazole-based AChE inhibitors.
Assuntos
Acetilcolinesterase , Inibidores da Colinesterase , Indazóis , Simulação de Acoplamento Molecular , Tiazóis , Inibidores da Colinesterase/farmacologia , Inibidores da Colinesterase/química , Inibidores da Colinesterase/síntese química , Simulação de Acoplamento Molecular/métodos , Indazóis/farmacologia , Indazóis/química , Acetilcolinesterase/metabolismo , Acetilcolinesterase/efeitos dos fármacos , Humanos , Tiazóis/farmacologia , Tiazóis/química , Desenho de Fármacos , Relação Estrutura-AtividadeRESUMO
In a recent issue of The Journal of Pathology, Chen and colleagues established novel patient-derived ex vivo models of NTRK fusion-positive soft tissue sarcoma to characterize resistance mechanisms against targeted therapy with tyrosine kinase inhibitors. Prolonged exposure to escalating concentrations of the tyrosine kinase inhibitor, entrectinib, ultimately led to the occurrence of resistant clones that harbored an inactivating mutation in the NF2 gene, not previously described in this context, accompanied by increased PI3K/AKT/mTOR and Ras/Raf/MEK/ERK signaling. Finally, an inhibitor screen identified, among others, MEK and mTOR inhibitors as potential combination agents. © 2024 The Pathological Society of Great Britain and Ireland.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Inibidores de Proteínas Quinases , Humanos , Resistencia a Medicamentos Antineoplásicos/genética , Inibidores de Proteínas Quinases/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Neurofibromina 2/genética , Proteínas de Fusão Oncogênica/genética , Benzamidas/uso terapêutico , Benzamidas/farmacologia , Receptor trkA/genética , Receptor trkA/metabolismo , Transdução de Sinais/genética , Indazóis/uso terapêutico , Indazóis/farmacologia , Mutação , Sarcoma/genética , Sarcoma/tratamento farmacológico , Sarcoma/patologia , Antineoplásicos/uso terapêutico , Antineoplásicos/farmacologia , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
Pazopanib (PAZ), an oral multi-tyrosine kinase inhibitor, demonstrates promising cytostatic activities against various human cancers. However, its clinical utility is limited by substantial side effects and therapeutic resistance. We developed a nanoplatform capable of delivering PAZ for enhanced anti-breast cancer therapy. Nanometer-sized PAZ@Fe-MOF, compared to free PAZ, demonstrated increased anti-tumor therapeutic activities in both syngeneic murine 4T1 and xenograft human MDA-MB-231 breast cancer models. High-throughput single-cell RNA sequencing (scRNAseq) revealed that PAZ@Fe-MOF significantly reduced pro-tumorigenic M2-like macrophage populations at tumor sites and suppressed M2-type signaling pathways, such as ATF6-TGFBR1-SMAD3, as well as chemokines including CCL17, CCL22, and CCL24. PAZ@Fe-MOF reprogramed the inhibitory immune microenvironment and curbed tumorigenicity by blocking the polarization of M2 phenotype macrophages. This platform offers a promising and new strategy for improving the cytotoxicity of PAZ against breast cancers. It provides a method to evaluate the immunological response of tumor cells to PAZ-mediated treatment.
Assuntos
Antineoplásicos , Neoplasias da Mama , Indazóis , Macrófagos , Estruturas Metalorgânicas , Nanopartículas , Pirimidinas , Sulfonamidas , Animais , Feminino , Estruturas Metalorgânicas/química , Estruturas Metalorgânicas/farmacologia , Neoplasias da Mama/tratamento farmacológico , Humanos , Macrófagos/efeitos dos fármacos , Indazóis/farmacologia , Indazóis/química , Camundongos , Pirimidinas/farmacologia , Pirimidinas/química , Linhagem Celular Tumoral , Nanopartículas/química , Sulfonamidas/farmacologia , Sulfonamidas/química , Antineoplásicos/farmacologia , Antineoplásicos/química , Camundongos Endogâmicos BALB C , Microambiente Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A series of indazole analogs, derived from the B,C-ring-truncated scaffold of deguelin, were designed to function as C-terminal inhibitors of heat shock protein 90 (HSP90) and investigated as novel antitumor agents against HER2-positive breast cancer. Among the synthesized compounds, compound 12d exhibited substantial inhibitory effects in trastuzumab-sensitive (BT474) and trastuzumab-resistant (JIMT-1) breast cancer cells, with IC50 values of 6.86 and 4.42 µM, respectively. Notably, compound 12d exhibited no cytotoxicity in normal cells. Compound 12d markedly downregulated the expression of the major HSP90 client proteins in both cell types, attributing its cytotoxicity to the destabilization and inactivation of HSP90 client proteins. Molecular docking studies using the homology model of an HSP90 homodimer demonstrated that inhibitor 12d fit nicely into the C-terminal domain, boasting a higher electrostatic complementary score than ATP. In vivo pharmacokinetic study indicated the high oral bioavailability of compound 12 d at F = 66.9 %, while toxicological studies indicated its negligible impact on hERG channels and CYP isozymes. Genotoxicity tests further confirmed its safety profile. The findings collectively position compound 12d as a promising candidate for further development as an antitumor agent against HER2-positive breast cancer.
Assuntos
Antineoplásicos , Proliferação de Células , Ensaios de Seleção de Medicamentos Antitumorais , Proteínas de Choque Térmico HSP90 , Indazóis , Simulação de Acoplamento Molecular , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Indazóis/farmacologia , Indazóis/química , Indazóis/síntese química , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Relação Estrutura-Atividade , Proliferação de Células/efeitos dos fármacos , Animais , Estrutura Molecular , Descoberta de Drogas , Relação Dose-Resposta a Droga , Linhagem Celular Tumoral , Feminino , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismoRESUMO
Homologous recombination deficiency (HRD) is prevalent in cancer, sensitizing tumor cells to poly (ADP-ribose) polymerase (PARP) inhibition. However, the impact of HRD and related therapies on the tumor microenvironment (TME) remains elusive. Our study generates single-cell gene expression and T cell receptor profiles, along with validatory multimodal datasets from >100 high-grade serous ovarian cancer (HGSOC) samples, primarily from a phase II clinical trial (NCT04507841). Neoadjuvant monotherapy with the PARP inhibitor (PARPi) niraparib achieves impressive 62.5% and 73.6% response rates per RECIST v.1.1 and GCIG CA125, respectively. We identify effector regulatory T cells (eTregs) as key responders to HRD and neoadjuvant therapies, co-occurring with other tumor-reactive T cells, particularly terminally exhausted CD8+ T cells (Tex). TME-wide interferon signaling correlates with cancer cells upregulating MHC class II and co-inhibitory ligands, potentially driving Treg and Tex fates. Depleting eTregs in HRD mouse models, with or without PARP inhibition, significantly suppresses tumor growth without observable toxicities, underscoring the potential of eTreg-focused therapeutics for HGSOC and other HRD-related tumors.
Assuntos
Terapia Neoadjuvante , Neoplasias Ovarianas , Piperidinas , Inibidores de Poli(ADP-Ribose) Polimerases , Linfócitos T Reguladores , Microambiente Tumoral , Feminino , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/imunologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Humanos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos , Animais , Camundongos , Terapia Neoadjuvante/métodos , Microambiente Tumoral/efeitos dos fármacos , Piperidinas/farmacologia , Piperidinas/uso terapêutico , Indazóis/uso terapêutico , Indazóis/farmacologia , Recombinação Homóloga , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular TumoralRESUMO
AIM: The objective was to investigate the specific role and the regulatory mechanism of vascular endothelial growth factor (VEGF) during wound healing in diabetic foot ulcer (DFU). METHODS: Streptozotocin-induced diabetic rats were used to establish a DFU animal model. VEGF and Axitinib (a specific inhibitor of VEGFR) were used for treatment in vivo. The wounds at different time points were imaged and histological analysis of the wounds were performed by haematoxylin and eosin (H&E) staining and Masson's trichrome staining. Immunohistochemical staining was conducted to examine CD31 and eNOS expression in the wounds. Immunofluorescence assay and quantitative real-time PCR were performed to examine macrophage markers. In addition, THP-1 was differentiated to macrophages, and then treated with interleukin (IL)-4 to induce M2 macrophages, followed by VEGF treatment. The conditional medium (CM) from VEGF-mediated macrophages were collected to culture human dermal fibroblasts (HDFs). Cell viability and migration were measured by Cell Counting Kit (CCK)-8, wound-healing and Transwell assays, respectively. RESULTS: VEGF treatment remarkably accelerated wound healing of DFU rats. VEGF promoted collagen deposition and elevated CD31 and eNOS expression, confirming the pro-angiogenesis of VEGF around diabetic wound in rats. Meanwhile, VEGF restricted pro-inflammatory cytokines and increased F4/80 and CD206 expression, highlighting the activated macrophages and enhanced M2 macrophages following VEGF treatment in diabetic wounds of DFU rats. However, Axitinib exerted an opposite function to VEGF in DFU rats. Moreover, VEGF directly promoted macrophage polarization toward M2 phenotype in vitro, and the CM from VEGF-mediated M2 macrophages markedly promoted HDFs proliferation, migration and collagen deposition. CONCLUSION: VEGF might accelerate the wound healing of DFU through promoting M2 macrophage polarization and fibroblast migration.
Assuntos
Axitinibe , Diabetes Mellitus Experimental , Pé Diabético , Macrófagos , Fator A de Crescimento do Endotélio Vascular , Cicatrização , Animais , Pé Diabético/metabolismo , Pé Diabético/patologia , Cicatrização/efeitos dos fármacos , Cicatrização/fisiologia , Ratos , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/complicações , Fator A de Crescimento do Endotélio Vascular/metabolismo , Macrófagos/metabolismo , Masculino , Axitinibe/farmacologia , Axitinibe/uso terapêutico , Humanos , Ratos Sprague-Dawley , Ativação de Macrófagos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Indazóis/farmacologia , Indazóis/uso terapêuticoRESUMO
The approval of immune checkpoint inhibitors (ICIs) has revolutionized the management of metastatic renal cell carcinoma (RCC), introducing several ICI-based combinations as the new standard of care for affected patients. Nonetheless, monotherapy with antiangiogenic tyrosine kinase inhibitors (TKIs), such as pazopanib or sunitinib, still represents a first-line treatment option for selected patients belonging to the favorable risk group according to the International mRCC Database Consortium (IMDC) model. After TKI monotherapy, the main second-line option is represented by ICI monotherapy with the anti-Programmed Death Receptor 1(PD-1) nivolumab. To date, the expected clinical outcomes are similar with pazopanib or sunitinib and there is no clear indication for selecting one TKI over the other. Moreover, their impact on subsequent ICI treatment outcomes is not well defined, yet. Based on these premises, we investigated the immunomodulatory activity of these drugs in vitro and in vivo.Both TKIs induced Programmed Cell Death Ligand-1 (PD-L1) expression and soluble PD-L1 release in RCC cells, and hampered T cell activation, reducing cytokine production and the proportion of activated T cells. Nevertheless, in a syngeneic co-culture system with peripheral blood mononuclear cells (PBMCs) and tumor cells, incubation with anti-PD-1 antibody following TKIs treatment significantly restored T cell function, potentiating the cytotoxic effects against tumor cells. Pazopanib and sunitinib followed by anti-PD-1 antibody produced a comparable inhibition of tumor growth in a RCC syngeneic mouse model. Our findings suggest that pazopanib and sunitinib, showing similar immunomodulatory effects, may have a comparable impact on the subsequent effectiveness of PD-1/PD-L1 blockade.
Assuntos
Inibidores da Angiogênese , Carcinoma de Células Renais , Neoplasias Renais , Receptor de Morte Celular Programada 1 , Inibidores de Proteínas Quinases , Pirimidinas , Sulfonamidas , Sunitinibe , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/patologia , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/patologia , Animais , Humanos , Camundongos , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêutico , Inibidores da Angiogênese/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Sunitinibe/farmacologia , Sunitinibe/uso terapêutico , Linhagem Celular Tumoral , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/metabolismo , Indazóis/farmacologia , Indazóis/uso terapêutico , Indazóis/administração & dosagem , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Feminino , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Nivolumabe/farmacologia , Nivolumabe/uso terapêutico , Nivolumabe/administração & dosagem , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/metabolismoRESUMO
PURPOSE: Pathological angiogenesis and vascular instability are observed in diabetic retinopathy (DR), diabetic macular edema (DME), and wet age-related macular degeneration (wAMD). Many receptor tyrosine kinases (RTKs) including vascular endothelial growth factor receptors (VEGFRs) contribute to angiogenesis, whereas the RTK TIE2 is important for vascular stability. Pan-VEGFR tyrosine kinase inhibitors (TKIs) such as vorolanib, sunitinib, and axitinib are of therapeutic interest over current antibody treatments that target only one or two ligands. This study compared the anti-angiogenic potential of these TKIs. METHODS: A kinase HotSpot™ assay was conducted to identify TKIs inhibiting RTKs associated with angiogenesis and vascular stability. Half-maximal inhibitory concentration (IC50) for VEGFRs and TIE2 was determined for each TKI. In vitro angiogenesis inhibition was investigated using a human umbilical vein endothelial cell sprouting assay, and in vivo angiogenesis was studied using the chorioallantoic membrane assay. Melanin binding was assessed using a melanin-binding assay. Computer modeling was conducted to understand the TIE2-axitinib complex as well as interactions between vorolanib and VEGFRs. RESULTS: Vorolanib, sunitinib, and axitinib inhibited RTKs of interest in angiogenesis and exhibited pan-VEGFR inhibition. HotSpot™ assay and TIE2 IC50 values showed that only axitinib potently inhibited TIE2 (up to 89%). All three TKIs effectively inhibited angiogenesis in vitro. In vivo, TKIs were more effective at inhibiting VEGF-induced angiogenesis than the anti-VEGF antibody bevacizumab. Of the three TKIs, only sunitinib bound melanin. TKIs differ in their classification and binding to VEGFRs, which is important because type II inhibitors have greater selectivity than type I TKIs. CONCLUSIONS: Vorolanib, sunitinib, and axitinib exhibited pan-VEGFR inhibition and inhibited RTKs associated with pathological angiogenesis. Of the three TKIs, only axitinib potently inhibited TIE2 which is an undesired trait as TIE2 is essential for vascular stability. The findings support the use of vorolanib for therapeutic inhibition of angiogenesis observed in DR, DME, and wAMD.
Assuntos
Inibidores da Angiogênese , Axitinibe , Células Endoteliais da Veia Umbilical Humana , Imidazóis , Indazóis , Indóis , Inibidores de Proteínas Quinases , Pirróis , Receptores de Fatores de Crescimento do Endotélio Vascular , Sunitinibe , Axitinibe/farmacologia , Humanos , Sunitinibe/farmacologia , Inibidores da Angiogênese/farmacologia , Imidazóis/farmacologia , Pirróis/farmacologia , Indóis/farmacologia , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Indazóis/farmacologia , Animais , Inibidores de Proteínas Quinases/farmacologia , Receptor TIE-2/metabolismo , Receptor TIE-2/antagonistas & inibidores , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismoRESUMO
BACKGROUND: We previously reported that metastases are generally characterized by a core program of gene expression that activates tissue remodeling/vascularization, alters ion homeostasis, induces the oxidative metabolism, and silences extracellular matrix interactions. This core program distinguishes metastases from their originating primary tumors as well as from their destination host tissues. Therefore, the gene products involved are potential targets for anti-metastasis drug treatment. METHODS: Because the silencing of extracellular matrix interactions predisposes to anoiks in the absence of active survival mechanisms, we tested inhibitors against the other three components. RESULTS: Individually, the low-specificity VEGFR blocker pazopanib (in vivo combined with marimastat), the antioxidant dimethyl sulfoxide (or the substitute atovaquone, which is approved for internal administration), and the ionic modulators bumetanide and tetrathiomolybdate inhibited soft agar colony formation by breast and pancreatic cancer cell lines. The individual candidate agents have a record of use in humans (with limited efficacy when administered individually) and are available for repurposing. In combination, the effects of these drugs were additive or synergistic. In two mouse models of cancer (utilizing 4T1 cells or B16-F10 cells), the combination treatment with these medications, applied immediately (to prevent metastasis formation) or after a delay (to suppress established metastases), dramatically reduced the occurrence of disseminated foci. CONCLUSIONS: The combination of tissue remodeling inhibitors, suppressors of the oxidative metabolism, and ion homeostasis modulators has very strong promise for the treatment of metastases by multiple cancers.
Assuntos
Indazóis , Pirimidinas , Sulfonamidas , Animais , Humanos , Camundongos , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Sulfonamidas/administração & dosagem , Linhagem Celular Tumoral , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Pirimidinas/administração & dosagem , Feminino , Indazóis/farmacologia , Indazóis/uso terapêutico , Indazóis/administração & dosagem , Metástase Neoplásica , Molibdênio/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
(1) Head and neck squamous cell carcinoma (HNSCC) is common, while treatment is difficult, and mortality is high. Kinase inhibitors are promising to enhance the effects of radiotherapy. We compared the effects of the PARP inhibitors talazoparib and niraparib and that of the DNA-PKcs inhibitor AZD7648, combined with ionizing radiation. (2) Seven HNSCC cell lines, including Cal33, CLS-354, Detroit 562, HSC4, RPMI2650 (HPV-negative), UD-SCC-2 and UM-SCC-47 (HPV-positive), and two healthy fibroblast cell lines, SBLF8 and SBLF9, were studied. Flow cytometry was used to analyze apoptosis and necrosis induction (AnnexinV/7AAD) and cell cycle distribution (Hoechst). Cell inactivation was studied by the colony-forming assay. (3) AZD7648 had the strongest effects, radiosensitizing all HNSCC cell lines, almost always in a supra-additive manner. Talazoparib and niraparib were effective in both HPV-positive cell lines but only consistently in one and two HPV-negative cell lines, respectively. Healthy fibroblasts were not affected by any combined treatment in apoptosis and necrosis induction or G2/M-phase arrest. AZD7648 alone was not toxic to healthy fibroblasts, while the combination with ionizing radiation reduced clonogenicity. (4) In conclusion, talazoparib, niraparib and, most potently, AZD7648 could improve radiation therapy in HNSCC. Healthy fibroblasts tolerated AZD7648 alone extremely well, but irradiation-induced effects might occur. Our results justify in vivo studies.
Assuntos
Apoptose , Indazóis , Ftalazinas , Piperidinas , Inibidores de Poli(ADP-Ribose) Polimerases , Radiossensibilizantes , Carcinoma de Células Escamosas de Cabeça e Pescoço , Humanos , Ftalazinas/farmacologia , Indazóis/farmacologia , Piperidinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Linhagem Celular Tumoral , Radiossensibilizantes/farmacologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/radioterapia , Apoptose/efeitos dos fármacos , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/radioterapia , Proteína Quinase Ativada por DNA/antagonistas & inibidores , Proteína Quinase Ativada por DNA/metabolismoRESUMO
The tumor microenvironment (TME) is crucial in tumor development, metastasis, and response to immunotherapy. DNA methylation can regulate the TME without altering the DNA sequence. However, research on the methylation-driven TME in clear-cell renal cell carcinoma (ccRCC) is still lacking. In this study, integrated DNA methylation and RNA-seq data were used to explore methylation-driven genes (MDGs). Immune scores were calculated using the ESTIMATE, which was employed to identify TME-related genes. A new signature connected with methylation-regulated TME using univariate, multivariate Cox regression and LASSO regression analyses was developed. This signature consists of four TME-MDGs, including AJAP1, HOXB9, MYH14, and SLC6A19, which exhibit high methylation and low expression in tumors. Validation was performed using qRT-PCR which confirmed their downregulation in ccRCC clinical samples. Additionally, the signature demonstrated stable predictive performance in different subtypes of ccRCC. Risk scores are positively correlated with TMN stages, immune cell infiltration, tumor mutation burden, and adverse outcomes of immunotherapy. Interestingly, the expression of four TME-MDGs are highly correlated with the sensitivity of first-line drugs in ccRCC treatment, especially pazopanib. Molecular docking indicates a high affinity binding between the proteins and pazopanib. In summary, our study elucidates the comprehensive role of methylation-driven TME in ccRCC, aiding in identifying patients sensitive to immunotherapy and targeted therapy, and providing new therapeutic targets for ccRCC treatment.