RESUMO
MDM2 and MDM4 are cancer drug targets validated in multiple models for p53-based cancer therapies. The RING domains of MDM2 and non-p53-binder MDM2 splice isoforms form RING domain heterodimer polyubiquitin E3 ligases with MDM4, which regulate p53 stability in vivo and promote tumorigenesis independent of p53. Despite the importance of the MDM2 RING domain in p53 regulation and cancer development, small molecule inhibitors targeting the E3 ligase activity of MDM2-MDM4 are poorly explored. Here, we describe the synthesis and characterization of quinolinol derivatives for the identification of analogs that are capable of targeting the MDM2-MDM4 heterodimer E3 ligase and inducing apoptosis in cells. The structure-activity-relationship (SAR) study identified structural moieties critical for the inhibitory effects toward MDM2-MDM4 E3 ligase, the targeted degradation of MDM4 and FTH1 in cells, and anti-proliferation activity. Lead optimization led to the development of compound MMRi71 with improved activity. In addition to accumulating p53 proteins in wt-p53 bearing cancer cells as expected of any MDM2 inhibitors, MMRi71 effectively kills p53-null leukemia cells, an activity that conventional MDM2-p53 disrupting inhibitors lack. This study provides a prototype structure for developing MDM4/FTH1 dual-targeting inhibitors as potential cancer therapeutics.
Assuntos
Leucemia , Neoplasias , Humanos , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteólise , Proteínas Proto-Oncogênicas/química , Ubiquitina-Proteína Ligases/metabolismo , Apoptose , Leucemia/tratamento farmacológico , Proteínas de Ciclo Celular/metabolismo , Ferritinas , Oxirredutases/metabolismoRESUMO
MDM2 and MDM4 proteins are key negative regulators of tumor suppressor p53. MDM2 and MDM4 interact via their RING domains and form a heterodimer polyubiquitin E3 ligase essential for p53 degradation. MDM4 also forms heterodimer E3 ligases with MDM2 isoforms that lack p53-binding domains, which regulate p53 and MDM4 stability. We are working to identify small-molecule inhibitors targeting the RING domain of MDM2-MDM4 (MMRi) that can inactivate the total oncogenic activity of MDM2-MDM4 heterodimers. Here, we describe the identification and characterization of MMRi62 as an MDM4-degrader and apoptosis inducer in leukemia cells. Biochemically, in our experiments, MMRi62 bound to preformed RING domain heterodimers altered the substrate preference toward MDM4 ubiquitination and promoted MDM2-dependent MDM4 degradation in cells. This MDM4-degrader activity of MMRi62 was found to be associated with potent apoptosis induction in leukemia cells. Interestingly, MMRi62 effectively induced apoptosis in p53 mutant, multidrug-resistant leukemia cells and patient samples in addition to p53 wild-type cells. In contrast, MMRi67 as a RING heterodimer disruptor and an enzymatic inhibitor of the MDM2-MDM4 E3 complex lacked MDM4-degrader activity and failed to induce apoptosis in these cells. In summary, this study identifies MMRi62 as a novel MDM2-MDM4-targeting agent and suggests that small molecules capable of promoting MDM4 degradation may be a viable new approach to killing leukemia cells bearing non-functional p53 by apoptosis.
RESUMO
[This corrects the article DOI: 10.1371/journal.pgen.1010171.].
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MDM2 and MDM4 are key regulators of p53 and function as oncogenes when aberrantly expressed. MDM2 and MDM4 partner to suppress p53 transcriptional transactivation and polyubiquitinate p53 for degradation. The importance of MDM2 E3-ligase-mediated p53 regulation remains controversial. To resolve this, we generated mice with an Mdm2 L466A mutation that specifically compromises E2 interaction, abolishing MDM2 E3 ligase activity while preserving its ability to bind MDM4 and suppress p53 transactivation. Mdm2L466A/L466A mice exhibit p53-dependent embryonic lethality, demonstrating MDM2 E3 ligase activity is essential for p53 regulation in vivo. Unexpectedly, cells expressing Mdm2L466A manifest cell cycle G2-M transition defects and increased aneuploidy even in the absence of p53, suggesting MDM2 E3 ligase plays a p53-independent role in cell cycle regulation and genome integrity. Furthermore, cells bearing the E3-dead MDM2 mutant show aberrant cell cycle regulation in response to DNA damage. This study uncovers an uncharacterized role for MDM2's E3 ligase activity in cell cycle beyond its essential role in regulating p53's stability in vivo.
Assuntos
Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53 , Animais , Ciclo Celular/genética , Dano ao DNA/genética , Camundongos , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/genéticaRESUMO
High frequency of KRAS and TP53 mutations is a unique genetic feature of pancreatic ductal adenocarcinoma (PDAC). TP53 mutation not only renders PDAC resistance to chemotherapies but also drives PDAC invasiveness. Therapies targeting activating mutant KRAS are not available and the outcomes of current PDAC treatment are extremely poor. Here, we report that MMRi62, initially identified as an MDM2-MDM4-targeting small molecule with p53-independent pro-apoptotic activity, shows anti-PDAC activity in vitro and in vivo. We show that MMRi62 inhibits proliferation, clonogenic, and spheroid growth of PDAC cells by induction of cell death. MMRi62-induced cell death in PDAC is characteristic of ferroptosis that is associated with increased autophagy, increased reactive oxygen species, and lysosomal degradation of NCOA4 and ferritin heavy chain (FTH1). In addition to induced degradation of FTH1, MMRi62 also induces proteasomal degradation of mutant p53. Interestingly, MMRi62-induced ferroptosis occurs in PDAC cell lines harboring either KRAS and TP53 double mutations or single TP53 mutation. In orthotopic xenograft PDAC mouse models, MMRi62 was capable of inhibiting tumor growth in mice associated with downregulation of NCOA4 and mutant p53 in vivo. Strikingly, MMRi62 completely abrogated metastasis of orthotopic tumors to distant organs, which is consistent with MMRi62's ability to inhibit cell migration and invasion in vitro. These findings identified MMRi62 as a novel ferroptosis inducer capable of suppressing PDAC growth and overcoming metastasis.
Assuntos
Carcinoma Ductal Pancreático , Ferroptose , Neoplasias Pancreáticas , Animais , Apoferritinas/uso terapêutico , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Camundongos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteína Supressora de Tumor p53/genéticaRESUMO
Hexamethylene bis-acetamide inducible protein 1 (HEXIM1) is identified as a novel inhibitor of estrogen stimulated breast cell growth, and it suppresses estrogen receptor-α transcriptional activity. HEXIM1 protein level has been found to be downregulated by estrogens. Recently, HEXIM1 has been found to inhibit androgen receptor transcriptional activity as well. Researchers have used Hexamethylene bis-acetamide (HMBA) for decades to stimulate HEXIM1 expression, which also inhibit estrogen stimulated breast cancer cell gene activation and androgen stimulated prostate cancer gene activation. However, the direct molecular targets of HMBA that modulate the induction of HEXIM1 expression in mammalian cells have not been identified. Based on HMBA and its more potent analog 4a1, we designed molecular probes to pull down the binding proteins of these compounds. Via proteomic approach and biological assays, we demonstrate that HMBA and 4a1 are actually heat shock protein 70 (HSP70) binders. The known HSP70 activator showed similar activity as HMBA and 4a1 to induce HEXIM1 expression, suggesting that HMBA and 4a1 might be putative HSP70 activators. Molecular target identification of HMBA and 4a1 could lead to further structural optimization of the parental compound to generate more potent derivatives to stimulate HEXIM1 expression, which could be a novel approach for hormone dependent breast cancer and prostate cancer treatment.
Assuntos
Acetamidas/química , Benzenoacetamidas/química , Estrogênios/metabolismo , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptores de Estrogênio/metabolismo , Biotinilação , Neoplasias da Mama/tratamento farmacológico , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Feminino , Humanos , Espectroscopia de Ressonância Magnética , Masculino , Espectrometria de Massas , Neoplasias da Próstata/tratamento farmacológico , Ligação Proteica , Proteômica , Espectrometria de Massas por Ionização por Electrospray , Fatores de TranscriçãoRESUMO
Peroxisome proliferator-activated receptor-γ (PPARγ) is a ligand-activated nuclear receptor which controls lipid and glucose metabolism. It is also the master regulator of adipogenesis. In adipocytes, ligand-dependent PPARγ activation is associated with proteasomal degradation; therefore, regulation of PPARγ degradation may modulate its transcriptional activity. Here, we show that neural precursor cell expressed developmentally down-regulated protein 4 (NEDD4), an E3 ubiquitin ligase, interacts with the hinge and ligand binding domains of PPARγ and is a bona fide E3 ligase for PPARγ. NEDD4 increases PPARγ stability through the inhibition of its proteasomal degradation. Knockdown of NEDD4 in 3T3-L1 adipocytes reduces PPARγ protein levels and suppresses adipocyte conversion. PPARγ correlates positively with NEDD4 in obese adipose tissue. Together, these findings support NEDD4 as a novel regulator of adipogenesis by modulating the stability of PPARγ.
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Adipócitos/citologia , Adipócitos/metabolismo , Diferenciação Celular , Ubiquitina-Proteína Ligases Nedd4/metabolismo , PPAR gama/metabolismo , Células 3T3-L1 , Adipogenia , Tecido Adiposo/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Ligantes , Lisina/metabolismo , Camundongos , Obesidade/metabolismo , PPAR gama/química , Ligação Proteica , Domínios Proteicos , Estabilidade Proteica , Proteólise , UbiquitinaçãoRESUMO
Inhibition of DNA methyltransferase 1 (DNMT1) can reverse the malignant behavior of cancer cells by restoring expression of aberrantly silenced genes that are required for differentiation, senescence, and apoptosis. Clinically used DNMT1 inhibitors decitabine and azacitidine inhibit their target by covalent trapping after incorporation into DNA as azacytidine analogs. These nucleoside compounds are prone to rapid enzymatic inactivation in blood, posing challenges to the development of purely epigenetic dosing schedules. Non-nucleoside compounds that suppress expression or function of DNMT1 may overcome this problem. Using a high-throughput PCR-based site specific chromatin condensation assay, we identified a compound that reactivated Cyclin-Dependent Kinase Inhibitor 2A (CDKN2A) in myeloma cells and suppressed expression of DNMT1 from a library of 5120 chemically diverse small molecules. Lead optimization was performed to generate 26 new analogs with lung cancer proliferation and DNMT1 expression as activity readout. Two of the new derivatives showed 2 fold improvement of growth inhibiting potency and also decreased DNMT1 protein levels in lung cancer cells.
Assuntos
Antineoplásicos/química , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Inibidores Enzimáticos/química , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Ligação ProteicaRESUMO
OBJECTIVES: There is an urgent need to develop a safe, effective, orally active, and inexpensive therapy for African trypanosomiasis due to the drawbacks of current drugs. Selective tubulin inhibitors have the potential to be promising drug candidates for the treatment of this disease, which is based on the tubulin protein structural difference between mammalian and trypanosome cells. We propose to identify novel tubulin inhibitors from a compound library developed based on the lead compounds that selectively target trypanosomiasis. METHODS: We used Trypanosoma brucei brucei as the parasite model, and human normal kidney cells and mouse microphage cells as the host model. Growth rates of both trypanosomes and mammalian cells were determined as a means to screen compounds that selectively inhibit the proliferation of parasites. Furthermore, we examined the cell cycle profile of the parasite and compared tubulin polymerization dynamics before and after the treatment using identified compounds. Last, in vivo anti-parasite activities of these compounds were determined in T. brucei-infected mice. RESULTS: Three compounds were selected that are 100 fold more effective against the growth of T. brucei cells than mammalian cells. These compounds caused cell cycle progression defects in T. brucei cells. Western analyses indicated that these compounds decreased tubulin polymerization in T. brucei cells. The in vivo investigation revealed that these compounds, when admitted orally, inhibited T. brucei cell proliferation in mouse blood. However, they were not potent enough to clear up the infection completely. CONCLUSIONS: These compounds are promising lead compounds as orally active agents for drug development of anti-trypanosome agents. A more detail structure activity relationship (SAR) was summarized that will be used to guide future lead optimization to improve the selectivity and potency of the current compounds.
Assuntos
Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Moduladores de Tubulina/farmacologia , Administração Oral , Animais , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citometria de Fluxo , Humanos , Camundongos , Proteínas de Protozoários/metabolismo , Relação Estrutura-Atividade , Tripanossomicidas/efeitos adversos , Tripanossomíase Africana/tratamento farmacológico , Moduladores de Tubulina/efeitos adversosRESUMO
Cyclooxygenase (COX) inhibitor Indomethacin analogs exhibited more potent cancer cell growth inhibition and apoptosis inducing activities than the parental compound. The anti-proliferative mechanism investigation of the analogs revealed that they inhibited tubulin polymerization at high concentrations whereas enhanced polymerization at low concentrations. The two opposite activities might antagonize each other and impaired the anti-proliferative activity of the derivatives eventually. In this study, we further performed lead optimization based on the structure activity relationship (SAR) generated. One of the new Indomethacin derivatives compound 11 {2-(4-(benzyloxy)phenyl)-N-(1-(4-bromobenzoyl)-3-(2-((2-(dimethylamino)ethyl)amino)-2-oxoethyl)-2-methyl-1H-indol-5-yl)acetamide} inhibited the proliferation of a panel of cancer cell lines with IC50s at the sub-micromole levels. Further study revealed that the compound only enhanced tubulin polymerization and was a tubulin stabilizer.
Assuntos
Antineoplásicos/farmacologia , Indometacina/farmacologia , Neoplasias/metabolismo , Neoplasias/patologia , Tubulina (Proteína)/metabolismo , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Indometacina/síntese química , Indometacina/química , Estrutura Molecular , Neoplasias/tratamento farmacológico , Estabilidade Proteica/efeitos dos fármacos , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Tubulin and heat shock protein 27 (Hsp27) are well-characterized molecular targets for anti-cancer drug development. We previously identified lead compounds that inhibited both Hsp27 and tubulin. These compounds exhibited extensive anti-cancer activities against the proliferation of various human cancer cell lines. In the current study, a systematic ligand based structural optimization led to new analogs that significantly inhibited the growth of a panel of breast cancer cell lines. Furthermore, the most potent compounds were examined with tubulin polymerization assay and Hsp27 chaperone activity assay. The compounds showed potent tubulin polymerization inhibition but no Hsp27 inhibitory effect. The structural optimization dissected the dual activity and improved the selectivity of the compounds for tubulin. The results revealed several structural moieties of the lead compounds that are critical for Hsp27 inhibition. The modification of these structural fragments eliminated Hsp27 inhibition, but did not harm tubulin-targeting effects of the compounds. This result further defined the structure-activity relationship between the tubulin and Hsp27 effects of these compounds.
Assuntos
Desenho de Fármacos , Proteínas de Choque Térmico HSP27/antagonistas & inibidores , Moduladores de Tubulina/farmacologia , Tubulina (Proteína)/química , Linhagem Celular Tumoral , Humanos , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Multimerização Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína , Relação Estrutura-Atividade , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/química , Moduladores de Tubulina/metabolismoRESUMO
The potency of a series of Hexamethylene bis-acetamide (HMBA) derivatives inducing Hexamethylene bis-acetamide inducible protein 1 (HEXIM1) was determined in LNCaP prostate cancer cells. Several compounds with unsymmetrical structures showed significantly improved activity. Distinct from HMBA, these analogs have increased hydrophobicity and can improve the short half-life of HMBA, which is one of the factors that have limited the application of HMBA in clinics. The unsymmetrical scaffolds of the new analogs provide the basis for further lead optimization of the compounds using combinatorial chemistry strategy.
Assuntos
Acetamidas/química , Proteínas de Ligação a RNA/metabolismo , Acetamidas/metabolismo , Acetamidas/farmacologia , Linhagem Celular Tumoral , Desenho de Fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Meia-Vida , Humanos , Fatores de TranscriçãoRESUMO
Heat shock protein 27 (Hsp27) is a chaperone protein, and its expression is increased in response to various stress stimuli including anticancer chemotherapy, which allows the cells to survive and causes drug resistance. We previously identified lead compounds that bound to Hsp27 and tubulin via proteomic approaches. Systematic ligand based optimization in the current study significantly increased the cell growth inhibition and apoptosis inducing activities of the compounds. Compared to the lead compounds, one of the new derivatives exhibited much better potency to inhibit tubulin polymerization but a decreased activity to inhibit Hsp27 chaperone function, suggesting that the structural modification dissected the dual targeting effects of the compound. The most potent compounds 20 and 22 exhibited strong cell proliferation inhibitory activities at subnanomolar concentration against 60 human cancer cell lines conducted by Developmental Therapeutic Program at the National Cancer Institute and represented promising candidates for anticancer drug development.
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Antineoplásicos/farmacologia , Proteínas de Choque Térmico HSP27/antagonistas & inibidores , Moduladores de Tubulina/farmacologia , Tubulina (Proteína)/metabolismo , Antineoplásicos/síntese química , Antineoplásicos/química , Western Blotting , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células HCT116 , Proteínas de Choque Térmico HSP27/metabolismo , Células HT29 , Humanos , Células K562 , Células MCF-7 , Modelos Químicos , Chaperonas Moleculares/antagonistas & inibidores , Chaperonas Moleculares/metabolismo , Estrutura Molecular , Polimerização/efeitos dos fármacos , Relação Estrutura-Atividade , Moduladores de Tubulina/síntese química , Moduladores de Tubulina/químicaRESUMO
It has been demonstrated that two-dimensional (2D) monolayer cancer cell proliferation assay for anti-cancer drug screening is a very artificial model and cannot represent the characteristics of three-dimensional (3D) solid tumors. The multi-cellular in vitro 3D tumor spheroid model is of intermediate complexity, and can provide a bridge to the gap between the complex in vivo tumors and simple in vitro monolayer cell cultures. In this study, a simple and cost-effective cancer 3D spheroid assay suitable for small molecule anti-cancer compound screening was developed, standardized and validated on H292 non-small lung cancer cell line. A pilot screening with this assay was performed utilizing a compound library consisting of 41 anti-cancer agents. The traditional 2D monolayer cell proliferation assay was also performed with the same cell line and compounds. A correlational study based on the IC(50) values from the 2D and 3D assays was conducted. There is low correlation with the two sets of biological data, suggesting the two screening methods provide different information regarding the potency of the tested drug candidates.
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Antineoplásicos/toxicidade , Esferoides Celulares/efeitos dos fármacos , Técnicas de Cultura de Células , Sobrevivência Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Humanos , Cinética , Paclitaxel/química , Paclitaxel/toxicidade , Sulfonamidas/química , Sulfonamidas/toxicidade , Moduladores de Tubulina/toxicidade , Células Tumorais CultivadasRESUMO
Cyclooxygenase (COX) inhibitors Indomethacin and its structural analogs Sulindac exhibit cell growth inhibition and apoptosis inducing activities in various cancer cell lines via COX independent mechanisms. In this study, the molecular structures of Indomethacin and Sulindac were used as starting scaffolds to design novel analogs and their effects on the proliferation of human cancer cells were evaluated. Compared to Indomethacin and Sulindac inhibiting cancer cell proliferation with IC(50)s of more than 1 mM, the derivatives displayed significantly increased activities. Especially, one of the Indomethacin analogs inhibited the growth of a series of cancer cell lines with IC(50)s around 0.5 µM-3 µM. Mechanistic investigation revealed that the new analog was in fact a tubulin inhibitor, although the parental compound Indomethacin did not show any tubulin inhibitory activity. Tubulin polymerization assay indicated this compound inhibited tubulin assembly at high concentrations, but promoted this process at low concentrations which is a very unique mechanism. The binding mode of this compound in tubulin was predicted using the molecular docking simulation.
Assuntos
Inibidores de Ciclo-Oxigenase/farmacologia , Indometacina/farmacologia , Sulindaco/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase/síntese química , Inibidores de Ciclo-Oxigenase/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Indometacina/síntese química , Indometacina/química , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Sulindaco/síntese química , Sulindaco/químicaRESUMO
The potency of a series of sulfonamide tubulin inhibitors against the growth of Trypanosoma brucei (T. brucei), as well as human cancer and primary fibroblast cells were evaluated with the aim of determining whether compounds that selectively inhibit parasite proliferation could be identified. Several compounds showed excellent selectivity against T. brucei growth, and have the potential to be used for the treatment of Human African trypanosomiasis. A T. brucei tubulin protein homology model was built based on the crystal structure of the bovine tubulin. The colchicine-binding domain, which is also the binding site of the tested sulfonamide tubulin inhibitors, showed clear differences between the tubulin structures and presumably explained the selectivity of the compounds.
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Tripanossomicidas/química , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacologia , Tubulina (Proteína)/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Homologia Estrutural de Proteína , Sulfonamidas/química , Sulfonamidas/farmacologia , Trypanosoma brucei brucei/crescimento & desenvolvimento , Tripanossomíase Africana/tratamento farmacológico , Tripanossomíase Africana/parasitologia , Tubulina (Proteína)/químicaRESUMO
JCC76 is a derivative of cyclooxygenase-2(COX-2) selective inhibitor nimesulide and exhibits potent anti-breast cancer activity. It selectively induces apoptosis of Her2 positive breast cancer cells. However, the specific molecular targets of JCC76 still remain unclear, which significantly withdraw the further drug development of JCC76. To identify the molecular targets of JCC76, a six carbon linker and biotin conjugated JCC76 probe was designed and synthesized. The anti-proliferation activity of the probe and its analogs was evaluated.