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1.
Bioorg Chem ; 116: 105273, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34474304

RESUMO

The ubiquitin-specific protease 7 (USP7)-murine double minute 2 (MDM2)-p53 network plays an important role in the regulation of p53, a tumor suppressor which plays critical roles in regulating cell growth, proliferation, cell cycle progression, apoptosis and immune response. The overexpression of USP7 and MDM2 in human cancers contributes to cancer initiation and progression, and their inhibition reactivates p53 signalings and causes cell cycle arrest and apoptosis. Herein, the current state of pharmacological characterization, potential applications in cancer treatment and mechanism of action of small molecules used to target and inhibit MDM2 and USP7 proteins are highlighted, along with the outcomes in clinical and preclinical settings. Moreover, challenges and advantages of these strategies, as well as perspectives in USP7-MDM2-p53 field are analyzed in detail. The investigation and application of MDM2 and USP7 inhibitors will deepen our understanding of the function of USP7-MDM2-p53 network, and feed in the development of effective and safe cancer therapies where USP7-MDM2-p53 network is implicated.

2.
Artigo em Inglês | MEDLINE | ID: mdl-34323202

RESUMO

BACKGROUND: As a FAD (Flavin Adenine Dinucleotide) - dependent histone demethylase discovered in 2004, LSD1 (lysine specific demethylase 1) was reported to be overexpressed in diverse tumors, regulating target genes transcription associated with cancer development. Hence, LSD1 targeted inhibitors may represent a new insight in anticancer drug discovery. For these reasons, researchers in both the pharmaceutical industry and academia have been actively pursuing LSD1 inhibitors in the quest for new anti-cancer drugs. OBJECTIVES: This review summaries patents about LSD1 inhibitors in recent 5 years in hope of providing a reference for LSD1 researchers to develop new modulators of LSD1 with higher potency and fewer adverse effects. METHODS: This review collects LSD1 inhibitors disclosed in patents since 2016. The primary ways of patent searching are Espacenet®, Google Patents, and CNKI. RESULTS: This review covers dozens of patents related to LSD1 inhibitors in recent five years. The compound structures are mainly divided into TCP (Tranylcypromine) derivatives, imidazole derivatives, pyrimidine derivatives, and other natural products and peptides. Meanwhile, the compounds that have entered the clinical phase are also described. CONCLUSION: Most of the compounds in these patents have been subjected to activity analysis with LSD1 and multi-cell lines, showing good antitumor activity in vitro and in vivo. These patents exhibited the structural diversity of LSD1 inhibitors and the potential of natural products as novel LSD1 inhibitors.

3.
Bioorg Chem ; 114: 105120, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34216895

RESUMO

Bromodomain 4 (BRD4) proteins play an important role in histone post-translational modifications and facilitate several important physiological and pathological processes, including cancers. The inhibition of BRD4 by small molecule inhibitors shows promise as a therapeutic strategy for cancer treatment. However, their clinical applications were limited, which is largely hampered by off-target effects-induced toxicity. We herein report the design, synthesis, and cellular imaging of a set of tumor-anchored and BRD4-targeted fluorescent ligands by introducing selective and potent BRD4 inhibitor into different fluorophores via variable linkers. One of the fluorescent conjugates (compound 6) was demonstrated to be cell-permeable and low cytotoxic, preferentially accumulated in cancer cells, and display pronounced fluorescent signal. More importantly, 6 was identified to show specific BRD4 engagement in the cellular content. Collectively, this study provides a pathway for developing labeled BRD4 ligands and highlights that compound 6 may represent a valuable tool for explorative learning and target delivery study of BRD4.

4.
EMBO Rep ; 22(8): e50922, 2021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34060205

RESUMO

Several studies have examined the functions of nucleic acids in small extracellular vesicles (sEVs). However, much less is known about the protein cargos of sEVs and their functions in recipient cells. This study demonstrates the presence of lysine-specific demethylase 1 (LSD1), which is the first identified histone demethylase, in the culture medium of gastric cancer cells. We show that sEVs derived from gastric cancer cells and the plasma of patients with gastric cancer harbor LSD1. The shuttling of LSD1-containing sEVs from donor cells to recipient gastric cancer cells promotes cancer cell stemness by positively regulating the expression of Nanog, OCT4, SOX2, and CD44. Additionally, sEV-delivered LSD1 suppresses oxaliplatin response of recipient cells in vitro and in vivo, whereas LSD1-depleted sEVs do not. Taken together, we demonstrate that LSD1-loaded sEVs can promote stemness and chemoresistance to oxaliplatin. These findings suggest that the LSD1 content of sEV could serve as a biomarker to predict oxaliplatin response in gastric cancer patients.


Assuntos
Vesículas Extracelulares , Neoplasias Gástricas , Histona Desmetilases/genética , Humanos , Lisina , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/genética
5.
J Hematol Oncol ; 14(1): 57, 2021 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-33827629

RESUMO

Ubiquitin-conjugating enzyme E2 M (UBE2M) and ubiquitin-conjugating enzyme E2 F (UBE2F) are the two NEDD8-conjugating enzymes of the neddylation pathway that take part in posttranslational modification and change the activity of target proteins. The activity of E2 enzymes requires both a 26-residue N-terminal docking peptide and a conserved E2 catalytic core domain, which is the basis for the transfer of neural precursor cell-expressed developmentally downregulated 8 (NEDD8). By recruiting E3 ligases and targeting cullin and non-cullin substrates, UBE2M and UBE2F play diverse biological roles. Currently, there are several inhibitors that target the UBE2M-defective in cullin neddylation protein 1 (DCN1) interaction to treat cancer. As described above, this review provides insights into the mechanism of UBE2M and UBE2F and emphasizes these two E2 enzymes as appealing therapeutic targets for the treatment of cancers.


Assuntos
Dano ao DNA/genética , Proteína NEDD8/metabolismo , Neoplasias/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Humanos , Neoplasias/patologia
8.
J Med Chem ; 64(5): 2466-2488, 2021 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-33619958

RESUMO

As a flavin adenine dinucleotide (FAD)-dependent monoamine oxidase, lysine specific demethylase 1 (LSD1/KDM1A) functions as a transcription coactivator or corepressor to regulate the methylation of histone 3 lysine 4 and 9 (H3K4/9), and it has emerged as a promising epigenetic target for anticancer treatment. To date, numerous inhibitors targeting LSD1 have been developed, some of which are undergoing clinical trials for cancer therapy. Although only two reversible LSD1 inhibitors CC-90011 and SP-2577 are in the clinical stage, the past decade has seen remarkable advances in the development of reversible LSD1 inhibitors. Herein, we provide a comprehensive review about structures, biological evaluation, and structure-activity relationship (SAR) of reversible LSD1 inhibitors.


Assuntos
Inibidores Enzimáticos/uso terapêutico , Histona Desmetilases/antagonistas & inibidores , Compostos Orgânicos/uso terapêutico , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Estrutura Molecular , Neoplasias/tratamento farmacológico , Compostos Orgânicos/química , Compostos Orgânicos/farmacologia , Relação Estrutura-Atividade
10.
J Med Chem ; 63(23): 14197-14215, 2020 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-32931269

RESUMO

Histone lysine-specific demethylase 1 (LSD1/KDM1A) has become an important and promising anticancer target since it was first identified in 2004 and specially demethylates lysine residues of histone H3K4me1/2 and H3K9me1/2. LSD1 is ubiquitously overexpressed in diverse cancers, and abrogation of LSD1 results in inhibition of proliferation, invasion, and migration in cancer cells. Over the past decade, a number of biologically active small-molecule LSD1 inhibitors have been developed. To date, six trans-2-phenylcyclopropylamine (TCP)-based LSD1 inhibitors (including TCP, ORY-1001, GSK-2879552, INCB059872, IMG-7289, and ORY-2001) that covalently bind to the flavin adenine dinucleotide (FAD) within the LSD1 catalytic cavity have already entered into clinical trials. Here, we provide an overview about the structures, activities, and structure-activity relationship (SAR) of TCP-based LSD1 inhibitors that mainly covers the literature from 2008 to date. The opportunities, challenges, and future research directions in this emerging and promising field are also discussed.


Assuntos
Inibidores Enzimáticos/farmacologia , Histona Desmetilases/antagonistas & inibidores , Tranilcipromina/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Desenho de Fármacos , Inibidores Enzimáticos/química , Humanos , Relação Estrutura-Atividade , Tranilcipromina/química
11.
Pharmacol Res ; 159: 104991, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32504836

RESUMO

LSD1 (histone lysine specific demethylase 1) takes part in the physiological process of cell differentiation, EMT (epithelial-mesenchymal transition) and immune response. In this study, we found LSD1 expression in metastatic gastric cancer tissues was significantly higher than that in normal tissues. Furthermore, LSD1 deletion was found to suppress gastric cancer migration by decreasing intracellular miR-142-5p, which further led to the upregulation of migration suppressor CD9, a newly identified target of miR-142-5p. While LSD1 was reported as a demethylase of H3K4me1/2, H3K9me1/2 and several non-histone proteins, this is a new evidence for LSD1 as a functional regulator of miRNA. On the other hand, our data suggested that promoting the secretion of miR-142-5p using small extracellular vesicles as vehicles is a new mechanism for LSD1 abrogation to down-regulate intracellular miR-142-5p. Taken together, this study uncovered a new mechanism for LSD1 that can contribute to gastric cancer migration by facilitating miR-142-5p to target CD9.


Assuntos
Movimento Celular , Deleção de Genes , Histona Desmetilases/metabolismo , MicroRNAs/metabolismo , Neoplasias Gástricas/enzimologia , Tetraspanina 29/metabolismo , Animais , Linhagem Celular Tumoral , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Histona Desmetilases/genética , Humanos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , MicroRNAs/genética , Invasividade Neoplásica , Metástase Neoplásica , Transdução de Sinais , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Tetraspanina 29/genética
12.
Med Res Rev ; 40(5): 1920-1949, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32391596

RESUMO

The ubiquitin-proteasome system (UPS) is a complex process that regulates protein stability and activity by the sequential actions of E1, E2 and E3 enzymes to influence diverse aspects of eukaryotic cells. However, due to the diversity of proteins in cells, substrate selection is a highly critical part of the process. As a key player in UPS, E3 ubiquitin ligases recruit substrates for ubiquitination specifically. Among them, RING E3 ubiquitin ligases which are the most abundant E3 ubiquitin ligases contribute to diverse cellular processes. The multisubunit cullin-RING ligases (CRLs) are the largest family of RING E3 ubiquitin ligases with tremendous plasticity in substrate specificity and regulate a vast array of cellular functions. The F-box protein Skp2 is a component of CRL1 (the prototype of CRLs) which is expressed in many tissues and participates in multiple cellular functions such as cell proliferation, metabolism, and tumorigenesis by contributing to the ubiquitination and subsequent degradation of several specific tumor suppressors. Most importantly, Skp2 plays a pivotal role in a plethora of cancer-associated signaling pathways. It enhances cell growth, accelerates cell cycle progression, promotes migration and invasion, and inhibits cell apoptosis among others. Hence, targeting Skp2 may represent a novel and attractive strategy for the treatment of different human cancers overexpressing this oncogene. In this review article, we summarized the known roles of Skp2 both in health and disease states in relation to the UPS.

13.
Eur J Med Chem ; 192: 112161, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32155529

RESUMO

KDM5B (also known as PLU-1 and JARID1B) is 2-oxoglutarate and Fe2+ dependent oxygenase that acts as a histone H3K4 demethylase, which is a key participant in inhibiting the expression of tumor suppressors as a drug target. Here, we present the discovery of pyrazole derivatives compound 5 by structure-based virtual screening and biochemical screening with IC50 of 9.320 µM against KDM5B, and its subsequent optimization to give 1-(4-methoxyphenyl)-N-(2-methyl-2-morpholinopropyl)-3-phenyl-1H-pyrazole-4-carboxamide (27 ab), a potent KDM5B inhibitor with IC50 of 0.0244 µM. In MKN45 cells, compound 27 ab can bind and stabilize KDM5B and induce the accumulation of H3K4me2/3, bona fide substrates of KDM5B, while keep the amount of H3K4me1, H3K9me2/3 and H3K27me2 without change. Further biological study also indicated that compound 27 ab is a potent cellular active KDM5B inhibitor that can inhibit MKN45 cell proliferation, wound healing and migration. In sum, our finding gives a novel structure for the discovery of KDM5B inhibitor and targeting KDM5B may be a new therapeutic strategy for gastric cancer treatment.


Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Pirazóis/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Histona Desmetilases com o Domínio Jumonji , Estrutura Molecular , Proteínas Nucleares , Pirazóis/síntese química , Pirazóis/química , Proteínas Repressoras , Relação Estrutura-Atividade , Células Tumorais Cultivadas , Cicatrização/efeitos dos fármacos
14.
Bioorg Chem ; 97: 103648, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32065882

RESUMO

Natural protoberberine alkaloids were first identified and characterized as potent, selective and cellular active lysine specific demethylase 1 (LSD1) inhibitors. Due to our study, isoquinoline-based tetracyclic scaffold was identified as the key structural element for their anti-LSD1 activity, subtle changes of substituents attached to the core structure led to dramatic changes of the activity. Among these protoberberine alkaloids, epiberberine potently inhibited LSD1 (IC50 = 0.14 ± 0.01 µM) and was highly selective to LSD1 over MAO-A/B. Furthermore, epiberberine could induce the expression of CD86, CD11b and CD14 in THP-1 and HL-60 cells, confirming its cellular activity of inducing acute myeloid leukemia (AML) cells differentiation. Moreover, epiberberine prolonged the survival of THP-1 cells bearing mice and inhibited the growth of AML cells in vivo without obvious global toxicity. These findings give the potential application of epiberberine in AML treatment, and the isoquinoline-based tetracyclic scaffold could be used for further development of LSD1 inhibitors.


Assuntos
Antineoplásicos/uso terapêutico , Alcaloides de Berberina/uso terapêutico , Histona Desmetilases/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Animais , Antineoplásicos/química , Alcaloides de Berberina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Células HL-60 , Histona Desmetilases/metabolismo , Humanos , Camundongos , Camundongos SCID
15.
Curr Drug Metab ; 21(1): 67-76, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31902352

RESUMO

BACKGROUND: Cancer is one of the most serious diseases threatening human health with high morbidity and mortality in the world. For the treatment of cancer, chemotherapy is one of the most widely used strategies, for almost all kinds of tumors and diverse stages of tumor development. The efficacy of chemotherapy not only depends on the activity of the drug administrated but also on whether the compound could reach the effective therapeutic concentration in tumor cells. Therefore, expression and activity of drug-metabolizing enzymes (DMEs) in tumor tissues and metabolic organs of cancer patients are important for the dispositional behavior of anticancer drugs as well as the clinical response of chemotherapy. METHODS: This review summarizes the recent advancement of the DMEs expression and activity in various cancers, as well as the potential regulatory mechanisms of major DMEs in cancer and cancer therapy. RESULTS: Compared to normal tissues, expression and activity of major DMEs are significantly dysregulated in patients by various factors including epigenetic modification, ligand-activated transcriptional regulation and signaling pathways. Additionally, DMEs play an important role in anticancer drug efficacy, chemoresistance as well as the activation of prodrugs. CONCLUSION: This review reinforces a more comprehensive understanding of DMEs in cancer and cancer therapy, and provides more opportunities for cancer therapy.


Assuntos
Antineoplásicos/metabolismo , Antineoplásicos/uso terapêutico , Inativação Metabólica/efeitos dos fármacos , Inativação Metabólica/fisiologia , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Animais , Humanos , Transdução de Sinais/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos
16.
Life Sci ; 242: 117247, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31899223

RESUMO

AIMS: Programmed death ligand 1 (PD-L1, CD274) has been reported to be expressed abnormally in many cancers, nevertheless, effect of PD-L1 on tumor cells remains unclear, especially in gastric cancer (GC). This study aimed to investigate the role of PD-L1 in metastasis and differentiation in GC. MAIN METHODS: Immunohistochemistry was performed on 237 paired GC tissues. shPD-L1 cells were generated by lentivirus shRNA solution and PD-L1-overexpressing cells were constructed by pcDNA3.1. Expression of PD-L1 and E-cadherin in GC cells were detected by western blot. KEY FINDINGS: PD-L1 expression was significantly lower in GC than that in adjacent normal tissues, especially in poorly differentiated and metastatic GC, but was positively correlated to survival time of patients. Moreover, PD-L1 ablation could decrease E-cadherin expression, promote cell migration and wound repair ability. In turn, overexpression of PD-L1 increased E-cadherin expression and inhibited wound repair ability. At the same time, All-trans retinoic acid (ATRA), which has the properties of pro-differentiation and inhibition of invasion and metastasis, upregulated the expression of PD-L1 and E-cadherin. SIGNIFICANCE: These findings not only identify PD-L1 may have a positive role for the treatment of GC, but also implicate that ATRA combined PD-L1 antibody drugs may enhance anti-tumor Immunity in GC.


Assuntos
Antígeno B7-H1/metabolismo , Neoplasias Gástricas/patologia , Antígeno B7-H1/fisiologia , Western Blotting , Caderinas/metabolismo , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Gástricas/metabolismo
17.
Eur J Med Chem ; 175: 357-372, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31096156

RESUMO

Lysine-specific demethylase 1 (LSD1), demethylase against mono- and di - methylated histone3 lysine 4, has emerged as a promising target in oncology. More specifically, it has been demonstrated as a key promoter in acute myeloid leukemia (AML), and several LSD1 inhibitors have already entered into clinical trials for the treatment of AML. In this paper, a series of new indole derivatives were designed and synthesized based on a lead compound obtained by a high-throughput screening with our in-house compound library. Among the synthetic compounds, 9e was characterized as a potent LSD1 inhibitor with an IC50 of 1.230 µM and can inhibit the proliferation of THP-1 cells effectively. And most importantly, this is the first irreversible LSD1 inhibitor that is not derived from monoamine oxidase inhibitors. Hence, the discovery of 9e may serve as a proof of concept work for AML treatment.


Assuntos
Descoberta de Drogas , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Histona Desmetilases/antagonistas & inibidores , Indóis/síntese química , Indóis/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Furanos/química , Ensaios de Triagem em Larga Escala , Histona Desmetilases/metabolismo , Humanos , Indóis/química , Indóis/metabolismo , Concentração Inibidora 50 , Leucemia Mieloide Aguda/patologia , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade
18.
Bioorg Chem ; 87: 688-698, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30953888

RESUMO

Sanggenon O (SO) is a Diels-Alder type adduct extracted fromMorus alba, which has been used for its anti-inflammatory action in the Oriental medicine. However, whether it has regulatory effect on human cancer cell proliferation and what the underlying mechanism remains unknown. Here, we found that SO could significantly inhibit the growth and proliferation of A549 cells and induce its pro-apoptotic action through a caspase-dependent pathway. It could also impair the mitochondria which can be reflected by mitochondrial membrane permeabilization. Besides, SQSTM1 up-regulation and autophagic flux measurement demonstrated that exposure to SO led to autophagosome accumulation, which plays a protective role in SO-treated cells. In addition, knocking down of LC3B increased SO triggered apoptotic cell rates. These results indicated that SO has great potential as a promising candidate combined with autophagy inhibitor for the treatment of NSCLC. In conclusion, our results identified a novel mechanism by which SO exerts potent anticancer activity.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Flavonoides/farmacologia , Substâncias Protetoras/farmacologia , Células A549 , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Flavonoides/síntese química , Flavonoides/química , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Conformação Molecular , Simulação de Acoplamento Molecular , Substâncias Protetoras/síntese química , Substâncias Protetoras/química , Espécies Reativas de Oxigênio/análise , Espécies Reativas de Oxigênio/metabolismo , Relação Estrutura-Atividade , Células Tumorais Cultivadas
19.
J Med Chem ; 62(5): 2772-2797, 2019 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-30803229

RESUMO

The cullin-RING ubiquitin ligases (CRLs) are responsible for about 20% of cellular protein degradation and regulate diverse cellular processes, and the dysfunction of CRLs is implicated in human diseases. Targeting the CRLs has become an emerging strategy for the treatment of human diseases. Herein, we describe the discovery of a hit compound from our in-house library and further structure-based optimizations, which have enabled the identification of new triazolo[1,5- a]pyrimidine-based inhibitors targeting the DCN1-UBC12 interaction. Compound WS-383 blocks the DCN1-UBC12 interaction (IC50 = 11 nM) reversibly and shows selectivity over selected kinases. WS-383 exhibits cellular target engagement to DCN1 in MGC-803 cells. WS-383 inhibits Cul3/1 neddylation selectively over other cullins and also induces accumulation of p21, p27, and NRF2. Collectively, targeting the DCN1-UBC12 interaction would be a viable strategy for selective neddylation inhibition of Cul3/1 and may be of therapeutic potential for disease treatment in which Cul3/1 is dysregulated.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Pirimidinas/farmacologia , Triazóis/farmacologia , Enzimas de Conjugação de Ubiquitina/metabolismo , Células Cultivadas , Humanos , Estrutura Molecular , Ligação Proteica , Pirimidinas/química , Triazóis/química
20.
J Pharm Biomed Anal ; 162: 9-15, 2019 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-30219599

RESUMO

N6-methyladenosine (m6A) is the most prevalent internal modification of eukaryotic messenger RNA (mRNA). Until now, two RNA demethylases have been identified, including FTO (fat mass and obesity-associated protein) and ALKBH5 (α-ketoglutarate-dependent dioxygenase alkB homologue 5). As a mammalian m6A demethylase, ALKBH5 significantly affects mRNA export and RNA metabolism as well as the assembly of mRNA processing factors in nuclear speckles, and ALKBH5 may play a significant role in these biological processes. Nevertheless, no modulator of ALKBH5 has been reported. The reason for that may be the lack of in vitro assays for ALKBH5 inhibitor screening. Herein, we describe the development of two homogeneous assays for ALKBH5 using N6-methyladenosine as substrate with different principles. Using ALKBH5 recombinant, we developed a formaldehyde dehydrogenase coupled fluorescence based assay and an antibody based assay for the activity evaluation of ALKBH5. These robust coupled assays are suitable for screening ALKBH5 inhibitors in 384-well format (Z' factors of 0.74), facilitating the discovery of modulators in the quest for the regulation of biological processes.


Assuntos
Adenosina/análogos & derivados , Aldeído Oxirredutases/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Descoberta de Drogas/métodos , Imunofluorescência , Formaldeído/metabolismo , Ensaios de Triagem em Larga Escala/métodos , RNA Mensageiro/metabolismo , Adenosina/metabolismo , Aldeído Oxirredutases/antagonistas & inibidores , Homólogo AlkB 5 da RNA Desmetilase/antagonistas & inibidores , Desmetilação , Inibidores Enzimáticos/farmacologia , Humanos , Cinética , Reprodutibilidade dos Testes , Espectrometria de Fluorescência
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