RESUMO
Antagonism of the human adenosine A3 receptor (hA3R) has potential therapeutic application. Alchemical relative binding free energy calculations of K18 and K32 suggested that the combination of a 3-(2,6-dichlorophenyl)-isoxazolyl group with 2-pyridinyl at the ends of a carbonyloxycarboximidamide group should improve hA3R affinity. Of the 25 new analogues synthesized, 37 and 74 showed improved hA3R affinity compared to K18 (and K32). This was further improved through the addition of a bromine group to the 2-pyridinyl at the 5-position, generating compound 39. Alchemical relative binding free energy calculations, mutagenesis studies and MD simulations supported the compounds' binding pattern while suggesting that the bromine of 39 inserts deep into the hA3R orthosteric pocket, so highlighting the importance of rigidification of the carbonyloxycarboximidamide moiety. MD simulations highlighted the importance of rigidification of the carbonyloxycarboximidamide, while suggesting that the bromine of 39 inserts deep into the hA3R orthosteric pocket, which was supported through mutagenesis studies 39 also selectively antagonized endogenously expressed hA3R in nonsmall cell lung carcinoma cells, while pharmacokinetic studies indicated low toxicity enabling in vivo evaluation. We therefore suggest that 39 has potential for further development as a high-affinity hA3R antagonist.
Assuntos
Antagonistas do Receptor A3 de Adenosina , Receptor A3 de Adenosina , Humanos , Receptor A3 de Adenosina/metabolismo , Receptor A3 de Adenosina/química , Relação Estrutura-Atividade , Animais , Antagonistas do Receptor A3 de Adenosina/farmacologia , Antagonistas do Receptor A3 de Adenosina/química , Antagonistas do Receptor A3 de Adenosina/síntese química , Simulação de Dinâmica Molecular , Ratos , Células CHO , Linhagem Celular Tumoral , Cricetulus , Compostos Heterocíclicos/química , Compostos Heterocíclicos/farmacologia , Compostos Heterocíclicos/síntese químicaRESUMO
Identification and isolation of senescent cells is challenging, rendering their detailed analysis an unmet need. We describe a precise one-step protocol to fluorescently label senescent cells, for flow cytometry and fluorescence microscopy, implementing a fluorophore-conjugated Sudan Black-B analog, GLF16. Also, a micelle-based approach allows identification of senescent cells in vivo and in vitro, enabling live-cell sorting for downstream analyses and live in vivo tracking. Our protocols are applicable to cellular systems, tissues, or animal models where senescence is present. For complete details on the use and execution of this protocol, please refer to Magkouta et al.1.