RESUMO
To identify new potential therapeutic targets for neurodegenerative diseases, we initiated activity-based protein profiling studies with withanolide A (WitA), a known neuritogenic constituent of Withania somnifera root with unknown mechanism of action. Molecular probes were designed and synthesized, and led to the discovery of the glucocorticoid receptor (GR) as potential target. Molecular modeling calculations using the VirtualToxLab predicted a weak binding affinity of WitA for GR. Neurite outgrowth experiments in human neuroblastoma SH-SY5Y cells further supported a glucocorticoid-dependent mechanism, finding that WitA was able to reverse the outgrowth inhibition mediated by dexamethasone (Dex). However, further GR binding and transactivation assays found no direct interference of WitA. Further molecular modeling analysis suggested that WitA, although forming several contacts with residues in the GR binding pocket, is lacking key stabilizing interactions as observed for Dex. Taken together, the data suggest that WitA-dependent induction of neurite outgrowth is not through a direct effect on GR, but might be mediated through a closely related pathway. Further experiments should evaluate a possible role of GR modulators and/or related signaling pathways such as ERK, Akt, NF-κB, TRα, or Hsp90 as potential targets in the WitA-mediated neuromodulatory effects.
Assuntos
Receptores de Glucocorticoides/metabolismo , Vitanolídeos/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dexametasona/química , Dexametasona/metabolismo , Dexametasona/farmacologia , Glucocorticoides/química , Glucocorticoides/metabolismo , Glucocorticoides/farmacologia , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Simulação de Acoplamento Molecular , NF-kappa B/metabolismo , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de Glucocorticoides/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Vitanolídeos/farmacologia , Vitanolídeos/uso terapêuticoRESUMO
Evaluation of the importance of C18/C19 stereochemistry of azinomycin A/B epoxyamide partial structures with respect to DNA alkylation sequence selectivity is reported using a unique assay with a DNA oligomer containing imbedded normal (5'-GGC-3'/3'-CCG-5') and inverted (5'-CGG-3'/3'-GCC-5') azinomycin consensus cross-linking sequences. Both species were found to have unique selectivity profiles and alkylate DNA in a manner distinct from azinomycin B. Computational docking experiments support altered binding modes for the enantiomers.