Click chemistry-assisted identification of caffeic acid propargylamide (CAPA)-modified protein Keap1 demonstrates a general mechanism underlying the neuroprotective and neuritogenic activities of o-catechol-bearing natural products

Abstract

Objectives: Caffeic acid and its derivatives bear a typical o-catechol structural element and exhibit a broad spectrum of pharmacological activities including antioxidant, anti-inflammatory, chemopreventive, anticancer, antibacterial and neuroprotective effects. The present study was designed to characterize the neuritogenic and neuroprotective activities of caffeic acid derivatives and the underlying molecular mechanisms. Methods: We synthesized caffeic acid propargylamide (CAPA) as a probe to label the intracellular protein targets in a covalent manner. Caffeic acid-modified proteins are extracted from CAPA-treated cells and incubated with Azido-propyl biotin through a Click chemistry procedure. The biotin tag was detected by streptavidin-HRP conjugate. For neurite outgrowth assay, the neurites were visualized by neurite outgrowth staining kit and neuronal biomarkers (e.g. β3-tubulin and MAP2) were detected by Western blotting and immunostaining. Cell viability was assayed by a colorimetric MTT assay. ROS and RNS were determined by fluorescent probes, DCFH-DA and DAF-FM DA, respectively. Results: We isolated a predominant protein with a size of 72 kDa according to the detection with streptavidin-HRP conjugate. We further verified that CAPA covalently bound to Keap1, a key protein to regulate the activation of transcription factor Nrf2 and antioxidant enzyme heme oxygenase-1 (HO-1) pathway. CAPA was unexpectedly found to be a potent neuritogenic agent. CAPA potentiated nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells in a concentration- and time-dependent manner. Moreover, CAPA could also protect neuronal cells against 6-hydroxydopamine (6-OHDA) toxicity via suppressing 6-OHDA-induced production of reactive oxygen species (ROS)/reactive nitrogen species (RNS) and preserving the integrity of mitochondrial membrane. Importantly, HO-1 inhibitor almost abolished the neuritogenic and neuroprotective activities of CAPA. Conclusion: These results suggest that CAPA and related caffeic acid derivatives exhibit neuroprotective and neuritogenic activities via the modification of Keap1 and subsequent activation of Nrf2/HO-1 pathway.