Cellular
target identification plays an essential role in innovative
drug development and pharmacological mechanism elucidation. However,
very few practical experimental methodologies have been developed
for identifying target proteins for supercomplex molecular systems
such as biologically active phytochemicals or pharmaceutical compositions.
To overcome this limitation, we synthesized gold nanoparticles (AuNPs)
as solid scaffolds, which were bound with 4,4′-dihydroxybenzophenone
(DHBP) as a photo-cross-linking group on the surface. Then, DHBP-modified
AuNPs cross-linked various organic compounds from phytochemicals under
ultraviolet radiation via carbene reactions, H–C bond insertion,
for catalytic C–C bond formation. We next used the phytochemical-cross-linked
AuNPs (phytoAuNPs) to pull down potential binding proteins from brain
tissue lysate and identified 13 neuroprotective targets by mass spectrometry
analysis. As an exemplary study, we selected Hsp60 as a crucial cellular
target to further screen 14 target-binding compounds from phytochemicals
through surface plasmon resonance (SPR) analysis, followed by Hsp60
activity detection and neuroprotective effect assay in cells. Collectively,
this gold nanoparticle-based photo-cross-linking strategy can serve
as a useful platform for discovering novel cellular targets for supercomplex
molecular systems and help to explore pharmacological mechanisms and
active substances.