Unveiling the molecular dynamics of a nitrile-containing 5-lipoxygenase-activating protein antagonist in primary macrophages through Raman spectroscopy

The nitrile (–CN) functional group is a versatile pharmacophore motif that also serves as an intrinsic, bioorthogonal Raman tag in the silent wavenumber region (1800–2700 cm−1). Here, we exploit this dual functionality to track the potent nitrile-containing 5-lipoxygenase-activating protein (FLAP) antagonist BRP-685 in primary human macrophages using label-free spontaneous and stimulated Raman scattering. This approach enables direct intracellular localization at biologically relevant, low micromolar concentrations without chemical modification or external labels. Quantitative Raman imaging reveals that BRP-685 preferentially accumulates in lipid droplets, distinct from its membrane-bound target site at the nuclear envelope/endoplasmic reticulum. Multiplexed analysis with an alkyne-tagged lipid analog uncovers a unique distribution pattern, suggesting that lipid droplets act as intracellular reservoirs for highly lipophilic drugs.