Non-invasive pharmacological profiling of novel endomorphin-2 analogs by confocal Raman spectroscopy and imaging

Understanding how biased opioid agonists signal through different intracellular pathways, particularly in the context of pain, is still a major challenge. One reason is the lack of suitable imaging methods that can capture ligand-receptor interactions and their downstream effects in live cells with spatial resolution. In this study, we apply a Raman-based imaging technique that allowed us to observe and compare the intracellular distribution and signaling behavior of endomorphin-2 analogs, JPC-11 and JPC-13, shedding light on the distinct dynamics of G protein versus β-arrestin recruitment by the MOR. The data obtained in this study provide valuable insights into the pharmacological activity of opioid analogs biased toward G protein or β-arrestin 2. We observed changes in the Raman bands at 750, 782, 1003, 1035, 1092, 1126, 1254, 1302, 1310, 1339, 1444, 1583, and 1654 cm^-1, which correspond to molecular features of lipids, proteins, carbohydrates and DNA associated with specific cellular organelles. Structural rearrangements can impact the vibrational environment of both ligand- and receptor-associated aromatic residues, leading to measurable differences in the tyrosine-associated Raman bands, particularly in the 800-1100 cm^-1 region, with a prominent shift of the band at 1035 cm^-1. Here we develop a new method to evaluate and understand the consequences of biased agonism in terms of therapeutic potential and side effects of opioid receptor ligands.