Receptor-mediated internalization and endocytic trafficking pathways are attractive targets for new drug development. Although important for delivery of therapeutics into the cell, the endocytic route has not been extensively explored as a pharmacological target per se. Endocytic trafficking has been implicated in some pathologies and therapies, e.g., HIV infection of cells, amyloid/precursor uptake or membrane disruption, G-protein-receptors and analgesia, glutamate I receptors and neuroprotection, and generally in the control of cell signaling-mediated proliferation, death, differentiation. We report on experimental strategies for screening potential therapeutic activities of phytochemicals in this context. The receptor and transport assays considered are aimed at detecting modulators of (i) cell surface receptor (R) levels, (ii) early internalization rates of R/ligand (L), and (iii) recycling of internalized R/L. In particular, we are developing a cellular screen based on the use of L-enzyme conjugates for analysis of (ii) and (iii). The method is currently being tested with a human keratinocyte line. We are beginning to screen plant aq. and alc. extracts, and purified phytochemicals; in most experiments L is biotinylated-transferrin. To date, we have observed the greatest % inhibition of (ii) with Capsicum annuum (5 microL of 0.4g/mL aq. extract): 54.2±12.3 (p <0.05, n=4). The main phytochemical(s) responsible for this activity remain(s) to be identified. Ascorbate is one candidate; at 40 microM it exhibits strong inhibition, 49.1±7.3% (p<0.05, n=8). Moreover, the ascorbate level likely reaches 30–60 microM with Capsicum treatment. In conclusion, we have developed a high-throughput cellular screen for modulation of endocytic trafficking by natural products; strong inhibitors or stimulators are candidates for further analyses (e.g., in context of above pathologies).
