Identification of new LAT-1 transporter substrates for drug delivery - The blood-brain barrier (BBB) prevents entry of most drug molecules into the brain, an obstacle which adds time
and cost to the discovery and development of central nervous system (CNS) drugs. The BBB possesses
transporter proteins that allow biological molecules including amino acids, glucose, and hormones to cross as
needed. One of these transporter proteins, the L-type amino acid transporter (LAT-1) transports specific amino
acids (e.g. phenylalanine) as well as compounds that resemble them (e.g. the drug L-DOPA). This transporter
offers a potential route for targeted drug delivery to the brain; moreover, it has been demonstrated that drugs
chemically linked to amino acids can cross the BBB via LAT-1. It is known that some molecules are transported
(substrates), while others only bind to the transporter surface (inhibitors); however, the structural requirements
for either type of activity is poorly understood. We propose mapping the structure-activity relationship (SAR) for
molecules that interact with LAT-1 with the aspiration of making this transporter a practical and widely used
vehicle for drug delivery. Our approach involves using a computational model of the LAT-1 binding site to guide
the synthesis of compounds similar in structure to natural amino acids but with unique chemical properties that
could be used to design improved drug delivery agents. Our specific aims are three-fold. First, we will synthesize
amino acids chemically substituted with groups that our model predicts will form beneficial interactions with the
transporter. Second, we will use cell-based assays to test these compounds. To determine whether they are
transported, we will use cis-inhibition and trans-stimulation cell assays which have successfully identified
previously unknown substrates. SAR from these experiments will be used to refine our computational model in
order to generate hypotheses to guide further substrate optimization. The intracellular concentration of selected
compounds will be analyzed by LC-MS/MS to verify our hypothesis that LAT-1 activity increases cell uptake. A
third aim of this project will involve the synthesis and testing of amino acid prodrugs of a topoisomerase I inhibitor
for cancer (SN-38) to assess whether the SAR derived from optimization of LAT-1 substrates in specific aims 1
and 2 can be applied to improve prodrug cellular uptake. Additionally, the effect of the linker joining SN-38 and
the amino acid promoiety, which may have a significant impact on prodrug pharmacokinetics and efficacy, will
be examined. This project has broad applications for treating many different diseases both in the brain and other
tissues where LAT-1 is heavily expressed, including cancer.
