Structural Features Affecting the Interactions and Transportability of LAT1-Targeted Phenylalanine Drug Conjugates

Abstract

L-type amino acid transporter 1 (LAT1) transfers essential amino acids across cell membranes. Owing to its predominant expression in the blood−brain barrier and tumor cells, LAT1 has been exploited for drug delivery and targeting to the central nervous system (CNS) and various cancers. Although the interactions of amino acids and their mimicking compounds with LAT1 have been extensively investigated, the specific structural features for an optimal drug scaffold have not yet been determined. Here, we evaluated a series of LAT1-targeted drugphenylalanine conjugates (ligands) by determining their uptake rates by in vitro studies and investigating their interaction with LAT1 via induced-fit docking. Combining the experimental and computational data, we concluded that although LAT1 can accommodate various types of structures, smaller compounds are preferred. As the ligand size increased, its flexibility became more crucial in determining the compound's transportability and interactions. Compounds with linear or planar structures exhibited reduced uptake; those with rigid lipophilic structures lacked interactions and likely utilized other transport mechanisms for cellular entry. Introducing polar groups between aromatic structures enhanced interactions. Interestingly, compounds with a carbamate bond in the aromatic ring's para-position displayed very good transport efficiencies for the larger compounds. Compared to the ester bond, the corresponding amide bond had superior hydrogen bond acceptor properties and increased interactions. A reverse amide bond was less favorable than a direct amide bond for interactions with LAT1. The present information can be applied broadly to design appropriate CNS or antineoplastic drug candidates with a prodrug strategy and to discover novel LAT1 inhibitors used either as direct or adjuvant cancer therapy.

Structure–activity characteristics of phenylalanine analogs selectively transported by L-type amino acid transporter 1 (LAT1)

“…This phenomenon mirrors our previous findings that bulky side chains can increase LAT1 affinity while reducing transport velocity 22 , 23 . Such a trend is also observed in the case of melphalan, an anticancer drug known for its high LAT1 affinity and low transport velocity, attributed to its bulky side chain 22 – 24 , 35 , 36 . These findings underscore the importance of considering both affinity and transport efficiency when developing LAT1-targeted compounds.…”

“…This strategy utilizes LAT1 for enhanced delivery through the BBB to improve brain uptake. Gabapentin and l -DOPA have been shown to act as LAT1 substrates, though they possess relatively lower affinity compared to endogenous substrates like Phe 21 – 24 . In vivo studies revealed that a high protein diet or the infusion of large amounts of neutral amino acids before administering l -DOPA can reduce its transport to the brains of monkeys, implying that endogenous amino acids compete with l -DOPA for LAT1-mediated transport and high concentrations of these amino acids can reduce brain uptake of l -DOPA 25 .…”

“…LAT1's role in cancer therapy drug delivery is also highlighted by melphalan (Phe-mustard), an alkylating agent selectively transported by LAT1 due to its bulky side chain 22 , 35 , 36 , although its transport velocity is relatively low 22 – 24 . To improve LAT1 transport efficiency, various amino acid-mustards have been synthesized, including Phg-mustard, which is one carbon shorter than melphalan and more effective as a substrate 37 .…”

Transporter-Mediated Drug Delivery

“…Later, a quantitative structure-activity relationship (QSAR) model was developed for designing potent binders of LAT1, which indicated that meta-substituted amide derivatives of phenylalanine (i.e., with an amide bond at the meta-position of the aromatic ring) have the highest ability to utilize LAT1 [ 105 ]. Subsequently, a detailed in vitro study was performed on the structural features affecting the transportability of LAT1-targeted phenylalanine-drug conjugates, providing further insight into suitable drug scaffolds for selective and efficient delivery via this strategy [ 106 ]. It should be noted that prodrugs designed to be substrates for LAT1 can also be substrates for other uptake transporters, such as monocarboxylate transporters 8 and 10 (MCT8/SLC16A2 and MCT10/SLC16A10, respectively), and organic anion transporter proteins (OATPs, SLCO/SLC21 family) [ 107 , 108 ].…”

Insights into the Transport Cycle of LAT1 and Interaction with the Inhibitor JPH203

“…Moreover, LAT1 is a very good candidate for the pro-drug approach, in which a drug molecule is linked to the physiological substrate of the transporter for facilitating membrane crossing. This approach is particularly interesting since LAT1 is physiologically expressed at the BBB, thus giving a further possibility to deliver a drug into the brain [ 20 ]. Therefore, LAT1 is an important target for research in drug discovery.…”