Structure–activity relations of leucine derivatives reveal critical moieties for cellular uptake and activation of mTORC1-mediated signaling

Abstract

Among amino acids, leucine is a potential signaling molecule to regulate cell growth and metabolism by activating mechanistic target of rapamycin complex 1 (mTORC1). To reveal the critical structures of leucine molecule to activate mTORC1, we examined the structure-activity relationships of leucine derivatives in HeLa S3 cells for cellular uptake and for the induction of phosphorylation of p70 ribosomal S6 kinase 1 (p70S6K), a downstream effector of mTORC1. The activation of mTORC1 by leucine and its derivatives was the consequence of two successive events: the cellular uptake by L-type amino acid transporter 1 (LAT1) responsible for leucine uptake in HeLa S3 cells and the activation of mTORC1 following the transport. The structural requirement for the recognition by LAT1 was to have carbonyl oxygen, alkoxy oxygen of carboxyl group, amino group and hydrophobic side chain. In contrast, the requirement for mTORC1 activation was more rigorous. It additionally required fixed distance between carbonyl oxygen and alkoxy oxygen of carboxyl group, and amino group positioned at α-carbon. L-Configuration in chirality and appropriate length of side chain with a terminal isopropyl group were also important. This confirmed that LAT1 itself is not a leucine sensor. Some specialized leucine sensing mechanism with rigorous requirement for agonistic structures should exist inside the cells because leucine derivatives not transported by LAT1 did not activate mTORC1. Because LAT1-mTOR axis is involved in the regulation of cell growth and cancer progression, the results from this study may provide a new insight into therapeutics targeting both LAT1 and leucine sensor.

LAT1 activity of carboxylic acid bioisosteres: Evaluation of hydroxamic acids as substrates

“…Lately, this story has become further convoluted as Nagamori et al reported that several carboxylic esters and a hydroxamic acid derivative of L -leucine were LAT1 substrates. 32 Their conclusions, based in part on a trans -stimulation assay, 36 contradicted previous reports that esters do not bind LAT1. 4, 24, 37 …”

“…And to further probe the SAR for close-in derivatives of the HA’s (Table 2), we also decided to test the structurally-related primary amide Leucinamide 13e . Though ester 9e was recently reported to be a LAT1 substrate, 32 to our knowledge this was the first time that 13e or any amino acid primary amide has been tested for LAT1 activity. Interestingly, 13e did not demonstrate significant substrate activity (efflux rate: 0.69 fmol/min).…”

“…gabapentin) are also transported by LAT1. 8, 30–32 Despite some flexibility in the presentation of the amine and carboxylic acid functional groups, it has been maintained that both of these functional groups are essential for transporter recognition. 4, 24, 33 The primary evidence supporting a carboxylic acid requirement has been centered on observations that replacement with esters and a closely related sulfonic acid resulted in loss of activity.…”

“…32 We have been focused on modifying the carboxylic acid rather than the amine due to the potential metabolism and toxicity 41 liabilities of the former. Besides contributing to the knowledge of LAT1 SAR, replacing the carboxylic acid functional group has the potential for altering the pharmacokinetics of prodrugs 34, 35 intended for LAT1 transport.…”

“…Given the disparity in the literature 4, 32, 37 as to whether esters are LAT1 substrates, we also tested the Leu methyl ester 9e , which was an intermediate in the preparation of HA-Leu 12e (Scheme 1). And to further probe the SAR for close-in derivatives of the HA’s (Table 2), we also decided to test the structurally-related primary amide Leucinamide 13e .…”