The Development of LAT1 Efflux Agonists as Mechanistic Probes of Cellular Amino Acid Stress.

Amino acid restriction induces cellular stress and cells often respond via the induction of autophagy. Autophagy or 'self-eating' enables the recycling of proteins and provides the essential amino acids needed for cell survival. Of the naturally occurring amino acids, methionine restriction has pleiotropic effects on cells because methionine also contributes to the intracellular methyl pools required for epigenetic controls as well as polyamine biosynthesis. In this report, we describe the chemical synthesis of four diastereomers of a methionine depletion agent and demonstrate how controlled methionine efflux from cells significantly reduces intracellular methionine, S-adenosylmethionine (SAM), S-adenosyl homocysteine (SAH), and polyamine levels. We also demonstrate that human pancreatic cancer cells respond via a lipid signaling pathway to induce autophagy. The methionine depletion agent causes the large amino acid transporter 1 (LAT1) to preferentially work in reverse and export the cell's methionine (and leucine) stores. The four diastereomers of the lead methionine/leucine depletion agent were synthesized and evaluated for their ability to (a) efflux 3H-leucine from cells, (b) dock to LAT1 in silico, (c) modulate intracellular SAM, SAH, and phosphatidylethanolamine (PE) pools, and (d) induce the formation of the autophagy-associated LC3-II marker. The ability to modulate the intracellular concentration of methionine regardless of exogenous methionine supply provides new molecular tools to better understand cancer response pathways. This information can then be used to design improved therapeutics that target downstream methionine-dependent processes like polyamines.

Serendipitous Discovery of Leucine and Methionine Depletion Agents during the Search for Polyamine Transport Inhibitors.

Targeting polyamine metabolism is a proven anticancer strategy. Cancers often escape the polyamine biosynthesis inhibitors by increased polyamine import. Therefore, there is much interest in identifying polyamine transport inhibitors (PTIs) to be used in combination therapies. In a search for new PTIs, we serendipitously discovered a LAT-1 efflux agonist, which induces intracellular depletion of methionine, leucine, spermidine, and spermine, but not putrescine. Because S-adenosylmethioninamine is made from methionine, a loss of intracellular methionine leads to an inability to biosynthesize spermidine, and spermine. Importantly, we found that this methionine-depletion approach to polyamine depletion could not be rescued by exogenous polyamines, thereby obviating the need for a PTI. Using 3H-leucine (the gold standard for LAT-1 transport studies) and JPH-203 (a specific LAT-1 inhibitor), we showed that the efflux agonist did not inhibit the uptake of extracellular leucine but instead facilitated the efflux of intracellular leucine pools.