Acetylation turns leucine into a drug by membrane transporter switching

Abstract

Small changes to molecules can have profound effects on their pharmacological activity as exemplified by the addition of the two-carbon acetyl group to make drugs more effective by enhancing their pharmacokinetic or pharmacodynamic properties. N-acetyl-D,L-leucine is approved in France for vertigo and its L-enantiomer is being developed as a drug for rare and common neurological disorders. However, the precise mechanistic details of how acetylation converts leucine into a drug are unknown. Here we show that acetylation of leucine switches its uptake into cells from the L-type amino acid transporter (LAT1) used by leucine to organic anion transporters (OAT1 and OAT3) and the monocarboxylate transporter type 1 (MCT1). Both the kinetics of MCT1 (lower affinity compared to LAT1) and the ubiquitous tissue expression of MCT1 make it well suited for uptake and distribution of N-acetyl-L-leucine. MCT1-mediated uptake of a N-acetyl-L-leucine as a prodrug of leucine bypasses LAT1, the rate-limiting step in activation of leucine-mediated signalling and metabolic process inside cells such as mTOR. Converting an amino acid into an anion through acetylation reveals a way for the rational design of drugs to target anion transporters.

Trial registration: ClinicalTrials.gov NCT03759639 NCT03759665 NCT03759678.

Conflict of interest statement

I have read the journal's policy and the authors of this manuscript have the following competing interests: MS is Joint Chief Editor of the Journal of Neurology, Editor in Chief of Frontiers of Neuro-otology and Section Editor of F1000. He has received speaker’s honoraria from Abbott, Actelion, Auris Medical, Biogen, Eisai, Grünenthal, GSK, Henning Pharma, Interacoustics, Merck, MSD, Otometrics, Pierre-Fabre, TEVA, UCB. He is a shareholder of IntraBio. He acts as a consultant for Abbott, Actelion, AurisMedical, Heel, IntraBio and Sensorion. MF is a co-founder, shareholder, and Chairman of IntraBio. MP is a shareholder of IntraBio and has received consulting fees, honoraria, and research grants from Actelion Pharmaceuticals Ltd. and Biomarin. TBE received honoraria for lecturing from Actelion and Sanofi Genzyme. CF is a consultant to IntraBio. GCC, AG and FP are cofounders, shareholders and consultants to IntraBio. IntraBio Ltd is the applicant for patents WO2018229738 (Treatment For Migraine), WO2017182802 (Acetyl-Leucine Or A Pharmaceutically Acceptable Salt Thereof For Improved Mobility And Cognitive Function), WO2019078915 and WO2018029658 (Therapeutic Agents For Neurodegenerative Diseases), WO2018029657 (Pharmaceutical Compositions And Uses Directed To Lysosomal Storage Disorders), and WO2019079536 (Therapeutic Agents For Improved Mobility And Cognitive Function And For Treating Neurodegenerative Diseases And Lysosomal Storage Disorders).

© 2021. The Author(s).

Figures

Figure 1

The effects of N-acetylation on the chemical properties and pharmacological consequences of the drug N-acetyl-l-leucine. (a) Mechanisms of absorption illustrated by crossing a membrane by passive diffusion or carrier-mediated uptake. In general, hydrophobic uncharged molecules can cross lipid bilayer membranes through simple passive diffusion, whereas charged molecules including zwitterions (a positive and negative charge within the same molecule), anions and cations cannot cross membranes without a transporter. Over 400 Solute Carrier (SLC) transporters are known with broad but overlapping selectivities for substrate. l-leucine as an obligate zwitterion at all biologically relevant pH values has an absolute requirement for its carrier, the high-affinity l-type amino Acid Transporter (LAT1). In contrast, N-acetyl-l-leucine can exist as a neutral species and passively cross membranes at low pH, or as an anion recognized by other transporters. (b) Comparison of the physicochemical properties of l-leucine and N-acetyl-l-leucine relating to oral bioavailability. (c) Speciation curves for the protonation states of l-leucine and N-acetyl-l-leucine. The gross charge distribution of a molecule as a function of pH is calculated as well. The dominant species is indicated in several tissues relevant to drug absorption and distribution. (d) Chemical structures showing charge at pH 7 with the pKa of the amino and carboxylic acid groups labelled. (e) Lewis structures illustrating the effect of N-acetylation on the pKa of nitrogen atom. Resonance delocalization of the lone pair electrons in the amide greatly decreases the basicity of the nitrogen relative to the amine, making the molecule neutral or charged, respectively.

Figure 2

N-acetyl-l-leucine is not transported by either the l-type amino acid transporter (LAT1) nor the peptide transporter (PepT1). Concentration–response curves for the uptake of N-acetyl-l-leucine by (a) LAT1 and (b) PepT1. Concentration-inhibition curves for the inhibition of uptake of known substrates (c) gabapentin (10 µM) for LAT1 and (d) dipeptide Gly-Sar (50 µM) for PepT1. DMSO (0.5%) was the solvent control (SC) and the known inhibitor for the positive control (PC) was JPH203 (10 µM) for LAT1 and losartan (200 µM) for PepT1. Data were fit to either the Michaelis–Menten equation for uptake or the Hill equation for inhibition using the solvent control to define the top and the positive control inhibitor to define the bottom. Symbols represent the mean ± SEM, n = 3. When the error bars are smaller than the symbol, they are not visible.

Figure 3

N-acetyl-l-leucine is transported by organic anion transporters (OAT). Concentration–response curves for the uptake of N-acetyl-l-leucine by (a) OAT1 and (b) OAT3. Concentration-inhibition curves for the inhibition of uptake of known substrates (c) chlorothiazide for OAT1 (3 µM) and (d) estrone-3-sulfate for OAT3 (2 µM). DMSO (0.5%) was the solvent control (SC) and the known inhibitor diclofenac (100 µM) was the positive control (PC). Data were fit to either the Michalis-Menten equation for uptake or the Hill equation for inhibition using the solvent control to define the top and the positive control inhibitor to define the bottom. Symbols represent the mean ± SEM, n = 3. When the error bars are smaller than the symbol, they are not visible.

Figure 4

Both enantiomers of N-acetyl-leucine are transported by the monocarboxylate transporter (MCT1) but only the l-enantiomer is metabolized. Concentration–response curves for the uptake of (a) N-acetyl-l-leucine and (b) N-acetyl-d-leucine. Concentration-inhibition curves for the inhibition of uptake of the known substrate (c, d) 2-thiophene glyoxylate (500 µM). DMSO (0.5%) was the solvent control (SC) and the known inhibitor phloretin (500 µM) was the positive control (PC). (e) Chemical structure of deuterated N-acetyl-d,l-leucine incubated with cellular fraction S9 from liver to determine metabolism using liquid chromatography and mass spectrometry. (f, g) Time courses for loss of deuterated N-acetyl-d,l-leucine (1 µM initial concentration) and appearance of deuterated L-leucine for extracts derived from human and mouse livers. Data are colour-coded according to the chemical structures and names shown in e with deuterated N-acetyl-d,l-leucine in blue and deuterated l-leucine in red. (h) Concentration versus initial velocities relationship for metabolism yielded a Km values of 216 and 69 µM and Vmax values of 6.8 and 2.6 µmol/min/mg protein for human and mouse, respectively. Data were fit to the Michalis–Menten equation for transporter uptake and metabolism or the Hill equation for transporter inhibition using the solvent control to define the top and the positive control inhibitor to define the bottom. Symbols represent the mean ± SEM, n = 3. When the error bars are smaller than the symbol, they are not visible.