Biology subjects

Factor, M.

Publications and source records attributed to Factor, M..

3 recordsLinked to original sources

bioRxiv2025

N-acetyl-L-leucine (Levacetylleucine) normalizes Transcription Factor EB (TFEB) activity by stereospecific bidirectional modulation

Levacetylleucine (AqneursaTM), a chemically modified amino acid, is the only US Food and Drug Administration-approved monotherapy for the treatment of Niemann-Pick disease type C (NPC) (Beninger, 2024; Mullard, 2024; van Gool et al., 2025). This acetylated derivative of L-leucine functions as a pro-drug, with the acetyl group rendering it a substrate for the monocarboxylate transporter (MCT) family of transporters to allow appreciable penetration of the blood-brain barrier and its efficient uptake into cells (Churchill et al., 2021). Inside cells, levacetylleucine undergoes metabolism catalysed by acylases, and the resultant high quantities of L-leucine enter metabolic pathways which enhance mitochondrial bioenergetics and, as previously demonstrated, indirectly ameliorate lysosomal function (Kaya et al., 2020). Here, we show a novel aspect of levacetylleucines mechanism of action, demonstrating a direct effect on lysosomal function through its rapid modulation of the translocation of the transcription factor TFEB, a master regulator of lysosomal biogenic and autophagic genes (Napolitano and Ballabio, 2016), from cytoplasm to nucleus. Uniquely, we have demonstrated a biphasic action whereby levacetylleucine normalizes TFEB activity, consistent with levacetylleucines previously shown ability to regulate cellular homeostasis: in wild-type HeLa cells, levacetylleucine enhances and activates the translocation of TFEB to the nucleus. In contrast, in cellular models of NPC type 1 disease, where TFEB is already over-expressed in the nucleus (as the cell attempts to compensate for the primary defect by activating TFEB as a natural cellular response to the lysosomal substrate accumulation and associated cellular stress), treatment with levacetylleucine down-regulates and restores the distribution of TFEB to a more normalized cytoplasmic: nuclear ratio. Importantly, both effects of levacetylleucine occur at concentrations consistent with plasma concentrations in therapeutic dosing (Churchill et al., 2020). The effects were also confirmed to be stereospecific to the L-enantiomer, as neither the D-enantiomer (N-acetyl-D-leucine) or racemate (N-acetyl-DL-leucine) had any effect, The presence of the D-enantiomer in the racemic mixture inhibited the ability of levacetylleucine to promote TFEB bidirectional translocation, consistent with previous studies, which have established antagonism of N-acetyl-L-leucine by N-acetyl-D-leucine in the racemic mixture (rendering the racemic mixture without effect). This bidirectional mechanism of action of levacetylleucine to impact lysosomal function directly and normalize, either by activating basal TFEB signalling or reducing aberrant TFEB function in NPC1 knockout cells, thereby modulating lysosomal and autophagic functions, lends itself to the treatment of a broad range of neurological and neurodevelopment disorders.

pharmacology and toxicology↗

bioRxiv2025

Stereospecific rapid activation of Transcription Factor EB (TFEB) by Levacetylleucine (NALL)

N-acetyl L-leucine (NALL, USAN or levacetylleucine, INN or trade name Aqneursa) is an FDA-approved agent for the treatment of Niemann-Pick disease type C (NPC). The N-acetyl group renders the compound a prodrug of L-leucine, making it a substrate for membrane-spanning monocarboxylate transporters (MCTs), which are ubiquitously expressed delivering NALL to all tissues with high capacity, including to the central nervous system. NALL enters enzyme-controlled pathways that correct metabolic dysfunction and enhance mitochondrial bioenergetics. Because NALL improves energy production (adenosine triphosphate, ATP) and ameliorates lysosomal function, it is potentially a therapy for a broad range of neurodegenerative and neurodevelopmental disorders (in addition to lysosomal storage disorders) in which energy homeostasis and lysosomal function are impaired. Here, we have performed a series of in vitro studies which reveal an additional aspect of NALLs polypharmacological mechanism of action. The studies demonstrate a direct lysosomal effect whereby NALL rapidly activates the translocation of the Transcription Factor EB (TFEB, a master regulator of lysosomal biogenesis and autophagy) from the cytoplasm to the nucleus in HeLa cells. The activation of TFEB is known to trigger the activation of specific genes that restore lysosomal biogenesis and function, as well as autophagy. Consistent with this, we show that NALL increases production of a TFEB target gene LAMP1, an integral lysosomal membrane protein responsible for maintaining lysosomal integrity, function and pH. This in vitro effect occurs at concentrations consistent with concentrations in plasma in humans after standard therapeutic dosing. We further demonstrated that acetylation is critical to this aspect of NALLs mechanism of action, as L-leucine itself had no effect on the activation of TFEB. Consistent with previous studies N-acetyl-D-leucine was inactive and also had no effect. Similarly, N-acetyl-DL-leucine also had only a modest effect, providing further evidence that N-acetyl-D-leucine is even antagonistic and inhibits the effects of the active L-enantiomer. This mechanism of action of NALL to activate TFEB signalling, thereby enhancing lysosomal and autophagic function, further elucidates the ways by which this compound targets the fundamental etiology of rare and common neurodegenerative disorders, from Niemann-Pick disease type C to Parkinsons disease. Based on its mechanism of action by improving the mitochondrial-lysosomal axis, NALL has the potential to be an effective therapy for a broad range of neurological and neurodevelopment conditions.

pharmacology and toxicology↗

bioRxiv2020

Acetylation of L-leucine switches its carrier from the L-amino acid transporter (LAT) to organic anion transporters (OAT)

N-acetyl-DL-leucine is an analogue of the alpha amino acid leucine with a chiral stereocenter. The active L-enantiomer of the racemate is currently under development for rare neurological disorders. Here we present evidence that a selective recognition of N-acetyl-L-leucine versus L-leucine by different uptake transporters significantly contributes to the therapeutic effects of N-acetyl-L-leucine. A previous study of the pharmacokinetics of racemic N-acetyl-DL-leucine and N-acetyl-L-leucine revealed D-L enantiomer competition and saturation kinetics, best explained by carrier-mediated uptake. The strategy we used was to first analyze the physicochemical properties associated with good oral bioavailable drugs and how these are alerted by N-acetylation by comparing N-acetyl-L-leucine with L-leucine. Using in silico computational chemistry we found that N-acetylation has a profound impact on certain physicochemical properties that can rationalize why N-acetyl-L-leucine is drug-like compared to L-leucine. Our calculations show that at physiological pH, L-leucine is a zwitterion, whereas N-acetyl-L-leucine is present as mainly an anion. Specifically, N-acetylation removes a charge from the nitrogen at physiological pH and N-acetyl-L-leucine is an anion that is then a substrate for the organic anion transporters. We examined N-acetyl-L-leucine uptake in human embryonic kidney cells overexpression candidate organic anion transporters (OAT) and pharmacological inhibitors. We found that N-acetyl-L-leucine is a translocated substrate for OAT1 and OAT3 with low affinity (Km ~10 mM). In contrast, L-leucine is known to be transported by the L-type Amino Acid Transporter (LAT) with high affinity (Km ~0.2 mM) and low capacity. The clinical consequence is that L-leucine uptake becomes saturated at 50-fold lower concentration than N-acetyl-L-leucine. These results demonstrate a mechanism of action that explains why N-acetyl-L-leucine is effective as a drug and L-leucine itself is not.

pharmacology and toxicology↗