Biology subjects

Galione, A.

Publications and source records attributed to Galione, A..

16 recordsLinked to original sources

bioRxiv2026

A novel 5-HT2A receptor-linked signalling pathway involving NAADP-mediated lysosomal Ca2+ release

Currently there is major research interest in the therapeutic potential of serotonergic psychedelic drugs across a range of neuropsychiatric disorders1-3. Such drugs exert psychedelic and neuroplastic effects, both likely linked to therapeutic outcomes, via agonist action at the 5-HT2A receptor4-7. This receptor is assumed to generate downstream intracellular signalling via an interaction with either Gq protein coupling and formation of the second messenger inositol trisphosphate (IP3), or through triggering the effector {beta}-arrestin2. Central to the IP3 pathway is mobilisation of Ca2+ stored in the endoplasmic reticulum (ER). Here, we report the unexpected discovery that the 5-HT2A receptor also signals via an additional second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP), which mobilises Ca2+ release from lysosomal stores. This effect was revealed through measurement of changes in cellular NAADP levels, pharmacological inhibition of NAADP and direct biosensor measurement of lysosomal Ca2+ release and was produced not only by 5-HT but also by a range of psychedelic drugs in a receptor-specific manner. Moreover, genetic ablation of the NAADP pathway reduced markers of both hallucinogenic and neuroplastic effects of psychedelic drug administration in mouse models. These findings reveal the involvement of a novel, therapeutically relevant signalling pathway in psychedelic drug action.

pharmacology and toxicology↗

bioRxiv2026

A High-Affinity Nanobody Recognizing mNeonGreen Enables Versatile Biochemical, Cellular, and in vivo Applications.

mNeonGreen (mNG) is among the brightest and most photostable monomeric green fluorescent proteins and is widely used for protein tagging. Here, we present sdAb(mNG), a high-affinity single-domain antibody (sdAb) that enables biochemical capture, imaging, and manipulation of mNG-tagged proteins. A 1.26 [A] crystal structure reveals an extensive interaction surface between mNG and sdAb(mNG), accounting for its high affinity (KD = 0.39 nM) and robust target recognition across diverse experimental conditions. This allows a single sdAb to support applications that typically require multiple specialized tools. We demonstrate the utility of sdAb(mNG) in several example applications including highly specific immunoprecipitation, direct immunofluorescence, and super-resolution imaging. Importantly, sdAb(mNG) retains high-performance target recognition even in intracellular environments. When expressed as an intrabody in living mammalian cells, sdAb(mNG) enables relocalization of mNG-tagged proteins to defined compartments or visualization of synaptic vesicle transport in primary neurons. In zebrafish, fusion of sdAb(mNG) to an F-box degradation domain induces cell-autonomous depletion of an endogenous mNG-tagged transcription factor and produces a clear developmental phenotype. These findings establish sdAb(mNG) as a versatile and robust affinity reagent that converts mNG from a passive fluorescent reporter into a multifunctional handle for imaging, proteomics, and programmable manipulation of endogenous and engineered proteins.

biochemistry↗

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

Alternate Splicing Directs PMCA2 to Lysosomes and is Linked to Neurodegeneration

Plasma membrane calcium ATPases (PMCAs) are believed to function exclusively at the plasma membrane where they expel calcium from the cytosol. We have unexpectedly identified a splice variant-dependent localisation of the PMCA isoform PMCA2 to the lysosome, where it forms an evolutionarily conserved complex with NPC1, the lysosomal membrane protein defective in the rare lysosomal storage disease Niemann-Pick disease type C (NPC). This interaction is required for lysosomal Ca2+ homeostasis and implicates PMCA2 as a mediator of Ca2+ uptake into lysosomes. Disruption of the NPC1-PMCA2 complex contributes to the pathophysiology of both Niemann-Pick disease type C and Parkinsons disease, revealing an unrecognised intracellular function for PMCA2 and a shared mechanism linking lysosomal Ca2+ and lipid regulation in neurodegeneration.

cell biology↗

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↗

bioRxiv2024

Lysosomal calcium signalling contributes to the acute α-adrenergic response via calcium-stimulated adenylyl cyclase 1 and 8

Inositol trisphosphate (IP3), a calcium (Ca2+)-mobilizing second messenger, releases Ca2+ from the sarcoplasmic reticulum (SR) via IP3 receptors and modulates adenylyl cyclase (AC) activity in atrial myocytes. Lysosomes participate in Ca2+ homeostasis by mobilising Ca2+ in response to Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP). We postulate that both downstream activation of Ca2+ sensitive AC (AC1 and AC8) and lysosomal Ca2+ signalling in response to IP3R activation contribute to atrial myocyte function and pacemaking. Ectopic application of phenylephrine (PE) increased chronotropy and inotropy and this response was reduced in the presence of NAADP pathway inhibitors (BZ-194 and SAN4825) and Bafilomycin A1. PE increased cyclic adenosine 3-5 monophosphate (cAMP) activity in neonatal rat atrial myocytes (NRAMs) and this was inhibited by NAADP pathway inhibitors. This inhibition was not observed in neonatal rat ventricular myocytes (NRVMs), revealing specificity of this response to NRAMs. We investigated expression of AC1 and AC8 as a possible explanation to these observations. Genetic perturbation of AC1 and AC8 by double-knockout of Adcy1 and Adcy8 in a mouse model showed a decrease in positive chronotropic and inotropic response upon cumulative dose of PE in atrial tissue, reduced PE stimulated amplitude of Ca2+ transient in isolated atrial myocytes and presented decreased cytosolic cAMP levels in response to PE in neonatal atrial myocytes that was not inhibited by NAADP pathway inhibitors. Our data identifies a link between NAADP and -adrenergic signalling pathways in atrial myocytes, highlighting that lysosomal Ca2+ is an important component of -adrenergic stimulation in the cardiac atria and warrants further investigation.

physiology↗

bioRxiv2024

Lysosomal signalling pathways influence heart rhythm, and regulate atrial function

In the heart, endogenous nicotinic acid adenine dinucleotide phosphate (NAADP) triggers lysosomal calcium (Ca2+) release to augment sarcoplasmic reticulum (SR) Ca2+ sequestration, producing larger Ca2+ transients. However, the role of lysosomal Ca2+ signals in pacemaker activity, a distinct Ca2+-operated function of the sinoatrial node (SAN), or in the atrial myocardium has not been investigated. Pharmacological or genetic ablation of the NAADP pathway inhibits the spontaneous beating rate response to beta-adrenergic stimulation in intact SAN. We found intracellular signaling microdomains between lysosomes and neighboring SR or mitochondria in mouse, and goat tissue. The spatial relationship between lysosomes and other Ca2+-handling organelles are altered in goat atrial fibrillation. Furthermore, we demonstrate atrial myocytes produce cAMP in response to lysosomal signaling, adding a novel trigger for cyclic nucleotide signaling. Our findings support the hypothesis that lysosomal Ca2+ signaling contributes to regulation of cardiomyocyte cAMP levels and pacemaker activity.

physiology↗

bioRxiv2023

Compartmentalisation proteomics revealed endolysosomal protein network changes in a goat model of atrial fibrillation

Endolysosomes (EL) are known for their role in regulating both intracellular trafficking and proteostasis. EL help facilitate elimination of damaged membrane and cytosolic proteins, protein aggregates, membranous organelles and also play an important role in calcium signalling. Despite the importance of EL, their specific role in cardiovascular disease is not well understood. In particular, its unclear how EL contribute to atrial pathology over longer time frames. To shed light on this question, we conducted a comprehensive analysis that involved proteomics, transcriptomics, integrated analysis, electron tomography, western blotting, and enzyme assays. To identify the role of EL in atrial fibrillation (AF), we applied a recently published organelle protein isolation method. We used this method to study biopsies from AF goat model and analyse the EL-specific proteins and pathways involved in this condition. Our results revealed the upregulation of the AMPK pathway and the expression of EL-specific proteins that were not found in whole tissue lysates (TL), including GAA, DYNLRB1, CLTB, SIRT3, CCT2, and muscle-specific HSPB2. We also observed structural anomalies, such as autophago-vacuole formation, irregularly shaped mitochondria, and glycogen deposition, which provide insights into the ELs contribution to AF and related pathways and molecular mechanisms. Overall, our findings suggest that EL play an important role in the development of AF over longer time frames, and provide a more detailed understanding of the underlying molecular processes involved.

molecular biology↗

bioRxiv2022

Lysosomal calcium loading promotes arrhythmias by potentiating ryanodine receptor release

Spontaneous calcium release by ryanodine receptors (RyRs) due to intracellular calcium overload results in delayed afterdepolarisations, closely associated with life-threatening arrhythmias. In this regard, inhibiting lysosomal calcium release by two-pore channel 2 (TPC2) knockout has been shown to reduce the incidence of ventricular arrhythmias under {beta}-adrenergic stimulation. However, mechanistic investigations into the role of lysosomal function on RyR spontaneous release remain missing. We investigate the calcium handling mechanisms by which lysosome function modulates RyR spontaneous release, and determine how lysosomes are able to mediate arrhythmias by its influence on calcium loading. Mechanistic studies were conducted using a population of biophysically-detailed mouse ventricular models including for the first time modelling of lysosomal function, and calibrated by experimental calcium transients modulated by TPC2. We demonstrate that lysosomal calcium uptake and release can synergistically provide a buffering pathway of fast calcium transport, by which lysosomal calcium release primarily modulates sarcoplasmic reticulum (SR) calcium reuptake and RyR release. Enhancement of this lysosomal transport pathway promoted RyR spontaneous release by elevating the SR-junction calcium gradient. In contrast, blocking either lysosomal calcium uptake or release revealed an antiarrhythmic impact. Under conditions of calcium overload, our results indicate that these responses are strongly modulated by intercellular variability in L-type calcium current, RyR release, and SERCA reuptake. Altogether, our investigations identify that lysosomal calcium handling directly influences RyR spontaneous release by regulating the SR-junction calcium gradient, suggesting antiarrhythmic strategies and identifying key modulators of lysosomal proarrhythmic action. Statement of SignificanceDelayed afterdepolarisations arising from spontaneous RyR calcium release are an important risk factor for arrhythmogenesis. Inhibiting lysosomal calcium release by TPC2-KO reduces the propensity for ventricular arrhythmias. However, understanding downstream effects of lysosomal calcium release on spontaneous RyR release is lacking. Understanding lysosomes as arrhythmia sources requires alternative approaches to controlled laboratory techniques: these restrain variability experimentally and statistically. Our study presents two methodological novelties by focusing on calibration with experimental findings using a population of biophysically-detailed models and incorporating lysosomal mechanisms. Lysosomal calcium handling promotes RyR spontaneous release by elevating the SR-junction calcium gradient. Blocking lysosomal function uncovers an antiarrhythmic strategy. Lysosome-release proarrhythmic risk is determined by synergistic enhancements of lysosomal uptake with RyR release or L-type calcium current.

biophysics↗

bioRxiv2022

The efficiency of Grignard Pure™ to inactivate airborne SARS-CoV-2 surrogate

Grignard Pure (GP) is a unique and proprietary blend of Triethylene Glycol (TEG) and inert ingredients designed for continuous antimicrobial treatment of air. GP received approval from the US EPA under its Section 18 Public Health Emergency Exemption program for use in seven states. This study characterizes the efficacy of GP for inactivating MS2 bacteriophage - a non-enveloped virus widely used as a surrogate for SARs-CoV-2. Experiments measured the decrease in the airborne viable MS2 concentration in the presence of different concentrations of GP from 60 to 90 minutes, accounting for both natural die-off and settling of MS2. Experiments were conducted both by introducing GP aerosol into air containing MS2 and by introducing airborne MS2 into air containing GP aerosol. GP is consistently able to rapidly reduce viable MS2 bacteriophage concentration by 2-3 logs at GP concentrations of 0.02 mg/m3 to 0.5 mg/m3 (corresponding to TEG concentrations of 0.012 mg/m3 to 0.287 mg/m3). Related GP efficacy experiments by the US EPA, as well as GP (TEG) safety and toxicology, are also discussed. SynopsisLimited research on the germicidal properties of triethylene glycol against airborne pathogens was conducted during the 1940s and 50s. This paper investigates the inactivation rate of airborne bacteriophage MS2 by Grignard Pure product, containing a unique and proprietary blend of Triethylene Glycol (TEG) and inert ingredients.

microbiology↗

bioRxiv2022

Release of acidic store calcium is required for effective priming of the NLRP3 inflammasome

The lysosome is a dynamic signaling organelle that is critical for cell functioning. It is a regulated calcium store that can contribute to Ca2+-regulated processes via both local calcium release and more globally by influencing ER Ca2+ release. Here, we provide evidence from studies of an authentic mouse model of the lysosomal storage disease Niemann-Pick Type C (NPC) that has reduced lysosomal Ca2+ levels, and genetically modified mice in which the two-pore lysosomal Ca2+ release channel family are deleted that lysosomal Ca2+ signaling is required for normal pro-inflammatory responses. We demonstrate that production of the pro-inflammatory cytokine IL-1{beta} via the NLRP3 inflammasome is significantly reduced in murine Niemann-Pick Type C, the inhibition is selective because secretion of TNF is not diminished and it is a consequence of inefficient inflammasome priming. Synthesis of precursor ProIL-1{beta} is significantly reduced in macrophages genetically deficient in the lysosomal protein Npc1, which is mutated in most clinical cases of NPC, and in wild type cells in which Npc1 activity is pharmacologically inhibited. Comparable reductions in ProIL-1{beta} generation were measured in vitro and in vivo by macrophages (M{Phi}) genetically altered to lack expression of the two-pore lysosomal Ca2+ release channels Tpcn1 or Tpcn2. These data demonstrate a requirement for lysosome-dependent Ca2+ signaling in the generation of specific pro-inflammatory responses.

immunology↗

bioRxiv2021

Cardiac Atrial Compartmentalisation Proteomics: A Modified Density Gradient Method to Analyse Endo-lysosomal Proteins

The importance of lysosomes in cardiac physiology and pathology are well established, and evidence for roles in calcium signalling are emerging. We describe a label-free proteomics method suitable for small cardiac tissue biopsies based on density-separated fractionation, which allows study of endo-lysosomal (EL) proteins. Density gradient fractions corresponding to tissue lysate; sarcoplasmic reticulum (SR), mitochondria (Mito) (1.3 g/ml); and EL with negligible contamination from SR or Mito (1.04 g/ml), were analysed using Western Blot, enzyme activity assay and LC-MS/MS analysis (adapted discontinuous Percoll, and sucrose differential density gradient). Kyoto Encyclopedia of Genes and Genomes, Reactome, Panther and Gene Ontology pathway analysis showed good coverage of RAB proteins and lysosomal cathepsins (including cardiac-specific cathepsin D) in the purified EL fraction. Significant EL proteins recovered included catalytic activity proteins. We thus present a comprehensive protocol and dataset of guinea-pig atrial EL organelle proteomics using techniques also applicable for non-cardiac tissue.

molecular biology↗

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↗

bioRxiv2020

Glucose and NAADP trigger elementary intracellular β cell Ca2+ signals

Pancreatic {beta}-cells release insulin upon a rise in blood glucose. The precise mechanisms of stimulus-secretion coupling, and its failure in Diabetes Mellitus Type 2, remain to be elucidated. The consensus model, as well as a class of currently prescribed anti-diabetic drugs, are based around the observation that glucose-evoked ATP production in {beta}-cells leads to closure of cell membrane ATP-gated potassium (KATP) channels, plasma membrane depolarisation, Ca2+ influx, and finally the exocytosis of insulin granules (Ashcroft et al., 1984; Cook and Hales, 1984). However, it has been demonstrated by the inactivation of this pathway using genetic and pharmacological means that closure of the KATP channel alone may not be sufficient to explain all {beta}-cell responses to glucose elevation (Henquin, 1998; Seghers et al., 2000). Here we show using total internal reflection fluorescence (TIRF) microscopy (Axelrod, 1981) that glucose as well as the Ca2+ mobilising messenger nicotinic acid adenine dinucleotide phosphate (NAADP), known to operate in {beta}-cells (Johnson and Misler, 2002; Masgrau et al., 2003), lead to highly localised elementary intracellular Ca2+ signals. These were found to be obscured by measurements of global Ca2+ signals and the action of powerful SERCA-based sequestration mechanisms at the endoplasmic reticulum (ER). This is the first demonstration of elemental Ca2+ signals in response to NAADP, although they have been suspected (Davis et al., 2020). Optical quantal analysis of these events reveals a unitary event amplitude equivalent to that of known elementary Ca2+ signalling events, inositol trisphosphate (IP3) receptor mediated blips (Parker et al., 1996; Parker and Ivorra, 1990), and ryanodine receptor mediated sparks (Cheng et al., 1993). We propose that a mechanism based on these highly localised intracellular Ca2+ signalling events mediated by NAADP may initially operate in {beta}-cells when they respond to elevations in blood glucose.

physiology↗

bioRxiv2019

Effects of N-Acetyl-Leucine and its enantiomers in Niemann-Pick disease type C cells

N-Acetyl-DL-Leucine is an acetylated derivative of the essential amino acid leucine and a racemate (1:1) of N-Acetyl-L-Leucine and N-Acetyl-D-Leucine enantiomers. Previous observational clinical studies have demonstrated that N-Acetyl-DL-Leucine is effective in improving ataxia in patients with Niemann-Pick disease type C (NPC), a lysosomal storage disorder characterized at the cellular level by increased relative volume of the endosomal/lysosomal system. In this study, we sought to further characterize the potential therapeutic benefit of N-acetyl-DL-leucine and its enantiomers for the treatment of NPC. We investigated the effectiveness of N-Acetyl-DL-Leucine, N-Acetyl-D-Leucine, and N-Acetyl-L-Leucine in reducing lysosomal volume in non-neuronal NPC1 cells using LysoTracker, a fluorescent dye that accumulates in acidic organelles. We report that both N-Acetyl-DL-Leucine and N-Acetyl-L-Leucine reduced relative lysosomal volume in NPC1-/- Chinese Hamster Ovary cells in a dose-dependent manner. Subsequently, we confirmed that N-Acetyl-L-Leucine was most effective at reducing relative lysosomal volumes in fibroblasts derived from NPC patients with severe disease (***p <0.001), followed by N-Acetyl-DL-Leucine (**p <0.01). Treatment with N-Acetyl-D-Leucine did not achieve statistical significance. Taken together, these results suggest that N-Acetyl-L-Leucine is the most effective enantiomer in correcting relative lysosomal volume in non-neuronal NPC cells, and support further research and development of the L-enantiomer.

cell biology↗

bioRxiv2019

Unexpected differences in the pharmacokinetics of N-acetyl DL-leucine enantiomers after oral dosing and their clinical relevance

These results suggest that during chronic administration of the racemate, the D-enantiomer would accumulate, which could have negative effects. The enantiomers of many chiral drugs not only exhibit different pharmacological effects in regard to targets that dictate therapeutic and toxic effects, but are also handled differently in the body due to pharmacokinetic effects. We investigated the pharmacokinetics of the enantiomers of N-acetyl-leucine after administration of the racemate (N-acetyl-DL-leucine) or purified, pharmacologically active L-enantiomer (N-acetyl-L-leucine). Compounds were administered orally to mice. Plasma and tissue samples were collected at predetermined time points (0.25 to 8 h), quantified with liquid chromatography/mass spectrometry, and pharmacokinetic constants were calculated using a noncompartmental model. When administered as the racemate, both the maximum plasma concentration (Cmax) and the area under the plasma drug concentration over time curve (AUC) were much greater for the D-enantiomer relative to the L-enantiomer. When administered as the L-enantiomer, the dose proportionality was greater than unity compared to the racemate, suggesting saturable processes affecting uptake and/or metabolism. Elimination (ke and T1/2) was similar for both enantiomers. These results are most readily explained by inhibition of uptake at an intestinal carrier of the L-enantiomer by the D-enantiomer, and by first-pass metabolism of the L-, but not D-enantiomer, likely by deacetylation. In brain and muscle, N-acetyl-L-leucine levels were lower than N-acetyl-D-leucine, consistent with rapid conversion into L-leucine and utilization by normal leucine metabolism. In summary, the enantiomers of N-acetyl-leucine exhibit large, unexpected differences in pharmacokinetics due to both unique handling and/or inhibition of uptake and metabolism of the L-enantiomer by the D-enantiomer. Taken together, these results have clinical implications supporting the use of N-acetyl-L-leucine instead of the racemate or N-acetyl-D-leucine, and support the research and development of isolated N-acetyl-L-leucine.

pharmacology and toxicology↗