Biology subjects

Churchill, G. C.

Publications and source records attributed to Churchill, G. C..

7 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↗

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↗

bioRxiv2024

Increased 5-HT2A receptor signalling efficacy differentiates serotonergic psychedelics from non-psychedelics

Background and PurposeSerotonergic psychedelic drugs are under renewed investigation for the potential treatment of several psychiatric disorders. While all serotonergic psychedelics have 5-HT2A receptor activity, the explanation for why some 5-HT2A receptor agonists are not psychedelic is unknown. To address this question, we investigated the 5-HT2A receptor signalling bias and efficacy of a panel of psychedelics and non-psychedelics. Experimental ApproachG -coupled (Ca2+ and IP) and {beta}-arrestin2 signalling effects of eight chemically diverse psychedelics (psilocin, 5-MeO-DMT, LSD, mescaline, 25B-NBOMe and DOI) and non-psychedelics (lisuride and TBG) were characterised using SH-SY5Y cells expressing recombinant human 5-HT2A receptors. Measurements of signalling efficacy and bias were derived from dose-responses curves for each agonist, compared to 5-HT. Follow-up experiments sought to confirm the generality of findings using rat C6 cells expressing endogenous 5-HT2A receptors. Key ResultsIn SH-SY5Y cells, all psychedelics were partial agonists at both 5-HT2A receptor signalling pathways and none showed significant signalling bias. In comparison, in SH-SY5Y cells the non-psychedelics lisuride and TBG were not distinguishable from psychedelics in terms of biased agonist properties, but both exhibited the lowest 5-HT2A receptor signalling efficacy of all drugs tested, a result confirmed in C6 cells. Conclusion and ImplicationsIn summary, all psychedelics tested were unbiased, partial 5-HT2A receptor agonists. Importantly, the non-psychedelics lisuride and TBG were discriminated from psychedelics, not through biased signalling but rather by relatively low efficacy. Thus, 5-HT2A receptor signalling efficacy and not bias provides a possible explanation for why some 5-HT2A receptor agonists are not psychedelic.

pharmacology and toxicology↗

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↗

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↗

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↗