Molecular target: prescribing information

“The distinct molecular target for levacetylleucine in the treatment of NPC or A-T is unknown.” (opens the prescribing information at this quote)

Mechanism as described by the European regulator

“The exact way the active substance in Aqneursa, levacetylleucine, works is not known. However, studies in animals suggest that it may help correct the cells’ ability to generate energy. This includes the cells’ ability to make adenosine triphosphate, the main source of energy for cells in the brain that control movement and balance.” (opens the source at this quote in a new tab)

“Aqneursa contains levacetylleucine that targets underlying processes of neurological dysfunction. Nonclinical studies demonstrated that levacetylleucine corrects energy metabolism, including improving adenosine triphosphate production.” (opens the source at this quote in a new tab)

“No studies researched the mechanism of action on the target.” (opens the source at this quote in a new tab)
“The mode of action of levacetylleucine in NPC disease is unclear.” (opens the source at this quote in a new tab)
“Mechanism of action is described very generally. Symptomatic, neuroprotective and disease-modifying effect is claimed. Based on results from period of 12 weeks of treatment, improvements have been observed, which may indicate symptomatic treatment rather than a disease modifying effect.” (opens the source at this quote in a new tab)

Proposed mechanism stated by the investigators of the A-T phase 3 trial

“Levacetylleucine (N-acetyl-L-leucine) is a modified amino acid that enters enzyme-controlled pathways to correct metabolic dysfunction, improve function of the lysosomal-mitochondrial axis, and restore membrane potential and cellular signaling.” (opens the source at this quote in a new tab)

Rationale in A-T: mitochondrial and metabolic function

“The increasingly recognized role of ATM in oxidative stress was also considered and how it was pertinent to mitochondrial dysfunction, metabolic abnormalities and energy metabolism in A-T.” (opens the source at this quote in a new tab)
“An important contribution to the meeting was the description of pathways /mechanisms that have led to the development of therapeutic approaches for A-T including the use of specific antisense oligonucleotides to restore ATM function in patients; delivery of full-length ATM cDNA to A-T cells; eDSP (formerly EryDex) that encapsulates dexamethasone sodium phosphate in a patient's own red blood cells and the use of small molecules (triheptanoin, nicotinamide riboside and N-acetyl leucine) to correct mitochondrial and metabolic function.” (opens the source at this quote in a new tab)

Shared neurological manifestations as the basis for one development protocol across NPC, GM2 gangliosidoses, and A-T

“Despite their different etiologies, NPC, GM2, and A-T are all characterized by progressive neurodegeneration of the cerebellum and cerebrum, resulting in physical and cognitive decline, and premature death.” (opens the source at this quote in a new tab)
“Owing to their common neurological manifestations, and the mechanism of action of the investigational product N-acetyl-l-leucine, a single master protocol has been developed for the three disorders.” (opens the source at this quote in a new tab)

Cellular uptake: acetylation switches transport from LAT1 to MCT1 and organic anion transporters

“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).” (opens the source at this quote in a new tab)
“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.” (opens the source at this quote in a new tab)
“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.” (opens the source at this quote in a new tab)
“Our calculations show that at physiological pH, l-leucine is a zwitterion, whereas N-acetyl-l-leucine is present as mainly an anion.” (opens the source at this quote in a new tab)
“We found that N-acetyl-l-leucine was a substrate of MCT1 with a Km of 3.0 mM (Fig. 4a) and an inhibitor of MCT1 (Fig. 4c) with an IC50 of 15 mM.” (opens the source at this quote in a new tab)

Transport of the D-enantiomer by the same carrier

“N-acetyl-D-leucine was also a substrate, with Km of 1.0 mM (Fig. 4b), and an inhibitor of MCT1 with an IC50 of 11 mM (Fig. 4d).” (opens the source at this quote in a new tab)
“which yielded an affinity (Ki) of 4.3 mM for the l-enantiomer and 1.6 mM for the d-enantiomer, similar to the respective Km values.” (opens the source at this quote in a new tab)
“This enantiomeric selectivity during transport by MCT1 provides a mechanistic explanation for the lower bioavailability of the l-enantiomer when administered as a racemate which impacts its therapeutic efficacy” (opens the source at this quote in a new tab)

Prodrug hypothesis: intracellular deacetylation to L-leucine

“are consistent with N-acetyl-l-leucine acting as prodrug, such as l-dopa, in which both uptake and metabolism are crucial components of its mechanism of action” (opens the source at this quote in a new tab)
“MCT1 can deliver leucine via the prodrug form N-acetyl-l-leucine without interference from, and disruption to, the uptake of other essential amino acids.” (opens the source at this quote in a new tab)

“Taken together, these data showing low amounts of N-acetyl-leucine in the brain and muscle suggest that the mechanism of action of N-acetyl-L-leucine requires metabolism.” (opens the source at this quote in a new tab)

MCT1-dependent uptake in human induced neurons

“We demonstrated that NALL exhibits neuroprotective properties in MS- and HC-derived hiPNs subjected to acute damage induced by the microtubule-destabilizing agent nocodazole determined by neurite length. This effect can be blocked by inhibition of transporter-specific NALL uptake.” (opens the source at this quote in a new tab)
“The specific uptake of NALL via MCT1 as here firstly demonstrated in hiPNs suggests that the proposed transporter switch is functionally relevant in neurons.” (opens the source at this quote in a new tab)

Lysosomal regulation through transcription factor EB

“Here, we describe a direct effect of levacetylleucine on lysosomal regulation through modulation of TFEB, the master transcription factor for lysosomal and autophagy genes.” (opens the source at this quote in a new tab)
“In wild-type HeLa cells, levacetylleucine promotes TFEB activation and nuclear localisation. However, in NPC1 disease models, where we show that TFEB is over-activated and enriched in the nucleus due to lysosomal stress, levacetylleucine reduces nuclear TFEB and restores a more normal cytoplasmic-to-nuclear balance.” (opens the source at this quote in a new tab)
“The effects of the drug are stereospecific: while the L-enantiomer is active, the D-enantiomer and racemate show no effect, revealing the antagonistic properties of the D-enantiomer.” (opens the source at this quote in a new tab)

Mechanisms summarized in a published review

“Preclinical studies demonstrated that NALL enhances mitochondrial bioenergetics, reduces oxidative stress, restores lysosomal and autophagic function, and modulates neuroinflammation.” (opens the source at this quote in a new tab)