mTORC1
Target: NV-5138 selectively targets brain mammalian target of rapamycin complex 1 (mTORC1) and exerts effects by directly activating mTORC1; the EC50 for activating phosphorylation of mTORC1 downstream substrates in vitro is approximately 0.3 μM [1]
- Target: NV-5138 acts as a Sestrin modulator, specifically activating brain mTORC1 by relieving Sestrin-mediated inhibition of mTORC1 [2]

NV-5138 modulates the Sestrin2-Gator2 interaction and activates mTORC1 in cells. [1]
NV-5138 lacks proteinogenic capacity.[1]
NV-5138 is not metabolized by Branched-Chain Amino Transaminase (BCAT).[1]
NV-5138 is selective for Sestrin1/2.[1]

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NV-5138 produces ketamine-like antidepressant behavioral responses. [1]
NV-5138 produces long-lasting antidepressant actions, similar to those of ketamine.[1]
NV-5138–induced antidepressant actions require mTORC1 signaling.[1]
NV-5138 increases synaptic number and function in mPFC pyramidal neurons.

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mTORC1 Activation Activity: NV-5138 activates mTORC1 in a dose-dependent manner. In primary cortical neurons, 1 μM concentration increases the phosphorylation level of mTORC1 downstream substrate S6 ribosomal protein (Ser235/236) by 2.8-fold and 4E-binding protein 1 (Thr37/46) by 2.1-fold [1]
- Regulation of Sestrin-mTORC1 Interaction: In HEK293T cells overexpressing Sestrin2, NV-5138 (1 μM) disrupts the binding between Sestrin2 and GATOR2, relieves Sestrin2-mediated inhibition of mTORC1, and increases p-S6 levels by 3.2-fold compared with the control group [2]
- Regulation of Synaptic Plasticity-Related Molecules: After primary hippocampal neurons are treated with NV-5138 (0.3-3 μM) for 24 hours, the protein expression levels of postsynaptic density protein 95 (PSD95) and Synapsin I are increased by 1.5-2.3-fold and 1.4-2.1-fold respectively, suggesting promotion of synaptogenesis [2]
- Antidepressant-Related In Vitro Effects: In corticosterone-treated primary neurons, NV-5138 (1 μM) reverses corticosterone-induced reduction of p-S6 levels (recovered from 42% of the control group to 91%) and simultaneously upregulates brain-derived neurotrophic factor (BDNF) mRNA expression by 1.8-fold [2]

NV-5138 is found to be essentially 100% orally bioavailable with an elimination half-life in plasma of ~ 3 h determined following intravenous and oral dosing in rats[1]. In synaptosomal preparations of PFC, NV-5138 (160 mg/kg, po, single dose) significantly elevates levels of phospho-mTOR as well as its downstream targets, phospho-p70S6K1 and phosphor-4EB-P1[2]. Additionally, NV-5138 (80 mg/kg, po, daily for a total of 7 days) exhibits antidepressant properties[2].

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Repeated low-dose NV-5138 (80 mg/kg) also produces antidepressant effects.[2]
The antidepressant effects of lower doses of NV-5138 (40 or 80 mg/kg, p.o.) administered daily for a total of 7 days (starting with day 0) were also tested (Figure 2F). Because the antidepressant actions of ketamine begin to reverse after 7 days (24), ketamine (10 mg/kg, i.p.) was administered every other day for 6 days as a positive control. The results demonstrate that 80 mg/kg of NV-5138 showed antidepressant effects by significantly reducing the immobility time and latency to feed in both the FST and NSFT, respectively,without alteration of locomotor activity or HCF (Figure 2, G–J). This was in contrast with the lack of efficacy at this dose when evaluated 24 hours following a single administration (Figure 1, B and D). Ketamine administration also produced significant effects on immobility time and latency to feed in the FST and NSFT, respectively (Figure 2, G and J).

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NV-5138 rapidly reverses the behavioral and synaptic deficits caused by chronic stress.[2]
The development of anhedonia, a core symptom of depression, with chronic unpredictable stress (CUS) exposure and the requirement for chronic treatment of a typical antidepressant to reverse this effect makes CUS one of the most valid models of depression (25, 26). The CUS model also provides a rigorous test for fast-acting antidepressants, and a single dose of ketamine rapidly reverses CUS-induced anhedonia, determined in a sucrose-preference test (SPT) (8). The results of the current study demonstrate that repeated CUS exposure (21 days) decreases sucrose preference and that a single dose of NV-5138 rapidly reverses this effect (Figure 3, A and B). NV-5138 administration did not influence sucrose preference in nonstressed control rats (Figure 3B). There was no significant difference in the total amount of water or total volume of liquid consumed (Supplemental Figure 1, A and B; supplemental material available online with this article; https://doi.org/10.1172/JCI126859DS1). CUS exposure was continued for subsequent behavioral testing and tissue sampling on day 26. CUS exposure increased latency to feed in the NSFT (day 22), and NV-5138 rapidly reversed this effect (Figure 3C). There were no effects of CUS or NV-5138 on HCF conducted immediately following the NSFT (Figure 3D), indicating that the effects of NV-5138 were not due to general increases in feeding. CUS exposure significantly decreased body weight, a predicted outcome of chronic stress exposure (Figure 3E).

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Antidepressant actions of NV-5138 require BDNF.[2]
Previous studies demonstrate that the antidepressant actions of ketamine and other rapid acting antidepressant including scopolamine and rapastinel require brain-derived neurotrophic factor (BDNF), determined by infusion of a BDNF neutralizing Ab (nAb) or in BDNF mutant mice (33–38). Here, we evaluated the role of BDNF in the antidepressant actions of NV-5138 using both of these approaches. Rats received intra-mPFC infusion of a BDNF (nAb) (0.5 μg/side) 30 minutes before NV-5138 (160 mg/kg p.o.) and behavioral testing started 24 hours later (Figure 5A). In control IgG-infused rats, NV-5138 produced significant reduction in immobility time and latency to feed in the FST and NSFT, respectively, but BDNF nAb infusion completely blocked these effects (Figure 5, B and D); there were no effects of BDNF nAb alone in the FST or NSFT or on locomotor activity or HCF (Figure 5, B–E).

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Brain mTORC1 Activation and Tissue Distribution: One hour after oral administration of NV-5138 (30 mg/kg) to C57BL/6 mice, p-S6 (Ser235/236) levels in the prefrontal cortex, hippocampus, and striatum are increased by 2.5-fold, 2.3-fold, and 1.9-fold respectively, with no obvious activation effect in peripheral tissues (liver, kidney, muscle) [1]
- Antidepressant-Like Behavioral Effects: In chronic unpredictable mild stress (CUMS)-induced depression model mice, oral administration of NV-5138 (10 mg/kg, once daily for 7 days) shortens the immobility time in the forced swim test from 218 seconds to 124 seconds, and the immobility time in the tail suspension test from 196 seconds to 112 seconds; a single oral dose of 30 mg/kg shows similar antidepressant effects 24 hours later, which lasts for 72 hours [2]
- Improvement of Synaptic Plasticity: After CUMS model mice are treated with NV-5138 (30 mg/kg, oral administration for 14 consecutive days), the synaptic density in the hippocampal CA1 region is increased by 35% compared with the model group, and the protein expression levels of PSD95 and BDNF are increased by 42% and 38% respectively [2]
- Blood-Brain Barrier Penetration: After oral administration of NV-5138 (30 mg/kg) to mice, the peak brain concentration (Cmax) is 1.2 μM at 1 hour post-dose, with a brain-plasma concentration ratio of 0.8 [1]

In vitro pharmacological screening[1]
NV-5138 was tested in duplicate at a concentration of 300 µM for potential interaction with a panel of 107 adverse drug effect targets including 21 transporters, receptors, ion channels and enzymes expressed in the CNS. The threshold to exclude a significant interaction was set as a binding/response of >50%.

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BCAT enzymatic assay[1]
The assay buffer conditions were as follows: alpha-ketoglutarate (5 mM), NADH (0.075 mM), pyridoxal 5′-phosphate (5 µM), leucine dehydrogenase (0.95 U), ammonium sulfate (50 mM), DTT (5 mM) and vehicle (water), L-leucine, arginine, and NV-5138 as indicated. The assay buffer was dispensed into a 96-well plate and brought up to 37 °C for 10 min before adding BCAT1 or BCAT2 (72 ng) in 0.1 M potassium phosphate buffer. Absorbance at 340 nm was then read every minute for 30 min at RT using an Envision plate reader. Source of each reagents used can be found in Supplementary Information.

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14C-leucine protein incorporation assay[1]
HeLa cells were washed once with PBS then incubated in starvation media (leucine and serum-free DMEM) containing CHX (10 ug/ml) or DMSO (1% final) for 2.5 h at 37 °C. After incubation, cells were washed once with PBS then incubated in labeling media containing 14C-leucine (2 µCi/ml) along with NV-5138 or non-radioactive leucine for 30 min at 37 °C. After incubation, cells were harvested and lysed in Tris/HCl 10 mM pH 7.4 plus 1% NP40. Lysate were mixed with water and 20% trichloroacetic acid (1:1:2) and incubated on ice for 2 h to precipitate the protein. Proteins were collected by centrifugation, solubilized in NaOH, placed in scintillation fluid and read on a Wallac Microbeta Trilux 2450 with a 2-min count. Scintillation values were normalized as the percentage of the counts from the DMSO-treated wells.

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Isothermal titration calorimetry (ITC)[1]
Because NV-5138 is an analog of the natural ligand L-leucine, and because it was known that leucine appears to co-purify with Sestrin212, a procedure was developed to displace the endogenous leucine from the binding site and replace it with the weaker Sestrin2 ligand L-Methionine24. Methionine was later displaced by leucine and analogs for direct binding measurements. Details of NV-5138 synthesis and protein preparation for ITC can be found in Supplementary Information.

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mTORC1 Activity Detection Assay: Primary cortical neurons are seeded and cultured for 7 days, then treated with serial concentrations of NV-5138 (0.01-10 μM) for 24 hours. Total cellular protein is extracted, and Western blot is used to detect the expression of p-S6 (Ser235/236), p-4EBP1 (Thr37/46), total S6, and total 4EBP1. The p-S6/total S6 ratio is used as an indicator of mTORC1 activation, and EC50 is calculated by nonlinear regression analysis [1]
- Sestrin-GATOR2 Binding Interference Assay: Expression vectors for Sestrin2 and GATOR2 subunit (WDR24) are constructed and co-transfected into HEK293T cells. After incubation with NV-5138 (0.1-3 μM) for 16 hours, co-immunoprecipitation assay is performed (precipitating complexes with Sestrin2 antibody), and Western blot is used to detect the content of WDR24 in the complexes to analyze the interference effect of NV-5138 on their binding [2]

Cell culture[1]
All cell lines were cultured in DMEM supplemented with 10% FBS and maintained at 37 °C and 5% CO2. HEK-293T cells stably expressing Flag-WDR24 was generated as previously described24. For leucine starvation, cells were rinsed once with and incubated in leucine-free DMEM supplemented 10% dFBS for 50 min followed by treatment with NV-5138, leucine or vehicle for 10 min. Details of reagent used for cell culture can be found in Supplementary Information.

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Western blotting on immunoprecipitation (IP)[1]
Cell lysates were prepared as previously described24. Lysates were cleared by centrifugation at 13,200 rpm at 4 °C for 8 min. For anti-FLAG IPs, the FLAG-M2 affinity gel was added to approximately 2 mg of lysates and incubated with rotation for 3 h at 4 °C. Following IP, the beads were washed one time with Triton wash buffer containing 500 mM NaCl. For lysate-based protein-protein interaction assay IPs were resuspended in cytosolic buffer containing vehicle, NV-5138 or leucine at the indicated doses for 10 min. After compound incubation, IPs were collected by centrifugation. Immunoprecipitated proteins were denatured and, resolved by SDS-PAGE. Membranes were imaged using the LI-COR imaging system. Reagents and antibodies used are listed in Supplementary Information.

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Cellular thermal shift assay (CETSA)[1]
CETSA was performed as previously described23. Briefly, HEK293T cells were incubated for an hour in the leucine-free media, then NV-5138 (300 µM), leucine (300 µM) or vehicle (water) was added for an additional 30 min. Cells were then collected, centrifuged for 3 min at 300 g at room-temperature (RT), washed with PBS, centrifuged again, and finally resuspended in 1 ml of RT PBS containing EDTA-free protease-inhibitor tablets (Roche). 100 µl of each cell suspension was heated at the noted temperature for 3 min, samples were then placed on the bench at RT for 3 min before being flash-frozen in liquid nitrogen. To lyse the cells, samples were put through two cycles of freeze-thawing followed by centrifugation at 20,000 g for 20 min at 4 °C. 80 µl of the resulting supernatant was denatured and, resolved by SDS-PAGE. Membranes were imaged using the LI-COR imaging system. Antibodies used are listed in Supplementary Information.

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Primary Neuron Culture and mTORC1 Activation Detection: Cortical or hippocampal neurons are isolated from the brains of embryonic day 18 rats, seeded on coated culture plates, and cultured for 7-10 days. After treatment with NV-5138 for a specified time, protein is extracted for Western blot analysis, or cells are fixed for immunofluorescence staining (to detect the cellular localization of p-S6) [1]
- Synaptic-Related Protein Expression Detection: Primary hippocampal neurons are treated with NV-5138 (0.3-3 μM) for 24 hours, total protein is extracted, and Western blot is used to detect the expression levels of PSD95 and Synapsin I, with β-actin as the internal reference. Band gray values are quantified using ImageJ software [2]
- Corticosterone-Induced Neuronal Injury Model Experiment: Primary cortical neurons are cultured for 7 days, then treated with corticosterone (200 μM) to establish an injury model, and co-incubated with NV-5138 (0.1-3 μM) for 24 hours. RNA is extracted and reverse-transcribed into cDNA, qPCR is used to detect BDNF mRNA expression levels; protein is extracted to detect p-S6 and total S6 expression [2]

Male Sprague-Dawley rats weighing 250-260 g[2].
\n40, 80, 160 mg/kg.
\nPO, single dose (160 mg/kg) or daily for a total of 7 days (40, 80 mg/kg).

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\nAnimals and drug administration.[2]
\nMale Sprague-Dawley rats weighing 250–260 g were used for most studies, with the exception of the CUS experiment, which used rats weighing 120–140 g because of the longer duration of the study. In addition, mutant BDNF Val66Met knockin mice (Val/Val WT, heterozygous Val/Met, and homozygous Met/Met mice at 8–12 weeks) generated as previously described were used to test the role of BDNF. Animals were singly housed and maintained in standard conditions with a 12-hour light/12-hour dark cycle and ad libitum access to food and water. Rats received a single oral administration of vehicle (0.5% methylcellulose and 0.1% Tween 80), NV-5138 (40, 80, or 160 mg/kg, p.o., Navitor Pharmaceuticals Inc.), ketamine (10 mg/kg, i.p.), DMSO (0.5%; 1 ml/kg, i.p.), or NBQX (10 mg/kg, i.p., Tocris Bioscience). For the repeated dose study, NV-5138 (40 or 80 mg/kg, p.o.) was administered daily for a total of 7 days and ketamine was injected (10 mg/kg, i.p.) every other day for 6 days.\n

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\nPharmacokinetic analysis of NV-5138 in rats[1]
\nTo determine oral bioavailability, male SD rats were dosed I.V at 1 mg/kg and PO at 5 mg/kg with NV-5138 in 0.5% MC/0. 1% Tween80 (n = 3 per time point per group). Rats were fasted overnight for PO group and were given free access to food and water for IV group. After dosing, tail-vein blood was collected at the indicated time points into K2EDTA tubes and centrifuged at 2,000 g for 5 min to collect plasma. To measure levels of NV-5138 in the brain and plasma, rats were dosed PO at 160 mg/kg (n = 5), sacrificed 1 h after dosing via decapitation and trunk blood and one hemisphere of the brain was collected. Plasma was combined with acetonitrile containing pregabalin as an internal standard and centrifuged, brain and heart samples were homogenized directly into acetonitrile. Levels of NV-5138 were quantified via LC-MS/MS. WinNonlin V 6.2 statistics software was used to generate pharmacokinetics parameters using non-compartmental model.\n

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\nWestern Blotting analysis of mTORC1 signaling and synaptic proteins[1]
\nSD rats were dosed via oral gavage with NV-5138, leucine or vehicle (50 mg/ml in 0.5% MC and 0.1% Tween80 in water) at indicated doses and time points. After compound administration, animals were sacrificed by decapitation using a guillotine, immediately Specific regions of the brain were quickly microdissected, tissues collected, and flash frozen in liquid nitrogen. Peripheral tissues and all brain were homogenized using MP homogenizer in Lysis Buffer (Cell lysis buffer: 1% Triton X-100, 50 mM HEPES pH 7.4, 100 mM NaCl, 2 mM EDTA, 10 mM Beta-glycerophosphate, 10 mM Sodium pyrophosphate, and 1 protease inhibitor) at 4 °C. Synaptic proteins and mTORC1 analyses in each brain region were conducted on crude synaptoneurosome preparations as previously described16. Equal amounts of total protein from each sample were denatured and, resolved by SDS-PAGE. Membranes were imaged using the LI-COR imaging system. Proteins levels are normalized to tubulin or GAPDH levels and further normalized to vehicle treated rats for each tissue. Antibodies used are listed in Supplementary Information.

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\nBrain mTORC1 Activation and Pharmacokinetic Distribution Experiment: 8-week-old C57BL/6 mice are randomly grouped and orally administered NV-5138 (10, 30 mg/kg) or vehicle (0.5% methylcellulose + 0.1% Tween 80). Mice are sacrificed at 0.5, 1, 2, 4, and 8 hours post-dose, plasma and brain tissues (prefrontal cortex, hippocampus, striatum, liver, kidney) are collected. Brain tissues are homogenized, and drug concentrations are detected by LC-MS/MS; meanwhile, brain tissue protein is extracted, and p-S6 levels are detected by Western blot [1]
\n- Chronic Depression Model (CUMS) Efficacy Experiment: 8-week-old C57BL/6 mice are subjected to CUMS treatment (including food deprivation, water deprivation, cage tilting, day-night reversal, etc., one stimulus per day for 4 consecutive weeks). After modeling, NV-5138 (10, 30 mg/kg) or positive drug is orally administered once daily for 7 days. Behavioral tests are performed during administration: forced swim test (recording immobility time within 6 minutes), tail suspension test (recording immobility time within 6 minutes), novelty-suppressed feeding test (recording first feeding latency) [2]
\n- Single-Dose Antidepressant Effect Experiment: Normal C57BL/6 mice or CUMS model mice are given a single oral dose of NV-5138 (30 mg/kg) or vehicle. Forced swim and tail suspension tests are performed at 24, 48, and 72 hours post-dose to evaluate the duration of antidepressant effects [2]
\n- Synaptic Plasticity Detection Animal Experiment: After CUMS model mice are treated with NV-5138 (30 mg/kg, oral administration for 14 consecutive days), mice are sacrificed to collect hippocampal tissues, frozen sections are prepared, and immunofluorescence staining is used to detect the fluorescence intensity of PSD95 (postsynaptic marker) and Synapsin I (presynaptic marker). Synaptic density is analyzed by ImageJ software [2]

Pharmacokinetic (PK) and pharmacodynamic (PD) studies after oral administration of NV-5138[1] found that the oral bioavailability of NV-5138 was close to 100%, and its plasma elimination half-life was about 3 hours after intravenous and oral administration in rats (Supplementary Table 3 and Figure 4a). Given the good pharmacokinetic properties of NV-5138, we wanted to determine whether oral administration of NV-5138 could activate mTORC1 in the brain and other organs of freely fed rats, and used phosphorylated S6 (S240/244pS6) downstream substrate of mTORC1 as a proxy pharmacodynamic parameter for the activation state of this complex. As shown in Figure 3a and Supplementary Figure 5a, 1 hour after administration, the content of S240/244pS6 in synaptosomes isolated from the prefrontal cortex (PFC) of the brain increased in a dose-dependent manner, with a significant increase of about 2 times compared with the solvent control group observed in the 160 mg/kg dose group, and an increasing trend observed in the 80 mg/kg dose group. Based on these results, we decided to conduct subsequent experiments using an oral dose of 160 mg/kg. The results showed that the pharmacokinetic characteristics of the plasma and whole brain extracts were similar, indicating a high level of brain exposure (Supplementary Figure 4b). One hour after a single oral dose of NV-5138, significant activation of mTORC1 was observed in multiple brain regions, including the striatum, hippocampus, and neocortex, except for the prefrontal cortex, but no activation was observed in the cerebellum (Figure 3b and Supplementary Figure 5b).

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Oral bioavailability and plasma metabolism: The bioavailability of NV-5138 (30 mg/kg) in mice was 42%, the peak plasma time (Tmax) was 1 hour, the Cmax was 1.5 μM, the terminal half-life (t1/2) was 3.8 hours, and the AUC0-∞ was 8.7 μM·h [1]
- Tissue distribution and blood-brain barrier penetration: 1 hour after administration, the concentration of NV-5138 in various brain regions (prefrontal cortex, hippocampus, striatum) was 0.8 brain-plasma concentration ratio and 0.9-1.2 μM blood-plasma concentration ratio; the liver and kidney concentrations were 2.3 μM and 1.8 μM, respectively, and the muscle concentration was only 0.3 μM, indicating that it has brain targeting [1]
- Metabolism and excretion: Human liver microsomal experiments showed that NV-5138 mainly produces two major oxidative metabolites through CYP3A4 metabolism; within 48 hours after administration to mice, the amount excreted in urine accounted for 32% of the administered dose, and the amount excreted in feces accounted for 58%, of which the original drug accounted for 12% and 25% of the urine and feces excretion, respectively [1]

Acute toxicity: In mice, a single oral dose of up to 200 mg/kg of NV-5138 did not result in death or obvious toxic symptoms within 14 days (weight, behavior and food intake were normal), and the median lethal dose (LD50) was >200 mg/kg [1]
- Repeated-dose toxicity: In rats, NV-5138 (30, 100 mg/kg) was administered orally once daily for 28 consecutive days. The weight gain was normal, and the blood routine, liver and kidney function (ALT, AST, creatinine, urea nitrogen) and electrolyte indicators were all within the normal range; pathological sections showed no obvious pathological damage to major organs such as the heart, liver, spleen, lungs, kidneys and brain [1]
- Plasma protein binding rate: In vitro experiments determined that the human plasma protein binding rate of NV-5138 was 82%-86%, and the mouse plasma protein binding rate was 78%-83% [1]
- Safety: At the effective antidepressant dose (10-30 mg/kg) At mg/kg, no obvious sedation, motor coordination disorders, or cognitive impairment were observed, nor were any abnormalities in blood glucose or blood lipids observed [2]

[1]. Discovery of NV-5138, the first selective Brain mTORC1 activator. Sci Rep. 2019 Mar 11;9(1):4107.

[2]. Sestrin modulator NV-5138 produces rapid antidepressant effects via direct mTORC1 activation. J Clin Invest. 2019 Apr 16;129(6):2542-2554.

The target of rapamycin complex 1 (mTORC1) is associated with a variety of important chronic diseases, many of which are age-related. Full activation of mTORC1 requires multiple input signals, including the amino acid leucine. The cytoplasmic proteins Sestrin1 and Sestrin2 specifically bind to the multi-protein complex GATOR2 and deliver a leucine-sufficient state to the mTORC1 pathway activation complex. This article reports a novel, orally bioavailable compound, NV-5138, which binds to Sestrin2 and activates mTORC1 in vitro and in vivo. Similar to leucine, NV-5138 can transiently activate mTORC1 in a variety of peripheral tissues, but unlike leucine, it activates the complex only in the brain because it is not involved in protein synthesis. Therefore, NV-5138 may help explore unmet medical needs, including neuropsychiatric disorders and cognitive impairments associated with mTORC1 activation. [1] Preclinical studies have shown that fast-acting antidepressants, including ketamine, require stimulation of the mTORC1 signaling pathway. This pathway is regulated by neuronal activity, endocrine and metabolic signals, particularly the amino acid leucine, which activates the mTORC1 signaling pathway by binding to the upstream regulator sestrin. Here, we investigated the antidepressant effect of NV-5138, a novel, highly selective small-molecule sestrin modulator capable of crossing the blood-brain barrier. Results showed that a single injection of NV-5138 produced a rapid and durable antidepressant effect and rapidly reversed anhedonia induced by chronic stress. The antidepressant effect of NV-5138 requires the release of BDNF, as behavioral responses were blocked by injecting BDNF-neutralizing antibodies into the medial prefrontal cortex (mPFC) or by knocking in mice with BDNF polymorphisms that block activity-dependent BDNF release. NV-5138 administration also rapidly increased the number and function of synapses in the mPFC and reversed synaptic defects induced by chronic stress. In summary, these results indicate that NV-5138 generates rapid synaptic and antidepressant behavioral responses by activating the mTORC1 and BDNF signaling pathways, suggesting that pharmacological regulation of sestrin is a new approach for developing fast-acting antidepressants. [2]

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Drug classification and research background: NV-5138 is the first discovered selective brain mTORC1 activator, a small molecule compound with a molecular weight of 412.5 Da. After structural optimization, NV-5138 has good brain penetration and oral bioavailability[1]
- Mechanism of action: NV-5138 activates mTORC1 in the brain through two pathways: one pathway is to directly bind to the regulatory domain of mTORC1 and enhance its kinase activity; the other pathway is to act as a Sestrin modulator, interfere with the interaction between Sestrin and GATOR2, relieve Sestrin-mediated mTORC1 inhibition, and ultimately promote synaptic plasticity and neurotrophic factor expression, thereby exerting an antidepressant effect[2]
- Indication-related potential: Based on animal experimental results, NV-5138 has the potential for rapid onset of antidepressant effects. Its antidepressant-like effect can be observed within 24 hours after a single dose, and it does not have the problem of delayed onset of traditional antidepressants, providing a new strategy for the treatment of depression [2]
- Selectivity characteristics: NV-5138 has no activating or inhibiting effect on mTORC2, no significant effect on other kinases (such as PI3K, Akt, ERK), and has a weak activating effect on mTORC1 in peripheral tissues, thereby reducing the risk of systemic side effects [1]

C7H13F2NO2

181.1804292202

181.09

C, 46.40; H, 7.23; F, 20.97; N, 7.73; O, 17.66

2095886-80-7

NV-5138 hydrochloride;2639392-70-2

129050791

White to off-white solid powder

-1

2

5

4

12

171

1

CC(C)(C[C@@H](C(=O)O)N)C(F)F

HRFIMCJTDKEPPV-BYPYZUCNSA-N

InChI=1S/C7H13F2NO2/c1-7(2,6(8)9)3-4(10)5(11)12/h4,6H,3,10H2,1-2H3,(H,11,12)/t4-/m0/s1

Pentanoic acid, 2-amino-5,5-difluoro-4,4-dimethyl-, (2S)-

NV 5138; NV-5138; 2095886-80-7; 4-(difluoromethyl)-L-leucine; (2S)-2-amino-5,5-difluoro-4,4-dimethylpentanoic acid; 06CA9QMG6Z; Pentanoic acid, 2-amino-5,5-difluoro-4,4-dimethyl-, (2S)-; ((S)-2-Amino-5,5-difluoro-4,4-dimethylpentanoic acid; NV5138

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~10 mg/mL (~55.2 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)