Discovery of NV-5138, the first selective Brain mTORC1 activator

Abstract

The mechanistic target of rapamycin complex 1 (mTORC1) has been linked to several important chronic medical conditions many of which are associated with advancing age. A variety of inputs including the amino acid leucine are required for full mTORC1 activation. The cytoplasmic proteins Sestrin1 and Sestrin2 specifically bind to the multiprotein complex GATOR2 and communicate leucine sufficiency to the mTORC1 pathway activation complex. Herein, we report NV-5138, a novel orally bioavailable compound that binds to Sestrin2 and activates mTORC1 both in vitro and in vivo. NV-5138 like leucine transiently activates mTORC1 in several peripheral tissues, but in contrast to leucine uniquely activates this complex in the brain due lack of metabolism and utilization in protein synthesis. As such, NV-5138 will permit the exploration in areas of unmet medical need including neuropsychiatric conditions and cognition which have been linked to the activation status of mTORC1.

Figure 1

NV-5138 is a novel leucine analog that binds the leucine-binding pocket of Sestrin2. (a) Chemical structure of NV-5138. (b) Melt curve of Sestrin2 in the absence and presence of increasing amounts of NV-5138; pink = 1 µM, green = 10 µM, blue = 100 µM. (c) Measurement of the binding affinity of NV-5138 for Sestrin2 by isothermal calorimetry (ITC) predicts a binding K_d of 1.5 µM with a molar stoichiometry of 1. (d) X-ray crystal structure of NV-5138 bound to sestrin 2 at 3.3 Å resolution. (e) Interactions made by NV-5138 in the leucine-binding pocket of sestrin 2; side-chains of residues within 4 Å of NV-5138 are highlighted.

Figure 2

NV-5138 activates mTORC1 by modulating the interaction between Sestrin2 and GATOR2. (a) Immunoblotting of Sesn2 from the cell-free Sesn2/WDR24 interaction assay. Flag-WDR24 was immunoprecipitated from amino acid-starved Flag-WDR24 expressing HEK-293T cells, resuspended in cytosolic buffer and NV-5138 or leucine (10 µM) was added for 10 min. (b) Dose-dependent activation of mTORC1 by NV-5138 or leucine correlates with disruption of Sesn2 from Flag-WDR24. Flag-WDR24 expressing HEK-293T cells were starved of leucine for 50 min followed by addition of NV-5138 or leucine for 10 min. Immunoblot shows levels of Sestrin2 bound to immunoprecipitated Flag-WDR24 (lower contrast images are available in Supplementary Fig. 6a) and levels of phosphorylated S6K1 (³⁸⁹pS6K1). (c) Cell based treatment with NV-5138 shifts the melting temperature of intracellular Sestrin2 ~+2 °C. Percentage of Sestrin2 remaining compared to Sestrin2 amount at 40 °C is plotted as a function of temperature with non-linear regression performed to fit a melting curve. Values were determined from immunoblots of Sestrin2. (d) Cell based treatment with NV-5138 shifts the melting temperature of intracellular Sestrin1 ~+ 2 °C. Curve generated as described in (c). (e) Cell based treatment with NV-5138 does not impact the melting temperature of intracellular Sestrin3. Curve generated as described in (c). (f,g) Immunoblots of phosphorylated S6K1 (³⁸⁹pS6K1) to measure mTORC1 activity in unedited HEK-293T cells, HEK-293T cells deficient for sestrins 1,2 and 3 or HEK-293T cells deficient for the GATOR1 component Nprl3. Cells were starved for Leucine for 50 min followed by addition of NV-5138 or leucine for 10 min. In (g) phosphorylated S6K1 (Thr389) samples have been run on two separate gels, original images are available in Supplementary Fig. 6b.

Figure 3

NV-5138 transiently activates mTORC1 in the brain. (a) Quantification of immunoblots for phosphorylated S6 (S240/44) from homogenized whole brains isolated from SD rats 1 h after oral dosing with NV-5138 at 40 mg/kg, 80 mg/kg or 160 mg/kg (n = 5). (b) Quantification of immunoblots for phosphorylated S6 (S240/44) from synaptoneursomes from specific brain regions after dosing with vehicle or NV-5138 (160 mg/kg, PO) for 1 h (n = 20). (c) Quantification of immunoblots for phosphorylated S6 (S240/44) from homogenized tissues from ad libitum fed SD rats orally dosed with leucine (500 mg/kg) or NV-5138 (160 mg/kg) and sacrificed 1 h later (n = 10). (d) Quantification of immunoblots for phosphorylated S6 (S240/44) from homogenized whole brains isolated from SD rats 1, 3, 5, 7 and 24 h after oral dosing with NV-5138 (160 mg/kg) (n = 10). (e) Quantification of immunoblots for GluR1, PSD95 and Synapsin 1 from homogenized synaptoneurosomes from specific brain regions collected from SD rats 24 h after dosing with vehicle or NV-5138 (160 mg/kg, PO, n = 10). All data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 indicates a significant difference by an unpaired two-tailed students t-test.