Novel pharmacotherapy: NNI-362, an allosteric p70S6 kinase stimulator, reverses cognitive and neural regenerative deficits in models of aging and disease

Abstract

Aging is known to slow the neurogenic capacity of the hippocampus, one of only two mammalian adult neurogenic niches. The reduction of adult-born neurons with age may initiate cognitive decline progression which is exacerbated in chronic neurodegenerative disorders, e.g., Alzheimer's disease (AD). With physiologic neurogenesis diminished, but still viable in aging, non-invasive therapeutic modulation of this neuron regeneration process remains possible. The discovery of truly novel neuron regenerative therapies could be identified through phenotypic screening of small molecules that promote adult-born neurons from human neural progenitor cells (hNPCs). By identifying neuron-generating therapeutics and potentially novel mechanism of actions, therapeutic benefit could be confirmed through in vivo proof-of-concept studies. The key aging and longevity mTOR/p70S6 kinase axis, a commonly targeted pathway, is substrate for potential selective kinase modulators to promote new hippocampal neurons from NPCs. The highly regulated downstream substrate of mTOR, p70S6 kinase, directly controls pleiotropic cellular activities, including translation and cell growth. Stimulating this kinase, selectively in an adult neurogenic niche, should promote NPC proliferation, and cell growth and survival in the hippocampus. Studies of kinase profiling and immunocytochemistry of human progenitor neurogenesis suggest that the novel small molecule NNI-362 stimulates p70S6 kinase phosphorylation, which, in turn, promotes proliferation and differentiation of NPCs to neurons. NNI-362 promoted the associative reversal of age- and disease-related cognitive deficits in aged mice and Down syndrome-modeled mice. This oral, allosteric modulator may ultimately be beneficial for age-related neurodegenerative disorders involving hippocampal-dependent cognitive impairment, specifically AD, by promoting endogenous hippocampal regeneration.

Trial registration: ClinicalTrials.gov NCT04074837.

Keywords: Allosteric modulator; Alzheimer’s disease; BrdU+ neuron survival; Hippocampal neuron regeneration; Human neural progenitors; mTOR/p70S6 kinase axis.

Conflict of interest statement

JAK is a majority stockholder in Neuronascent. No other conflict exists.

Figures

Fig. 1

Discovery of neuron restorative small molecule therapy, NNI-362. Phenotypic screen to measure both proliferative and neuron maturity capacity from commercial novel libraries (a); proliferation using Alamar Blue, n = 5 (b). Ratio of mature neuron (MAP2a,b+) to total cell numbers (Hoechst dye) from human neural progenitors at DIV12, n = 6–12. Structure and formula weight of NNI-362 (d). Oxidative cellular metabolism directly related to cell numbers was measured by fluorescence increase in Alamar Blue using emission filter 530/580 nm. Data were analyzed using Kruskal-Wallis with Group as factor. *p < 0.05 vs. controls

Fig. 2

NNI-362 improves memory and increases adult-born neural cells in an aging mouse model (a–c) and a Down syndrome mouse model (d–f). Percent time spent with a novel object during acquisition and memory task, n = 8–11 (a). Number of BrdU-positive cells in the dentate gyrus of the hippocampus after 5–6 weeks oral treatment, n = 3 (b). Latency to fall from bridge test, n = 8–11 (c). Percent time spent with a novel object during memory task, n = 5–9 (d). Number of BrdU-positive cells in the dentate gyrus of the hippocampus after 4 weeks oral treatment, n = 2–4 (e). Distance traveled during an open field test, n = 9–14. BrdU staining and DS behavioral outcomes were analyzed using Kruskal-Wallis with group as factor. Behavioral measurements for the aging study were subjected to one-way ANOVA with group as factor. *p < 0.05 vs. young control; #p < 0.05 vs. old control; @p < 0.05 vs. WT-vehicle; +p < 0.05 vs. DS-vehicle

Fig. 3

NNI-362 putative mechanism via allosteric stimulation. Significant kinases that either are inhibited or stimulated by NNI-362 in CEREP panel of 151 kinases, significant with ± 25% inhibition (a). Measure of active site competition serial dilution to 10× (1000 nM) the effective concentration of NNI-362 (DiscoverX, KinomeScan) (b). Phosphorylation of p70S6 kinase in cultures prepared from 6 and 12DIV human neural progenitors treated with 0, 100, 300, and 1000 nM NNI-362 with each media change, n = 3 (c). Effect of NNI-362 on p70S6 kinase in two separate preparations of human neuronal progenitor cells at 5–6 and 11DIV (d). Phosphorylated kinases not consistently modulated by NNI-362: Akt1/2/3&pan, CREB, ERK1/2, GSK3beta, HSP 27, JNK1/2&pan, MKK3, MKK6, MSK2, p38alpha/beta/delta/gamma, p53, RSK1/2, and TOR. Effects of CDK5 inhibitors (indirubin and BML529), GSK3beta inhibitors (indirubin-3-monoxime and kenpaullone), and IKK2 inhibitor (SC-514) (e) on NNI-362-associated proliferation. Effects of p38 MAPK inhibitors on NNI-362-associated proliferation (f). Putative mechanism describing neuronal regeneration during aging and potential mechanism of action for NNI-362 (g). In vitro studies were analyzed using Kruskal-Wallis with group as factor. *p < 0.05 vs. vehicle, #p < 0.05 vs. NNI-362