Stem Cell Research & Therapy Publishes “Novel Pharmacotherapy: NNI-362, an Allosteric p70S6 Kinase Stimulator, Reverses Cognitive and Neural Regenerative Deficits in Models of Aging and Disease.”

S H O R T R E P O R T Open Access Novel pharmacotherapy: NNI-362, an allosteric p70S6 kinase stimulator, reverses cognitive and neural regenerative deficits in models of aging and disease Nathalie Sumien 1 , Matthew S. Wells 2,3, Akram Sidhu 1, Jessica M. Wong 1, Michael J. Forster 1, Qiao-Xi Zheng 2 and Judith A. Kelleher-Andersson 2* 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 mo lecules 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 co mmonly targeted pathway, is substrate for potential selective kinase modulators to promote new hippocampal neurons from NPCs. The highly regulated downstream substrate of mTOR, p70S6 k inase, directly controls pleiotropic cellular activities, including translation and cell growth. Stimulating this kinase, s electively 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, allos teric modulator may ultimately be beneficial for age- related neurodegenerative disorders involving hippocamp al-dependent cognitive impairment, specifically AD, by promoting endogenous hippocampal regeneration. Keywords: Alzheimer’s disease, mTOR/p70S6 kinase axis, Allosteric modulator, Human neural progenitors, Hippocampal neuron regeneration, BrdU + neuron survival © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. * Correspondence: [email protected] 2Neuronascent, Inc., 15601 Crabbs Branch Way, Rockville, MD 20855, USA Full list of author information is available at the end of the article Sumien et al. Stem Cell Research & Therapy (2021) 12:59 https://doi.org/10.1186/s13287-020-02126-3 Introduction Neurogenesis in adult humans is an accepted phenomenon following demonstration of BrdU + neurons in the hippocampal dentate gyrus (DG) [ 1]. However, neurogenesis, in aged individuals and chronic neurode- generative conditions, remains disputed [ 2]. An article suggested a lack of measurable human neurogenesis with age [ 3], while data support continuous neurogenesis throughout aging [4, 5] through neurogenic slowing where differentiating neural progenitors may have longer latency phase before becoming functional neurons [ 6]. Neural stem cell markers appeared age-independent, while prolif- erative and maturation factors diminished with age, but independently of each other, providing multiple neural targets for intervention [ 7]. In AD postmortem DG, neurogenesis was reduced and co-neuron maturation fac- tors dropped-off in relation to later Braak stages [ 8]. The reduced maturation of differentiated early neurons “in waiting” may be causative of age-related dementias [ 6] and suggests the later stages of neuron maturation are age-critical targets. Antidepressants promoting neuronal progenitor proliferation [ 9] have minimal benefit on de- pression in aged patients [ 10, 11], suggesting that en- hanced progenitor proliferation is insufficient to promote functioning new neurons. With the continued presence of neural progenitors in aged subgranular zone, this niche re- mains a putative pharmacotherapeutic target for promot- ing adult-born neurons. The standard therapeutic-tested Down syndrome (DS) model, Ts65Dn mice, exhibits many phenotypes of DS and AD [ 12]. Though Ts65Dn has no direct APP gene- dosage-linked pathology, other trisomy-related pheno- types provide abundant rationale for testing therapeutics to address memory and neurogenic dysfunction [ 13]. Re- versing the neurogenic deficit in young Ts65Dn mice using fluoxetine provided a memory benefit [ 14, 15], though the effects ’ durability in adults remains question- able [ 16]. New neuron formation in neurodegenerative disorders suggests potential for neuro-compensation as observed in postmortem AD tissue [ 17], Huntington ’sp a t i e n t s[18], and stroke in non-human primates [ 19]. Whether therap- ies targeting this compensatory neurogenesis are effective in age-related neurodegenerative disorders (ARND) re- mains undetermined. One non-pharmacotherapy promot- ing neurogenesis and cognition in aging is aerobic exercise. Human trials demonstrated a 6- or 12-month aerobic fitness program improved cognition, increased hippocampal volume [ 20, 21], and promoted adult-born neurons to compensate for the loss in the anterior hippo- campus [20]. Apparently, the brain retains an innate abil- ity for neuron regeneration even in ARND. Since aging is the greatest risk factor for AD [ 22], any purported disease- modifying AD pharmacotherapy must be effective in compensating for the slowing of adult-born neuron for- mation and the associated cognitive decline in aging and AD. The exact [non-therapeutic] trigger that initiates com- pensatory neurogenesis in ARND is unknown, though growth factors and downstream signaling pathways ap- pear likely candidates. Growth factor mimetics have been tested clinically, exhibiting side effect profiles out- weighing any direct gains. However, working down- stream at the mTORC1/p70S6 kinase axis would provide a more precise control of protein synthesis and neuron regeneration. Moreover, p70S6 kinase stimula- tion requires phosphorylation at eight sites, including four auto-inhibitory pseudo-substrate sites [ 23]. Inhibi- tors of kinases upstream of p70S6 kinase show benefit as age-related disorder therapeutics, though often associ- ated with toxic side-effects [ 24]. Without clear/safe tar- gets for promoting adult-born functional neurons, the use of a phenotypic screening platform using hNPCs, and small molecule libraries is a favorable option. The ability to measure neural progenitor proliferation and differentiation to mature neurons in culture has been suggested as necessary to identify novel therapies [ 25]. Our aim was to discover orally administrated, novel kin- ase modulators that penetrate the BBB, and promote the formation of functional adult-born neurons from en- dogenous neural progenitor cells. Results Neuronascent’s phenotypic screening platform using hNPCs (Fig. 1a) allowed the discovery of novel neuro- genic agents that are also neuroprotective. Due to in silico physicochemical characteristics provided by the commercial library vendor, the number of screened mol- ecules can be narrowed to only BBB-permeable and drug-like compounds. This is critical for small company drug discovery programs in order to quickly progress from the in vitro screening into in vivo efficacy testing. This platform description was expanded from that de- scribed by Kelleher-Andersson [ 25] and revealed a lead candidate: NNI-362 (Fig. 1d). NNI-362 promoted prolif- eration at 1000 nM versus vehicle ( p < 0.05) at DIV3 (Fig. 1b) and increased the ratio of mature neurons (MAP2a,b+) to total cells at DIV12/13 at ≥ 1000 nM ver- sus vehicle (all p < 0.05) (Fig. 1c). Young mice spent more time with the novel object than old mice ( p < 0.05) and old NNI-362-treated mice exhibited a similar pattern to the young mice ( p < 0.05 vs. old controls) (Fig. 2a). Old mice had lower new BrdU+ cells ( p < 0.05), while the old 10 mg/kg NNI-362 dose had higher BrdU + cells migrating/surviving than the old controls ( p < 0.05) and were not significantly dif- ferent from the young ( p = 0.08) (Fig. 2b). This effect was associated with an improvement in memory Sumien et al. Stem Cell Research & Therapy (2021) 12:59 Page 2 of 6 function only at the 10 mg/kg dose (data not shown) similar to Fig. 2a. NNI-362 did not reverse age-related motor dysfunction (Fig. 2c). Similarly, in Ts65Dn mice, NNI-362 treatment was associated with reversal of DS- modeled memory impairment ( p < 0.05) (Fig. 2d) and decreased number of mature BrdU + cells surviving in the DG ( p < 0.05) (Fig. 2e). NNI-362 did not reduce the hyperactivity observed in Ts65Dn mice (Fig. 2f). NNI- 351 was also discovered through the phenotypic screen (Fig. 1a); however, it lacked the neuroprotection capacity in vitro and did not improve memory during aging or consistently in the DS model (Fig. 2), yet did reduce hyperactivity (Fig. 2f). NNI-362 only significantly stimulated the p70S6 kin- ase from the 151 kinase panel (Fig. 3a). No competition at the active site of p70S6 nor any of the other pathway- related kinases were observed, providing evidence for allosteric targeting by NNI-362 (Fig. 3b). Only at the neuron regenerative concentration ( ≥ 1000 nM) and only during the early dividing and beginning differentiation phase was p70S6 kinase selectively phosphorylated (6DIV) significantly ( p < 0.05), while being absent in fully differentiated neurons at 12DIV (Fig. 3c, d). The switch- ing on of translation and increased neurogenesis is pos- sible through numerous targeting points of the mTOR/ p70S6 kinase axis [ 23], but selectivity to neural cells may be only possible by means of predominantly neuronal CDK5 kinase phosphorylation at p70S6 kinase auto- inhibitory site [ 26]. Results confirm only the addition of CDK5 inhibitors, indirubin and BML259, ameliorated NNI-362-induced proliferation, while the addition of other inhibitors, including p38 kinase inhibitor, was inef- fective (Fig. 3e, f). These results suggest that NNI-362 acts at the auto-inhibitory pseudo-substrate site, Ser411, where CDK5 phosphorylates p70S6 kinase (Fig. 3g) dur- ing the mitogenic translational stage, not the terminally differentiated neuron stage. Discussion The main findings from this study were that NNI-362 promoted proliferation and survival of adult-born neural cells associated with reversal of cognitive deficits, with- out toxic or off-target effects up to 6 weeks of adminis- tration, and acts allosterically and downstream of mTOR. The screening platform allowed for the discovery of small molecule therapies that promote proliferation and increase new mature neuron numbers in culture, through unique kinase modulating pathway(s). This phenotype is obligatory for the compound to succeed as 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 Sumien et al. Stem Cell Research & Therapy (2021) 12:59 Page 3 of 6 a human therapy for age-related degenerative disorders. A therapeutic candidate that is only neural progenitor- proliferative appears unable to reverse age-related cogni- tive impairment [ 11]. Fluoxetine had neurogenic and be- havioral benefit in young TS65Dn mice [ 14, 15], but not in adult mice [ 16]. Noteworthy, NNI-351, a compound with only a proliferative phenotype (Ki67 + cells greater than DS-vehicle, data not shown), reduced hyperactivity, but was ineffective in reversing memory deficits in aging and DS models, suggesting that maturation and survival of adult-born neural cells are necessary for cognitive im- provements in aging and neurodegenerative disease. In- deed, brain aging may be more complex especially in the adult hippocampal neurogenic niche, where “stem cell exhaustion” [27] may be less critical than promotion of maturation of dividing neural progenitors/early neurons to fully mature, functioning neurons. Boldrini et al. [ 4] suggest the human neural progenitor hippocampal pool remains available for neurogenesis into our 80s. This need for promotion of maturation of the adult-born neurons appeared more critical in AD than aging alone [8]. Further studies of differentiation and synaptic inte- gration will be needed to conclusively resolve that NNI- 362 has an effect on neurogenesis beyond proliferation, migration, and adult-born cell survival. Identifying the mechanism of action of NNI-362 by which neural cells proliferate, migrate, survive, and im- prove cognitive function could lead to novel target iden- tification for disease-modifying neuron regeneration. While a selective p70S6 kinase inhibitor is known [ 28], our data indicate that NNI-362 stimulates p70S6 kinase, suggesting activity at a critical auto-inhibitory allosteric site: Ser411 (conserved across many species [ 29]), a sub- strate for CDK5/activator phosphorylation, suggesting selectivity to neural tissue [ 30]. Inhibition of CDK5 ame- liorated NNI-362-induced proliferation accounting for NNI-362’s neuron selectivity, while other upstream ki- nases or their inhibitors played no definitive role in in- duced neuron generation. Conclusion Elucidating the CDK5/activator substrate Ser411 site on p70S6 kinase [ 26] that is selectively stimulated in neural cells by NNI-362 provides greater insights into how this clinical-stage therapeutic works in aging brain and neu- rodegenerative disorders. Further studies will look at 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 Sumien et al. Stem Cell Research & Therapy (2021) 12:59 Page 4 of 6 selective neuron regeneration markers and reversal of cognitive deficits in aging and neurodegenerative models and the level of allosteric site stimulation on p70S6 kin- ase by Western and MAP kinase profiling in vivo. Though mTOR inhibition has been suggested to pro- mote longevity, our data support a downstream target to improving brain “healthspan”: restorative capacity via neural-selective allosteric stimulation of p70S6 kinase. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s13287-020-02126-3. Additional file 1. Acknowledgements N/A. Authors’ contributions Therapy invention: JAK; concept and experimental design: JAK, NS, and MJF; experimental procedures: MW, Q-XZ, JMW, and AS. Analyses and writing: JAK and NS. The authors read and approved the final manuscript. Funding This work was supported by Neuronascent, Inc., and the National Institutes of Health [2P01 AG022550 (PI-NS)]. NIA has supported the development of NNI-362 through a translational safety program (Dr. Petanceska-directed pro- gram) and through the IND/phase 1a clinical trial (R01 AG056561, PI-JAK). NNI-362 is presently in phase 1a clinical trial for Alzheimer ’s disease NCT04074837. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate N/A. Consent for publication N/A. Competing interests JAK is a majority stockholder in Neuronascent . No other conflict exists. Author details 1Department of Pharmacology & Neuroscience, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX, USA. 2Neuronascent, Inc., 15601 Crabbs Branch Way, Rockville, MD 20855, USA. 3Novavax, 21 Firstfield Rd., Gaithersburg, MD 20878, USA. 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 Sumien et al. Stem Cell Research & Therapy (2021) 12:59 Page 5 of 6 Received: 6 August 2020 Accepted: 27 December 2020 References 1. Eriksson PS, et al. Neurogenesis in the adult human hippocampus. Nat Med. 1998;4(11):1313–7. 2. Kumar A, et al. Adult neurogenesis in humans: a review of basic concepts, history, current research, and clinical implications. Innov Clin Neurosci. 2019; 16(5–6):30–7. 3. Sorrells SF, et al. Human hippocampal neurogenesis drops sharply in children to undetectable levels in adults. Nature. 2018;555(7696):377 –81. 4. Boldrini M, et al. Human hippocampal neurogenesis persists throughout aging. Cell Stem Cell. 2018;22(4):589 –99 e5. 5. Spalding KL, et al. Dynamics of hippocampal neurogenesis in adult humans. Cell. 2013;153(6):1219–27. 6. Kempermann G, et al. Human adult neurogenesis: evidence and remaining questions. Cell Stem Cell. 2018;23(1):25 –30. 7. Mathews KJ, et al. Evidence for reduced neurogenesis in the aging human hippocampus despite stable stem cell markers. Aging Cell. 2017;16(5):1195 – 9. 8. Moreno-Jimenez EP, et al. Adult hippocampal neurogenesis is abundant in neurologically healthy subjects and drops sharply in patients with Alzheimer’s disease. Nat Med. 2019;25(4):554 –60. 9. Santarelli L, et al. Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants. Science. 2003;301(5634):805 –9. 10. Cowen DS, et al. Age-dependent decline in hippocampal neurogenesis is not altered by chronic treatment with fluoxetine. Brain Res. 2008;1228:14 –9. 11. Couillard-Despres S, et al. Ageing abolishes the effects of fluoxetine on neurogenesis. Mol Psychiatry. 2009;14(9):856 –64. 12. Gardiner KJ. Pharmacological approaches to improving cognitive function in Down syndrome: current status and considerations. Drug Des Devel Ther. 2015;9:103–25. 13. Hamlett ED, et al. Cognitive impairment, neuroimaging, and Alzheimer neuropathology in mouse models of Down syndrome. Curr Alzheimer Res. 2016;13(1):35–52. 14. Bianchi P, et al. Early pharmacotherapy restores neurogenesis and cognitive performance in the Ts65Dn mouse model for Down syndrome. J Neurosci. 2010;30(26):8769–79. 15. Clark S, et al. Fluoxetine rescues deficient neurogenesis in hippocampus of the Ts65Dn mouse model for Down syndrome. Exp Neurol. 2006;200(1): 256–61. 16. Heinen M, et al. Adult-onset fluoxetine treatment does not improve behavioral impairments and may have adverse effects on the Ts65Dn mouse model of Down syndrome. Neural Plast. 2012;2012:467251. 17. Jin K, et al. Increased hippocampal neurogenesis in Alzheimer ’s disease. Proc Natl Acad Sci U S A. 2004;101(1):343 –7. 18. Curtis MA, et al. Increased cell proliferation and neurogenesis in the adult human Huntington ’s disease brain. Proc Natl Acad Sci U S A. 2003;100(15): 9023–7. 19. Koketsu D, et al. Increased number of new neurons in the olfactory bulb and hippocampus of adult non-human primates after focal ischemia. Exp Neurol. 2006;199(1):92–102. 20. Erickson KI, et al. Exercise training increases size of hippocampus and improves memory. Proc Natl Acad Sci U S A. 2011;108(7):3017 –22. 21. Jonasson LS, et al. Aerobic exercise intervention, cognitive performance, and brain structure: results from the physical influences on brain in aging (PHIBRA) study. Front Aging Neurosci. 2016;8:336. 22. 2020 Alzheimer ’s disease facts and figures. 2020. Alzheimers Dement., 2020. 23. Magnuson B, Ekim B, Fingar DC. Regulation and function of ribosomal protein S6 kinase (S6K) within mTOR signalling networks. Biochem J. 2012; 441(1):1–21. 24. Hu S, et al. GSK3 inhibitors show benefits in an Alzheimer ’s disease (AD) model of neurodegeneration but adverse effects in control animals. Neurobiol Dis. 2009;33(2):193 –206. 25. Kelleher-Andersson J. Discovery of neurogenic, Alzheimer ’s disease therapeutics. Curr Alzheimer Res. 2006;3(1):55 –62. 26. Zhou Y, et al. Increased phosphorylation of p70 S6 kinase is associated with HPV16 infection in cervical cancer and esophageal cancer. Br J Cancer. 2007; 97(2):218–22. 27. Lopez-Otin C, et al. The hallmarks of aging. Cell. 2013;153(6):1194 –217. 28. Pearce LR, et al. Characterization of PF-4708671, a novel and highly specific inhibitor of p70 ribosomal S6 kinase (S6K1). Biochem J. 2010;431(2):245 –55. 29. Hou Z, He L, Qi RZ. Regulation of s6 kinase 1 activation by phosphorylation at ser-411. J Biol Chem. 2007;282(10):6922 –8. 30. Dhavan R, Tsai LH. A decade of CDK5. Nat Rev Mol Cell Biol. 2001;2(10): 749–59. Publisher’sN o t e Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Sumien et al. Stem Cell Research & Therapy (2021) 12:59 Page 6 of 6

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