We have previously demonstrated that DHA metabolism to N-docosahexaenoylethanolamine (synaptamide) is a significant endogenous mechanism for promoting neurogenesis, neuritogenesis and synaptogensis in a cAMP dependent manner. We further demonstrated that orphan G-protein coupled receptor 110 (GPR110, ADGRF1) is the synaptamide target receptor, triggering cAMP production with low nM potency. We generated GPR110 knock-out (KO) mice, and confirmed that synaptamide-induced bioactivity is GPR110-dependent. GPR110 KO mice exhibited reduced synaptic protein expression and memory deficits, providing in vivo evidence that GPR110 is a functional synaptamide receptor for promoting brain development and function. The gpr110 expression profile indicated that this gene is primarily a developmental gene at least in the brain. During this review period, we tested whether there is a critical period for omega-3 fatty acid supplementation in premature infants that are deprived of prenatal accumulation of brain DHA. We found in a mouse model that deficits of brain DHA level, hippocampal synaptic protein expression and spatial learning and memory were normalized only when the diet was changed at an early age (3 weeks), although DHA loss in the brain can be reversed within two months of omega-3 supplementation at any age. This finding corroborates the significance of synaptamide/GPR110 signaling in neurodevelopment, and underscores the importance of DHA administration to high risk populations like preterm infants in the immediate perinatal period, since developmental deprivation of brain DHA may have a lasting impact on cognitive function if not corrected at an early age. Although synaptamide is known to be derived from DHA, the relationship between DHA and synaptamide concentration has not been established in humans. We further extended this study to establish the correlation between DHA and synaptamide concentration in human milk, plasma and cerebrospinal fluid. During this review period, we also investigated whether the mechanism for developmental neurite outgrowth, namely synaptamide/GPR110-mediated cAMP/PKA signaling, is applicable to axon regeneration after injury in both in vitro and in vivo models. In an in vitro model using primary cortical neurons grown in a two-compartment chamber system, we found that synaptamide can promote axon regrowth from axotomized neurons. To test the effect of synaptamide in vivo, we used a mouse model of eye injury employing optic nerve crush (ONC), which can serve as an experimental disease model for traumatic optic neuropathy or glaucoma. We observed that intravitreal administration of synaptamide immediately after ONC enables the robust regeneration of the lesioned optic nerve, leading to partial functional recovery of vision in adult mice. Our findings suggest that synaptamide may have therapeutic potential for optic nerve injury, offering a new possibility for clinical translation. We have previously demonstrated that DHA-derived synaptamide is a potent suppressor of neuroinflammation in an LPS-induced model, by enhancing cAMP/PKA signaling and inhibiting NF-kappaB activation. Since synaptamide is a substrate for fatty acid amide hydrolase (FAAH), and can be converted to DHA in vivo, the observed anti-inflammatory effect of synaptamide may be due to its hydrolysis product DHA. To test this possibility, we examined anti-inflammatory effects of synaptamide in FAAH KO mice where hydrolysis of synaptamide to DHA does not occur. Upon injection of LPS (1 mg/kg, .p.) followed by synaptamide at a lower level (2 mg/kg) than that used for WT (5 mg/kg), LPS-induced increase of proinflammatory cytokines was almost completely blocked, indicating that synaptamide suppresses the expression of pro-inflammatory mediators more potently in FAAH KO mice compared to WT. These results established that the inhibitory effect of synaptamide on neuroinflammation does not involve its hydrolysis to DHA. We also found that anti-inflammatory effect of synaptamide is GPR110-dependent. GPR110 blocking antibody abolished the anti-inflammatory effects of synaptamide in microglia cells. In vivo, synaptamide was no longer able to inhibit proinflammatory cytokine production increased by LPS in GPR110 KO mice. Synaptamide-mediated inhibition of microglia activation may not only serve as a new DHA-derived neuroprotective mechanism, but also provide a potential therapeutic strategy for controlling neuroinflammation and related neurodegenerative conditions. Previously, we developed a cell-based assay using the time-resolved fluorescence resonance energy transfer (TR-FRET) technology to detect Akt S473 phosphorylation and performed high throughput screening (HTS) for 373,868 Molecular Libraries Screening Center Network compounds in collaboration with NCATS. After primary and secondary screening, approximately 100 compounds were selected during the previous review period. The tertiary screening completed during this review period revealed 4-phenylquinolin-2(1H)-one (G7) as a specific inhibitor that effectively decreased Akt phosphorylation at both T308 and S473, and inhibited Akt kinase activity (IC50=6 M) and downstream signaling. G7 did not alter the activity of upstream kinases including PI3K, PDK1, and mTORC2 as well as closely related kinases that affect cell proliferation and survival such as SGK1, PKA, PKC, or ERK1/2. The inactivity of G7 on closely related kinases (e.g. PKA and PKC) suggests that G7 does not bind to the conserved ATP binding pocket of the kinases. In fact, kinase profiling efforts using KINOMEscan revealed that G7 does not bind to the active site of over 380 human kinases including Akt. When active Akt was incubated with ATP and a substrate peptide in the presence of 20 M G7, only negligible change of the substrate phosphorylation was observed, confirming that G7 did not interfere with the ATP or substrate binding. Direct interaction of G7 with the PH domain of Akt was evident based on the biomolecular interaction analysis using microscale thermophoresis (MST). The changes in the mobility of the purified Akt PH domain molecules in microscale temperature gradients upon addition of G7 indicated that G7 binds to the PH domain with a Kd of 8 M. However, G7 did not compete with PIP3 for binding to the PH domain. Instead, G7 binding caused a local conformational change in the PH domain probed by chemical cross-linking coupled to mass spectrometry as described previously. It is therefore concluded that G7 is an allosteric inhibitor that causes conformational change of Akt and hinders its phosphorylation by upstream kinases PDK1 and mTORC2. This notion was confirmed by the impaired in vitro phosphorylation of Akt by PDK1 at T308 or by MAPKAP kinase 2 which can phosphorylate S473 in vitro, after binding to G7. Considering that G7 does not affect the activity of PDK1 activity or MAPKAP kinase 2, hindered accessibility of these upstream kinases to T308 and S473 of Akt was the only plausible explanation for the impaired phosphorylation of Akt. We found that G7 inhibits the proliferation of Neuro 2A neuroblastoma cancer cells as effectively as the ATP-competitive PI3K inhibitor LY or mTORC2 inhibitor Torin1. However, when the 3T3 cells were confluent and no longer proliferating, considerably less inhibition was observed with G7 in comparison to LY or Torin1. These data suggest that G7 is less cytotoxic to non-cancerous cells while effectively inhibiting the proliferation of cancer cells, thus making it an attractive candidate to develop as an anti-cancer agent with reduced side effects. In conclusion, we have identified an allosteric Akt inhibitor with excellent selectivity and less toxicity, presenting a promising lead compound for further optimization and development of novel therapeutic agents against cancers.

Project Start
Project End
Budget Start
Budget End
Support Year
28
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Alcohol Abuse and Alcoholism
Department
Type
DUNS #
City
State
Country
Zip Code
Kim, Hee-Yong; Spector, Arthur A (2018) N-Docosahexaenoylethanolamine: A neurotrophic and neuroprotective metabolite of docosahexaenoic acid. Mol Aspects Med :
Huang, Bill X; Newcomer, Kenny; Kevala, Karl et al. (2017) Identification of 4-phenylquinolin-2(1H)-one as a specific allosteric inhibitor of Akt. Sci Rep 7:11673
Lozada, Luis E; Desai, Abhishek; Kevala, Karl et al. (2017) Perinatal Brain Docosahexaenoic Acid Concentration Has a Lasting Impact on Cognition in Mice. J Nutr 147:1624-1630
Sidhu, Vishaldeep K; Huang, Bill X; Desai, Abhishek et al. (2016) Role of DHA in aging-related changes in mouse brain synaptic plasma membrane proteome. Neurobiol Aging 41:73-85
Rashid, Mohammad Abdur; Kim, Hee-Yong (2016) N-Docosahexaenoylethanolamine ameliorates ethanol-induced impairment of neural stem cell neurogenic differentiation. Neuropharmacology 102:174-85
Park, Taeyeop; Chen, Huazhen; Kevala, Karl et al. (2016) N-Docosahexaenoylethanolamine ameliorates LPS-induced neuroinflammation via cAMP/PKA-dependent signaling. J Neuroinflammation 13:284
Lee, Ji-Won; Huang, Bill X; Kwon, HeungSun et al. (2016) Orphan GPR110 (ADGRF1) targeted by N-docosahexaenoylethanolamine in development of neurons and cognitive function. Nat Commun 7:13123
Spector, Arthur A; Kim, Hee-Yong (2015) Cytochrome P450 epoxygenase pathway of polyunsaturated fatty acid metabolism. Biochim Biophys Acta 1851:356-65
Huang, Bill X; Lee, Rachel; Akbar, Mohammed et al. (2015) Threonine 34 phosphorylation by phosphoinositide-dependent protein kinase 1 facilitates dissociation of Akt from the plasma membrane. Int J Biochem Cell Biol 64:195-201
Kim, Hee-Yong (2015) Phospholipids: a neuroinflammation emerging target. Nat Chem Biol 11:99-100

Showing the most recent 10 out of 33 publications