Previously, we demonstrated a novel molecular mechanism for Akt activation where Akt interaction with PS-binding residues, particularly in the regulatory domain, is critical for Akt phosphorylation at S473 by mTORC2. Phosphorylation at S473 is an important modulator of Akt activation, which can serve as a new target, different from well-recognized PIP3- or ATP-binding, for drug development. As an effort to identify selective Akt inhibitors based on this new target, we performed high throughput screening (HTS) for approximately the 400,000 MLSCN (Molecular Libraries Screening Center Network) compound collections in collaboration with NCGC. Among high probability hits from an automated HTS, we narrowed down the list to compounds that only showed substantial and reproducible inhibition of Akt S473 phosphorylation confirmed by western blot analysis, without an effect on PI3K, PDK1, and SGK1. During this period, we further narrowed down the list to two compounds based upon Akt activity assays and continued to evaluate for cell viability. We found only one compound did not significantly affect the cell viability at 50 microM, and thus was selected as a lead compound for developing non-toxic Akt inhibitors. Despite inhibiting S473 phosphorylation, this compound did not affect fully activated Akt, indicating that the target is not the substrate- or ATP-binding site of Akt. The interaction between the lead compound and inactive Akt was probed by chemical cross-linking and mass spectrometry developed in our laboratory. This compound did not alter the crosslinking profile of Akt, or the interdomain conformational change of Akt induced by membrane interaction. It is possible that this compound causes only a subtle local conformational change of Akt which could not be detected by the chemical cross-linking approach. It is also possible that this compound does not interact with Akt directly, and instead it inhibits mTORC2, the upstream activator of Akt, leading to the inhibition of Akt. The mTORC2 activity assay currently available yielded no conclusive results. To determine the target for lead optimization, we are in the process of developing an mTORC2 assay method while evaluating local Akt-inhibitor interaction profile using hydrogen/deuterium exchange mass spectrometry. The small molecules that inhibit specifically Akt S473 phosphorylation/mTORC2 activity thus identified will not only serve as valuable research tools but also may have significant therapeutic potential with fewer side effects, especially for conditions involving hyperactive Akt signaling such as cancer and Alzheimers disease. We have previously demonstrated that DHA metabolism to N-docosahexaenoylethanolamine (synaptamide) is a significant endogenous mechanism for promoting neurogenesis, neuritogenesis, synaptogensis in a cAMP dependent manner. During this period, we demonstrate that orphan G-protein coupled receptor 110 (GPR110, ADGRF1) is the synaptamide receptor, mediating synaptamide-induced neurogenesis and neurite outgrowth in a cAMP-dependent manner. Mass spectrometry-based proteomic characterization and cellular fluorescence imaging probed by chemical analogues of synaptamide revealed specific binding of GPR110 to synaptamide. Synaptamide binding triggered cAMP production with low nM potency. Disruption of this binding or silencing GPR110 abolished while GPR110 overexpression enhanced synaptamide-induced neurogenesis and neurite outgrowth. GPR110 was highly expressed in neural stem cells and primary neurons in culture as well as fetal brains but its expression in the brain decreased significantly after birth. Nevertheless, substantial GPR110 expression remained in the adult hippocampal detate gyrus area where neurogenesis is known to occur throughout life. GPR110 deorphanized as a functional synaptamide receptor provides a novel target for neurodevelopmental control and offers new insight into mechanisms by which omega-3 fatty acids promote brain development and function. Axonogenesis, a process for the establishment of neuron connectivity, is central to brain function. During this period we tested potential interactions of synaptamide with neurodevelopmental and morphogen pathways. We found that synaptamide increased the average axon length, inhibited Gli1 transcription and Sonic Hedgehog (Shh) target gene expression while inducing cAMP elevation. Similar effects were produced by cyclopamine, a regulator of the Shh pathway. Conversely, Shh antagonized elevation of cAMP and blocked the synaptamide-mediated increase in axon length. Activation of Shh pathway by a Smoothened (SMO) agonist SAG or overexpression of SMO did not inhibit axon growth mediated by synaptamide or cyclopamine. Instead, adenylate cyclase inhibitor SQ22536 abolished synaptamide-mediated axon growth indicating requirement of cAMP elevation for this process. Our findings establish that synaptamide promotes axon growth while Shh antagonizes synaptamide-mediated cAMP elevation and axon growth by a SMO-independent, non-canonical pathway. In addition to developmental axon growth, we also tested whether synaptamide has positive effects on axon regeneration after injury. To investigate the axon regeneration in vitro, we first established an axon injury model using a microfluidic two-chamber system. In this model, cortical neurons were seeded in one side of the chamber and axons were grown through microfluidic grooves to reach the other side of the chamber. Axotomy was performed by scratching and rapid aspiration. The compartmentalized neuron culture system allowed evaluation of synaptamide effects separately on axons and neuronal soma from which they originate. Using this in vitro model, we found that synaptamide treatment dramatically induces axon regrowth after axotomy. For extension of these findings to in vivo axon regeneration, we established the optic nerve crush (ONC) mouse model, and synaptamide was administered by intravitreous injections into the eye. After 4 weeks, we analyzed the synaptamide effect by immunofluorescence staining. The synaptamide injected group showed significantly more axon growth in comparison to the DMSO-treated control group. These findings suggest therapeutic potential of synaptamide for axon regeneration after injury in the central nervous system (CNS). During this period, we have also established in vivo significance of the synaptamide-induced neurogenesis. To evaluate the effect of synaptamide on neurogenesis in offspring pup brains, pregnant mice were injected with BrdU (i.p. 150 mg/kg, on E12) and subsequently with synaptamide (i.p. 20 mg/kg, on E14 and E16). The delivery of synaptamide to fetal brains was verified by maternal injection of d4-synaptamide on E14 (i.p. 20 mg/kg) followed by mass spectrometric analysis. The expression of fatty acid amide hydrolase (FAAH) was negligible in fetal brain and increased only after birth, explaining the considerable level of d4-synaptamide detected in fetal brains. Both FACS and microscopic analyses of BrdU- and NeuN-positive nuclei from 3 day old pup brains consistently indicated that neurogenesis in offspring brains is significantly increased by the maternal supplement of synaptamide to the fetal brains. Using the same approach, we also found that prenatal exposure to ethanol significantly impairs fetal neurogenesis while synaptamide ameliorates ethanol-impaired in vivo neurogenesis during development. We continued to develop an isotope-assisted mass spectrometric metabolite profiling method using uniformly labeled 13C-DHA and high resolution mass spectormetry. The metabolites formed in cortical cultures, microglia and human granulocytes are being investigated to find novel DHA metabolites involved in neuronal cell function and neuroinflammation.
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