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Summary Many tumors become addicted to autophagy for survival, suggesting inhibition of autophagy as a potential broadly-applicable cancer therapy. ULK1/Atg1 is the only serine/threonine kinase in the core autophagy pathway and thus represents an excellent drug target. Despite recent advances in the understanding of ULK1 activation by nutrient deprivation, how ULK1 promotes autophagy remains poorly understood. Here, we screened degenerate peptide libraries to deduce the optimal ULK1 substrate motif and discovered fifteen phosphorylation sites in core autophagy proteins that were verified as in vivo ULK1 targets. We utilized these ULK1 substrates to perform a cell-based screen to identify and characterize a potent ULK1 small molecule inhibitor. The compound SBI-0206965 is a highly selective ULK1 kinase inhibitor in vitro and suppressed ULK1-mediated phosphorylation events in cells, regulating autophagy and cell survival. SBI-0206965 greatly synergized with mTOR inhibitors to kill tumor cells, providing a strong rationale for their combined use in the clinic.

Highly selective positive allosteric modulators (PAMs) of metabotropic glutamate receptor subtype 5 (mGluR5) have emerged as a potential approach to treat positive symptoms associated with schizophrenia. mGluR5 plays an important role in both long term potentiation (LTP) and long term depression (LTD), suggesting that mGluR5 PAMs may also have utility in improving impaired cognitive function. However, if mGluR5 PAMs shift the balance of LTP and LTD or induce a state in which afferent activity induces lasting changes in synaptic function that are not appropriate for a given pattern of activity, this could disrupt rather than enhance cognitive function. We determined the effect of selective mGluR5 PAMs on induction of LTP and LTD at the Schaffer collateral – CA1 synapse in the hippocampus. mGluR5-selective PAMs significantly enhanced threshold theta burst stimulation (TBS)-induced LTP. In addition, mGluR5 PAMs enhanced both DHPG-induced LTD and LTD induced by delivery of paired-pulse low frequency stimulation. Selective potentiation of mGluR5 had no effect on LTP induced by suprathreshold TBS or saturated LTP. The finding that potentiation of mGluR5-mediated responses to stimulation of glutamatergic afferents enhances both LTP and LTD supports the hypothesis that activation of mGluR5 by endogenous glutamate contributes to both forms of plasticity. Furthermore, two systemically active mGluR5 PAMs enhanced performance in the Morris water maze, a measure of hippocampus-dependent spatial learning. Discovery of small molecules that enhance both LTP and LTD in an activity-appropriate manner demonstrates a unique action on synaptic plasticity that may provide a novel approach for treatment of impaired cognitive function.

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5- (2) but not all hallucinogens (3, 4) and is the target of a number of commonly prescribed therapeutic agents, including atypical antipsychotics, antidepressants, and anxiolytics (5). As with other GPCRs, elucidating the mechanisms of signal transduction and regulation for 5-HT 2A receptors is likely to be of great relevance for the rational design of novel medications (6 -8). In particular, one point where the regulation of GPCR signaling converges is the endocytic pathway (8, 9), and therefore, this pathway is likely to play a role in various functions involving the 5-HT 2A receptor.Prototypically, GPCRs are internalized via an integrated process involving arrestins and G-protein receptor kinases, a process that has been most elegantly elucidated for the ␤-adrenergic receptor (10), although many other proteins are also involved in the intracellular trafficking of GPCRs (6, 11). For example, we have uncovered a cell type-specific, arrestin-independent, dynamin-dependent mechanism of 5-HT 2A receptor regulation (12, 13). In light of recent studies demonstrating that ␤-adrenergic receptors, in particular, are associated with caveolae and caveolin in their native milieu (14, 15), we hypothesized that 5-HT 2A receptors might also be associated with caveolae-enriched membrane specializations and that this interaction might functionally modulate 5-HT 2A -mediated signal transduction.Caveolae are small flask-shaped invaginations of the plasma membrane (16) containing high levels of cholesterol and glycosphingolipids and are initially characterized by the presence of the protein Cav-1 (17). Caveolae differ biochemically from other specialized subdomains of the plasma membrane (16). A family of caveolin proteins has been identified that includes caveolin-1, caveolin-2, and caveolin-3 (Cav-1, -2, and -3, respectively (18)), with Cav-1 and Cav-2 being expressed ubiquitously. Caveolae function is critically dependent on Cav-1 because caveolae are not formed in Cav-1 knockout mice (19); conversely, caveolin-deficient cells acquire caveolae when transfected with .In prior studies, we found that caveolae were not normally required for the membrane targeting of 5-HT 2A receptors heterologously expressed in either NIH 3T3 (21) or HEK-293 (12) cells, although a recent study (22) indirectly implicated caveolae in 5-HT 2A -mediated signal transduction in vascular smooth muscle cells. Even though prior studies have not implicated Cav-1 as a modulator of 5-HT 2A receptor signaling, Cav-1 has been indirectly implicated as a regulator of GPCR signaling via unknown mechanisms (19).Here we report that both transiently transfected 5-HT 2A

Salvinorin A is a naturally occurring hallucinogenic diterpenoid from the plant Salvia divinorumthat selectively and potently activates kappa-opioid receptors (KORs). Salvinorin A is unique in that it is the only known lipid-like molecule that selectively and potently activates a G-protein coupled receptor (GPCR), which has as its endogenous agonist a peptide; salvinorin A is also the only known non-nitrogenous opioid receptor agonist. In this paper, we identify key residues in KORs responsible for the high binding affinity and agonist efficacy of salvinorin A. Surprisingly, we discovered that salvinorin A was stabilized in the binding pocket by interactions with tyrosine residues in helix 7 (Tyr313 and Tyr320) and helix 2 (Tyr119). Intriguingly, activation of KORs by salvinorin A required interactions with the helix 7 tyrosines Tyr312, Tyr313, and Tyr320 and with Tyr139 in helix 3. In contrast, the prototypical nitrogenous KOR agonist U69593 and the endogenous peptidergic agonist dynorphin A (1-13) showed differential requirements for these three residues for binding and activation. We also employed a novel approach, whereby we examined the effects of cysteine-substitution mutagenesis on the binding of salvinorin A and an analogue with a free sulfhydryl group, 2-thiosalvinorin B. We discovered that residues predicted to be in close proximity, especially Tyr313, to the free thiol of 2-thiosalvinorin B when mutated to Cys showed enhanced affinity for 2-thiosalvinorin B. When these findings are taken together, they imply that the diterpenoid salvinorin A utilizes unique residues within a commonly shared binding pocket to selectively activate KORs.

M1 muscarinic acetylcholine receptors (mAChRs) represent a viable target for treatment of multiple disorders of the central nervous system (CNS) including Alzheimer’s disease and schizophrenia. The recent discovery of highly selective allosteric agonists of M1 receptors has provided a major breakthrough in developing a viable approach for discovery of novel therapeutic agents that target these receptors. Here, we describe the characterization of two novel M1 allosteric agonists VU0357017 and VU0364572 that display profound differences in their efficacy in activating M1 coupling to different signaling pathways including Ca++ and β-arrestin responses. Interestingly, the ability of these agents to differentially activate coupling of M1 to specific signaling pathways leads to selective actions on some but not all M1-mediated responses in brain circuits. These novel M1 allosteric agonists induced robust electrophysiological effects in rat hippocampal slices but showed lower efficacy in striatum and no measureable effects on M1-mediated responses in medial prefrontal cortical pyramidal cells in mice. Consistent with these actions, both M1 agonists enhanced acquisition of hippocampal-dependent cognitive function but did not reverse amphetamine-induced hyperlocomotion in rats. Together, these data reveal that M1 allosteric agonists can differentially regulate coupling of M1 to different signaling pathways and this can dramatically alter the actions of these compounds on specific brain circuits important for learning and memory and psychosis.

Previous studies suggest that selective antagonists of specific subtypes of muscarinic acetylcholine receptors (mAChRs) may provide a novel approach for the treatment of certain central nervous system (CNS) disorders, including epileptic disorders, Parkinson's disease, and dystonia. Unfortunately, previously reported antagonists are not highly selective for specific mAChR subtypes, making it difficult to definitively establish the functional roles and therapeutic potential for individual subtypes of this receptor subfamily. The M 1 mAChR is of particular interest as a potential target for treatment of CNS disorders. We now report the discovery of a novel selective antagonist of M 1 mAChRs, termed VU0255035 [N-(3-oxo-3-(4-(pyridine-4-yl) VU0255035 has excellent brain penetration in vivo and is efficacious in reducing pilocarpine-induced seizures in mice. We were surprised to find that doses of VU0255035 that reduce pilocarpine-induced seizures do not induce deficits in contextual freezing, a measure of hippocampus-dependent learning that is disrupted by nonselective mAChR antagonists. Taken together, these data suggest that selective antagonists of M 1 mAChRs do not induce the severe cognitive deficits seen with nonselective mAChR antagonists and could provide a novel approach for the treatment certain of CNS disorders.Muscarinic acetylcholine receptors (mAChRs) are G protein-coupled receptors (GPCRs) that are widely expressed in the central nervous system (CNS) and are critical for the modulation of activity in multiple brain circuits (Langmead et al., 2008). Previous studies suggest that mAChRs play important roles in a broad range of CNS functions, including