INTRODUCTIONFunctionally selective antagonists: The sequencing of the human genome has revealed that the GPCRsuperfamily represents one of the largest protein families in the human genome with 367 members (excluding the olfactory and tastant receptors; (Vassilatis et al., 2003). Family A the rhodopsin-like GPCR's-representsthe largest group with 274 estimated members (Kroeze et al., 2003b). GPCRs remain one of the most viabletargets for drug discovery efforts because of their easy 'drugability1 (Hopkins and Groom, 2002; Armbrusterand Roth, 2005). Indeed, when considering psychiatric drugs, nearly every psychiatric indication is populatedby drugs which primarily target GPCRs. Thus, atypical antipsychotic drugs target 5-HT2A-serotonin and D2-dopamine receptors in addition to a multiplicity of other GPCRs (Roth et al., 2004b) leading to both sideeffects(Kroeze et al., 2003a) and enhanced therapeutic efficacies (Roth et al., 2004a; Davies et ai., 2005a).Although many antidepressants preferentially target neurotransmitter transporters, they also have substantial'off-target' actions at 5-HT2A and 5-HT2C receptors (Palvimaki et al., 1996; Dziedzicka-Wasylewska et al.,2006). As well, the atypical antidepressants (e.g. mirtazepine, mianserin, nefazadone) exert their actions viainteractions with 5-HT2-family receptors and possibly other receptors (see http://pdsp.med.unc.edu/kidb.php ).Finally, several antidepressant, anti-insomnia, anti-anxiety and antipsychotic drugs currently in developmenttarget mainly GPCRs particularly 5-HT2A receptors where they function as inverse agonists (Roth et al.,2004b; Roth and Kroeze, in press; Roth, 2006).According to classical concepts of pharmacology when inverse agonists are chronically administered upregulationof receptor number and super-sensitivity occurs (Urban et al., 2007). 5-HT2-family serotoninreceptors are unusual in that inverse agonists paradoxically induce internalization and down-regulation in vitroand in vivo (Peroutka and Snyder, 1980; Blackshear and Sanders-Bush, 1982; Berry et al., 1996; Willins et al.,1999; Bhatnagar et al., 2001). A few years ago, we reviewed this general area and found nearly 100 exampleswherein 5-HT2A antagonists of varying specificities induced receptor down-regulation in vivo (Gray and Roth,2001). The mechanism responsible for receptor down-regulation appears to be receptor internalization andsubsequent degradation (Willins et al., 1999; Bhatnagar et al., 2001) and not due to changes in receptorsynthesis (Newton and Elliott, 1997; Roth and Ciaranello, 1991). For many GPCRs receptor internalizationoccurs via one of two distinct pathways: (a) internalization via endosomes (Ferguson et al., 1996) or (b) viacaveolae (Roettger et al., 1995). We have found internalized 5-HT2A receptors to be associated with bothendosomes (Berry et al., 1996; Bhatnagar et al., 2001) and caveolae (Bhatnagar et al., 2004), although theextent to which each pathway is used likely depends on the cellular milieu.The classical pathway for GPCR down-regulation involves arrestin binding and then translocation of thearrestin-receptor complex to clathrin-coated pits followed by internalization and either recycling to the plasmamembrane or down-regulation (Ferguson, 2001); (Gray and Roth, 2002); Fig 1). Many proteins are involved inthe post-endocytic decision-making process including GASP, SNX-1 and ubiquitin ligases. For 5-HT2Areceptors it is clear that internalization may occur via both arrestin-sensitive and insensitive pathways(Bhatnagar et al., 2001; Gray et al., 2001b; Hanley and Hensler, 2002; Gray et al., 2003) in vitro althoughwhich pathways are essential in vivo is unknown.A major goal of this application is to understand how the ability of various 5-HT2A antagonists to induceinternalization and down-regulation in vitro and in vivo affects their ability to treat schizophrenia and relateddisorders. As most of the previous preclinical studies were performed with psychiatric medications whichtarget other GPCRs and neurotransmitter transporters (Gray and Roth, 2001) it is impossible to ascribe anybeneficial action to effects on 5-HT2A receptor expression. We will evaluate relatively selective 5-HT2antagonists which differ in their abilities to induce receptor internalization and down-regulation.Extensive preclinical studies indicate that 5-HT2A antagonists may prove beneficial in conditions ofdopamine excess (Barr et al., 2004; Pehek et al., 2001) and NMDA-receptor hypofunction (Varty et al., 1999)as has been postulated to occur in schizophrenia. Salutary effects on excessive dopamine release are likelymediated via both midbrain and cortical 5-HT2A receptors (Nocjar et al., 2002; Pehek et al., 2006) whilebeneficial effects on glutamatergic function are likely mediated via direct actions on pyramidal neurons (Willinset al., 1997, Martin-Ruiz et al., 2001, Gonzalez-Maeso et al., 2007). Clinically, mixed results of 5-HT2Apreferringantagonists in schizophrenia have appeared. Early promising development of ritanserin (a 5-HT2A/2C antagonist) (Strauss and Klieser, 1991; Krystal et al., 1992) was halted due to cardiovascular safetyissues. A large study, which has never been completely published, with M100907 showed beneficial effectsgreater than placebo but less than haloperidol (de Paulis, 2001; Potkin et al., 2001). A more recent study withSR46349B showed effects greater than placebo and equivalent to haloperidol (Meltzer et al., 2004). Theseequivalent results are reminiscent of a large trial comparing several atypical antipsychotic drugs andperphenazine (Lieberman et al., 2005). Currently, several 5-HT2A antagonists are in clinical trials as standaloneor add-on medications for schizophrenia of which ACP-103 is furthest along. Phase II results for ACP-103 demonstrated that co-therapy with low-dose risperidone was (a) more efficacious that risperidone alone;(b) was faster in onset; and (c) was associated with fewer side-effects (www.acadiapharm.com/programs/schizophrenia.htm ) as predicted by preclinical studies (Bayes et al., 2006; Vanover etal., 2006) Clinical trial data with selective 5-HT2A antagonists (SR46349B, M100907, ACP-103) as standalonetherapies also reveals no significant cardiovascular or metabolic side-effects (Meltzer et al., 2004; Potkinet al., 2001) actions which distinguish them from all currently available atypical antipsychotic drugs.Intriguingly, SR46349B, along with MDL11,939, are among the only 5-HT2A antagonists which causereceptor supersensitivity and up-regulation in vitro and in vivo with chronic administration (Rinaldi-Carmona etal., 1993; Rinaldi-Carmona et al., 1994; Gray and Roth, 2001; Aloyo et al., 2001; Harvey et al., 2004).SR46349B is also the only known 5-HT2A antagonist which has been demonstrated to be effective in alarge-scale double-blind placebo controlled trial, suggesting that its unique ability to induce receptorup-regulation might be responsible for its clinical utility. It is unknown to what extent other selective 5-HT2A antagonists induce receptor redistribution in vivo although our prior studies with M100907 indicated thatit efficiently promotes receptor internalization. As previously mentioned, receptor regulation is profoundlymodulated by cellular milieu (Gray and Roth, 2001; Gray et al., 2001 a) and it is important that we will beprofiling our compounds in native environments in vitro and in vivo.In this project we will examine the hypothesis that 5-HT2A inverse agonists which differ in theirabilities to induce receptor redistribution in vivo will also differ in antipsychotic efficacy in various invivo models. Because 5-HT2A receptors modulate the activity of glutamatergic pyramidal neurons(Aghajanian and Marek, 1997) (Martin-Ruiz et al., 2001) and cortical dopaminergic inputs (Nocjar et al.,2002; Pehek et al., 2005) either up- or down-regulation of 5-HT2A receptor number could conceivablyhave salutary effects on psychosis (Roth and Xia, 2004) and cognition (Harvey, 2003; Harvey et al.,2004).Designer receptors to probe the in vivo relevance of functional selectivity: It is likely that the actions of afunctionally-selective ligand ultimately depends on the relative ability of a ligand to activate signal transductionpathways in discrete neuronal populations. Thus, in Fig 2 (top panels) different partial agonists have the abilityto differentially activate the same receptor to a different extent depending upon the cellular milieu. In the panelon the left, Partial agonist 'A' selectively activates the blue neurons and the summed action would reflect to asomewhat greater extent the actions of these neurons (A10 dopamine neurons for example). In the panel onthe right, Partial agonist 'B' selectively activates the green neurons and the summed activity would reflect to asomewhat greater extent the actions of these neurons (A9 dopamine neurons, for example). As can be seen,however, it is likely that any given drug will have some activity at both neuronal populations and that thesummed behavioral activity will represent an integration response between the two neuronal populations.Currently, there is no technology which allows us to distinguish to what extent the behavioral actions of afunctionally-selective ligand depend upon the discrete activation of different neuronal populations.The approach we've taken has been to create a technological platform which will allow us to, ultimately,precisely regulate the activity of discrete neuronal populations via drug-like compounds. What we havebegun to do is to design families of receptors which can only be activated by pharmacologically-inert ligandsand then to selectively express them in discrete neuronal populations. In the case above, these engineeredreceptors would induce a response upon activation that was solely 'blue' or 'green' in color. One couldimagine, for instance, selectively modulating the activity of A9 or A10 dopamine neurons and determine thefunctional readouts of these sorts of activities. For this project, we will provide proof-of-concept studieswhereby chemical switchs which selectively modulates the activity of single neurons or populations ofneurons in vivo will be created.
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