Schizophrenia is a complex and highly heterogeneous psychiatric disorder whose precise etiology remains elusive. While genome-wide association studies (GWAS) have identified risk genes, they have failed to determine if rare coding single nucleotide polymorphisms (nsSNPs) contribute in schizophrenia. Recently, two independent studies identified 12 rare, deleterious nsSNPS in the GRM1 gene, which encodes the metabotropic glutamate receptor subtype 1 (mGlu1), in schizophrenic patients. Here, we generated stable cell lines expressing the mGlu1 mutant receptors and assessed their pharmacology. Using both the endogenous agonist glutamate and the synthetic agonist DHPG, we found that several of the mutant mGlu1 receptors displayed a loss of function that was not due to a loss in plasma membrane expression. Due to a lack of mGlu1 positive allosteric modulators (PAM) tool compounds active at human mGlu1, we optimized a known mGlu4 PAM/mGlu1 NAM chemotype into a series of potent and selective mGlu1 PAMs by virtue of a double “molecular switch”. Employing mGlu1 PAMs from multiple chemotypes, we demonstrate that the mutant receptors can be potentiated by small molecules and in some cases efficacy restored to that comparable to wild type mGlu1 receptors, suggesting deficits in patients with schizophrenia due to these mutations may be amenable to intervention with an mGlu1 PAM. However, in wild type animals, mGlu1 negative allosteric modulators (NAMs) are efficacious in classic models predictive of antipsychotic activity, whereas we show that mGlu1 PAMs have no effect to slight potentiation in these models. These data further highlight the heterogeneity of schizophrenia and the critical role of patient selection strategies in psychiatric clinical trials to match genotype with therapeutic mechanism.

Hepatitis C virus (HCV) induces microtubule aggregates in infected hepatocytes. To determine if cytoskeletal elements are important for HCV RNA synthesis, we examined the effect of cytoskeleton inhibitors on HCV replicon transcription in Huh7 cells. The data demonstrate that HCV replication complex-mediated RNA synthesis requires microtubule and actin polymerization.Hepatitis C virus (HCV) infection is the leading cause of liver transplantation in the United States, with sequelae including liver fibrosis, cirrhosis, and hepatocellular carcinoma (reviewed in reference 19). Identified in 1989 as a plus-strand RNA virus (4), HCV causes an infection that was originally distinguished by its characteristic induction of microtubule (MT) aggregates in infected hepatocytes, implicating MTs in HCV-associated disease (2,11,23,28,29,32,36). Studies with a related flavivirus (Kunjin virus) have demonstrated that MT aggregates similar to those induced by HCV are also important for Kunjin RNA synthesis (15,22). Moreover, MT paracrystals have recently been detected in cultured cells transfected with an HCV subgenomic replicon (21). Taken together, these studies suggest that cytoskeletal elements may be required for HCV replication. Yet, despite the historical emphasis on MTs and HCV from a clinical perspective, the role of MTs in the HCV life cycle at a molecular level remains poorly understood.To determine if HCV RNA synthesis requires functional actin or MT networks, we examined the effects of cytoskeleton inhibitors on the efficiency of HCV RNA synthesis in the HCV replicon cell system ( Fig. 1) (18). In this system, Huh7 cells stably transfected with an HCV replicon RNA are used to mimic the RNA synthesis that occurs in an ongoing, persistent infection with HCV. Because the replicon construct encodes a neomycin resistance gene for G418 (Geneticin) selection, HCV RNA synthesis in the replicon cells can be detected by quantitative PCR using a primer-probe set specific for the neomycin sequence. We have validated this approach by demonstrating a dose-dependent decrease in replicon RNA levels upon alpha interferon treatment (data not shown), using alpha interferon concentrations similar to those cited previously by other laboratories (1, 10, 18). In addition, we have confirmed that transcription of the replicon in our HCV replicon cells is resistant to actinomycin D, as originally demonstrated by Lohmann et al. (18), verifying that transcription of replicon RNA is specific to the RNA-dependent RNA polymerase activity of the HCV replication complex. A single HCV replicon cell line-a serially passaged line originally generated by stable transfection of Huh7 cells with a replicon with a sequence identical to that used by Lohmann et al. (18) (Fig. 1)-was used for all experiments.Because vinblastine sulfate (VS) is a well-characterized inhibitor of MT polymerization and has been shown to alter Kunjin virus replication (15, 22), we first determined the effect of VS on HCV replicon RNA synthesis. One day prior to addition of the inhib...

Previous preclinical work has demonstrated the therapeutic potential of antagonists of the group II metabotropic glutamate receptors (mGlus). Still, compounds that are selective for the individual group II mGlus (mGlu2 and mGlu3) have been scarce. There remains a need for such compounds with the balance of properties suitable for convenient use in a wide array of rodent behavioral studies. We describe here the discovery of a selective mGlu3 NAM 106 (VU0650786) suitable for in vivo work. Compound 106 is a member of a series of 5-aryl-6,7-dihydropyrazolo[1,5-a]pyrazine-4(5H)-one compounds originally identified as a mGlu5 positive allosteric modulator (PAM) chemotype. Its suitability for use in rodent behavioral models has been established by extensive in vivo PK studies, and the behavioral experiments presented here with compound 106 represent the first examples in which an mGlu3 NAM has demonstrated efficacy in models where prior efficacy had previously been noted with nonselective group II antagonists.

ABSTRACT:Negative allosteric modulation (NAM) of metabotropic glutamate receptor subtype 5 (mGlu 5 ) represents a therapeutic strategy for the treatment of childhood developmental disorders, such as fragile X syndrome and autism. VU0409106 emerged as a lead compound within a biaryl ether series, displaying potent and selective inhibition of mGlu 5 . Despite its high clearance and short half-life, VU0409106 demonstrated efficacy in rodent models of anxiety after extravascular administration. However, lack of a consistent correlation in rat between in vitro hepatic clearance and in vivo plasma clearance for the biaryl ether series prompted an investigation into the biotransformation of VU0409106 using hepatic subcellular fractions. An in vitro appraisal in rat, monkey, and human liver S9 fractions indicated that the principal pathway was NADPH-independent oxidation to metabolite M1 (؉16 Da). Both raloxifene (aldehyde oxidase inhibitor) and allopurinol (xanthine oxidase inhibitor) attenuated the formation of M1, thus implicating the contribution of both molybdenum hydroxylases in the biotransformation of VU0409106. The use of 18 Olabeled water in the S9 experiments confirmed the hydroxylase mechanism proposed, because 18O was incorporated into M1 (؉18 Da) as well as in a secondary metabolite (M2; ؉36 Da), the formation of which was exclusively xanthine oxidase-mediated. This unusual dual and sequential hydroxylase metabolism was confirmed in liver S9 and hepatocytes of multiple species and correlated with in vivo data because M1 and M2 were the principal metabolites detected in rats administered VU0409106. An in vitro-in vivo correlation of predicted hepatic and plasma clearance was subsequently established for VU0409106 in rats and nonhuman primates.