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Extensive evidence supports the hypothesis that deficits in inhibitory GABA transmission in the prefrontal cortex (PFC) may drive pathophysiological changes underlying symptoms of schizophrenia that are not currently treated by available medications, including cognitive and social impairments. Recently, the mGlu

Leishmania parasites are the causative agents of a wide spectrum of human diseases. The clinical manifestations of leishmaniasis range from self-healing skin lesions to fatality. The World Health Organization has classed leishmaniasis as a category 1 neglected tropical disease. Leishmaniasis represents a major international health challenge, affecting 12 million people per year and with nearly 310 million people at risk. The first-line chemotherapies used to treat leishmaniasis are intravenous pentavalent antimonials; however, these drugs are highly toxic. As the use of oral treatment options such as paromomycin and miltefosine has increased, the incidence of disease relapse has increased and drug resistance to antimonials has developed, emphasizing the importance of identifying new chemotherapies. A novel, target-free fluorometric high-throughput screen with an average Z-score of 0.73 +/- 0.13 has been developed to identify small molecules with antileishmanial activity. Screening of 10,000 small molecules from the ChemBridge DIVER-set™ library cassette #5 yielded 210 compounds that killed 80% of parasites, resulting in a hit rate of 2.1%. One hundred and nine molecular scaffolds were represented within the hit compounds, and one scaffold that exhibited potent antileishmanial activity was 2,4-diaminoquinazoline. Host cell toxicity was determined prior to in-vitro infection of human THP-1 macrophages with Leishmania donovani mCherry expressing promastigotes; successful drug treatment was considered when the half maximal inhibitory concentration was10 µM. BALB/c mice were infected with Leishmania major mCherry promastigotes and treated with small molecules that were successful during in-vitro infections. Several small molecules tested were as efficacious at resolving cutaneous leishmaniasis lesions in mice as known antimonial treatments.

This letter describes progress towards an M4 PAM preclinical candidate inspired by an unexpected aldehyde oxidase (AO) metabolite of a novel, CNS penetrant thieno[2,3-c]pyridine core to an equipotent, non-CNS penetrant thieno[2,3-c]pyrdin-7(6H)-one core. Medicinal chemistry design efforts yielded two novel tricyclic cores that enhanced M4 PAM potency, regained CNS penetration, displayed favorable DMPK properties and afforded robust in vivo efficacy in reversing amphetamine-induced hyperlocomotion in rats.

This letter describes progress towards an M4 PAM preclinical candidate that resulted in the discovery of VU6005806/AZN-00016130. While the thieno[2,3-c]pyridazine core has been a consistent feature of key M4 PAMs, no work had previously been reported with respect to alternate functionality at the C3 position of the pyridazine ring. Here, we detail new chemistry and analogs that explored this region, and quickly led to VU6005806/AZN-00016130, which was profiled as a putative candidate. While, the β-amino carboxamide moiety engendered solubility limited absorption in higher species precluding advancement (or requiring extensive pharmaceutical sciences formulation), VU6005806/AZN-00016130 represents a new, high quality preclinical in vivo probe.

This letter describes a focused exercise to explore the role of the β-amino carboxamide moiety found in all of the first generation M4 PAMs and question if the NH2 group served solely to stabilize an intramolecular hydrogen bond (IMHB) and enforce planarity. To address this issue (and to potentially find a substitute for the β-amino carboxamide that engendered P-gp and contributed to solubility liabilities), we removed the NH2, generating des-amino congeners and surveyed other functional groups in the β-position. These modifications led to weak M4 PAMs with poor DMPK properties. Cyclization of the β-amino carboxamide moiety by virtue of a pyrazole ring re-enforced the IMHB, led to potent (and patented) M4 PAMs, many as potent as the classical bicyclic β-amino carboxamide analogs, but with significant CYP1A2 inhibition. Overall, this exercise indicated that the β-amino carboxamide moiety most likely facilitates an IMHB, and is essential for M4 PAM activity within classical bicyclic M4 PAM scaffolds.

Niemann-Pick disease type C1 (NPC1) is a rare genetic cholesterol storage disorder caused by mutations in the NPC1 gene. Mutations in this transmembrane late endosome protein lead to loss of normal cholesterol efflux from late endosomes and lysosomes. It has been shown that broad spectrum histone deacetylase inhibitors (HDACi's) such as Vorinostat correct the cholesterol accumulation phenotype in the majority of NPC1 mutants tested in cultured cells. In order to determine the optimal specificity for HDACi correction of the mutant NPC1s, we screened 76 HDACi's of varying specificity. We tested the ability of these HDACi's to correct the excess accumulation of cholesterol in patient fibroblast cells that homozygously express NPC1 I1061T , the most common mutation. We determined that inhibition of HDACs 1, 2, and 3 is important for correcting the defect, and combined inhibition of all three is needed to achieve the greatest effect, suggesting a need for multiple effects of the HDACi treatments. Identifying the specific HDACs involved in the process of regulating cholesterol trafficking in NPC1 will help to focus the search for more specific druggable targets.

Further optimization of the VU0486321 series of highly selective and CNS-penetrant mGlu

This letter describes synthesis and evaluation of two series of dual mGlu2/mGlu3 positive allosteric modulators with moderate mGlu3 potency and robust mGlu2 potency in thallium flux assays. These compounds were profiled their ability to modulate mGlu3-mediated signaling in central neurons by co-application of a selective mGlu2 NAM to isolate mGlu3-selective effects. Using acute mouse brain slices from the prefrontal cortex, potentiation of group II mGlu receptor agonist Ca2+ signaling in PFC pyramidal cells with either the dual mGlu2/mGlu3 PAM 16e or 23d demonstrated effects mediated selectively via mGlu3.

This letter describes the further chemical optimization of the 5-amino-thieno[2,3-c]pyridazine series (VU0467154/VU0467485) of M4 positive allosteric modulators (PAMs), developed via iterative parallel synthesis, culminating in the discovery of the non-human primate (NHP) in vivo tool compound, VU0476406 (8p). VU0476406 is an important in vivo tool compound to enable translation of pharmacodynamics from rodent to NHP, and while data related to a Parkinson’s disease model has been reported with 8p, this is the first disclosure of the optimization and discovery of VU0476406, as well as detailed pharmacology and DMPK properties.

This letter describes the chemical optimization of a novel series of M4 positive allosteric modulators (PAMs) based on a 5-amino-thieno[2,3-c]pyridazine core, developed via iterative parallel synthesis, and culminating in the highly utilized rodent in vivo tool compound, VU0467154 (5). This is the first report of the optimization campaign (SAR and DMPK profiling) that led to the discovery of VU0467154, and details all of the challenges faced in allosteric modulator programs (steep SAR, species differences in PAM pharmacology and subtle structural changes affecting CNS penetration).

Herein, we report the structure–activity relationships within a series of potent, selective, and orally bioavailable muscarinic acetylcholine receptor 4 (M4) positive allosteric modulators (PAMs). Compound 6c (VU0467485) possesses robust in vitro M4 PAM potency across species and in vivo efficacy in preclinical models of schizophrenia. Coupled with an attractive DMPK profile and suitable predicted human PK, 6c (VU0467485) was evaluated as a preclinical development candidate.

This letter describes progress towards an M(4) PAM preclinical candidate driven by an unexpected aldehyde oxidase (AO) metabolite of a novel, CNS penetrant thieno[2,3-c]pyridine core to an equipotent, non-CNS penetrant thieno[2,3-c]pyrdin-7(6H)-one core. Medicinal chemistry design efforts yielded two novel tricyclic cores that enhanced M(4) PAM potency, regained CNS penetration, displayed favorable DMPK properties and afforded robust in vivo efficacy in reversing amphetamine-induced hyperlocomotion in rats.

Niemann-Pick type C1 (NPC1) disease is a fatal neurovisceral disease for which there are no FDA approved treatments, though cyclodextrin (HPβCD) slows disease progression in preclinical models and in an early phase clinical trial. Our goal was to evaluate the mechanism of action of a previously described combination-therapy, Triple Combination Formulation (TCF) - comprised of the histone deacetylase inhibitor (HDACi) vorinostat/HPβCD/PEG - shown to prolong survival in Npc1 mice. In these studies, TCF's benefit was attributed to enhanced vorinostat pharmacokinetics (PK). Here, we show that TCF reduced lipid storage, extended lifespan, and preserved neurological function in Npc1 mice. Unexpectedly, substitution of an inactive analog for vorinostat in TCF revealed similar efficacy. We demonstrate that the efficacy of TCF was attributable to enhanced HPβCD PK and independent of NPC1 protein expression. We conclude that although HDACi effectively reduce cholesterol storage in NPC1-deficient cells, HDACi are ineffective in vivo in Npc1 mice.

This letter describes progress towards an M

AT13387 is an orally bioavailable clinical candidate developed to inhibit theheat shock protein 90 (Hsp90). This article describes a modified synthetic route for the multi-gram production of AT13387 in 46% overall yield. The modified synthetic route is short, avoids stringent reaction conditions and difficult purifications, which led to increase in an overall yield.

This letter describes a focused exercise to explore the role of the β-amino carboxamide moiety found in all of the first generation M(4) PAMs and question if the NH(2) group served solely to stabilize an intramolecular hydrogen bond (IMHB) and enforce planarity. To address this issue (and to potentially find a substitute for the β-amino carboxamide that engendered P-gp and contributed to solubility liabilities), we removed the NH(2), generating des-amino congeners and surveyed other functional groups in the β-position. These modifications led to weak M(4) PAMs with poor DMPK properties. Cyclization of the β-amino carboxamide moiety by virtue of a pyrazole ring re-enforced the IMHB, led to potent (and patented) M(4) PAMs, many as potent as the classical bicyclic β-amino carboxamide analogs, but with significant CYP1A2 inhibition. Overall, this exercise indicated that the β-amino carboxamide moiety most likely facilitates an IMHB, and is essential for M(4) PAM activity within classical bicyclic M(4) PAM scaffolds.

IMPACT: The first-line chemotherapies used to treat leishmaniasis are highly toxic intravenous antimonials yet drug resistance has begun to develop, causing the use of oral treatment options with high price tags; there is a strong need for new, safe, and effective chemotherapeutic agents to treat leishmaniasis. OBJECTIVES/GOALS: This study was conducted in order to identify novel chemical compounds that exhibit anti-leishmanial activity and to further characterize their efficacy and toxicity in in vitro and in vivo systems in the hopes of future chemotherapeutic developments. METHODS/STUDY POPULATION: A total of 28 unique 1,4-diaryl-pyrazolo-pyridinone (1,4-DAPP) compounds were synthesized and anti-leishmanial efficacy and host cell toxicity were determined using L. donovani mCherry-expressing amastigotes and THP-1 macrophages. Additional pharmacokinetic analyses of a potent 1,4-DAPP compound were conducted, revealing a potential metabolite structure. A select group of the novel compounds were screened in a cutaneous leishmaniasis (CL) murine model using L. major mCherry-expressing parasites and female Balb/C mice. The treatment consisted of 10 intralesional injections of compound over a period of 4 weeks, while lesion growth was monitored via fluorescence and manual measurements. RESULTS/ANTICIPATED RESULTS: Four experimental compounds had IC50 values less than 5 micromolar, providing similar anti-leishmanial activity to Miltefosine. Compound 9279817 had a clearance almost twice the rate of normal hepatic blood flow and had a relatively high volumes of distribution, indicating this compound is rapidly cleared and distributes into tissues. In vitro rat liver microsome assays suggest a rapid metabolism of 9279817 and MS/MS results suggest this metabolite is most likely formed via oxidation of the sulfur on the lower aryl ring. This sulfoxide metabolite has similar efficacy as the parent compound and does not exhibit toxicity in vitro. Three of the experimental compounds behaved similarly to the antimony positive control in the murine CL model. DISCUSSION/SIGNIFICANCE OF FINDINGS: This study revealed a novel structural class of compounds that have anti-leishmanial activity. Experiments show compounds with similar efficacy to Miltefosine while having significantly less cytotoxicity, suggesting that the 1,4-DAPP structural class could be further developed as a potential chemotherapeutic.

Further optimization of the VU0486321 series of highly selective and CNS-penetrant mGlu1 PAMs identified unique 'molecular switches' on the central aromatic ring that engendered positive cooperativity with multiple mGlu subtypes across the receptor family, resulting in compounds with comparable activity at Group I (mGlu1/5) and Group III (mGlu4/6/7/8) mGlu receptors, receptors. These exciting data suggests this PAM chemotype appears to bind to multiple mGlu receptors, and that subtype selectivity is dictated by the degree of cooperativity, not a subtype selective, unique allosteric binding site. Moreover, there is interesting therapeutic potential for mGlu1/4/7/8 PAMs, as well as the first report of a GPCR allosteric 'privileged structure'.

We report crystal structures of zebrafish histone deacetylase 6 (HDAC6) catalytic domains in tandem or as single domains in complex with the (R) and (S) enantiomers of trichostatin A (TSA) or with the HDAC6-specific inhibitor nexturastat A. The tandem domains formed, together with the inter-domain linker, an ellipsoid-shaped complex with pseudo-twofold symmetry. We identified important active site differences between both catalytic domains and revealed the binding mode of HDAC6 selective inhibitors. HDAC inhibition assays with (R)- and (S)-TSA showed that (R)-TSA was a broad-range inhibitor, whereas (S)-TSA had moderate selectivity for HDAC6. We identified a uniquely positioned α-helix and a flexible tryptophan residue in the loop joining α-helices H20 to H21 as critical for deacetylation of the physiologic substrate tubulin. Using single-molecule measurements and biochemical assays we demonstrated that HDAC6 catalytic domain 2 deacetylated α-tubulin lysine 40 in the lumen of microtubules, but that its preferred substrate was unpolymerized tubulin.

OBJECTIVES/GOALS: This study was conducted in order to identify novel chemical compounds that exhibit anti-leishmanial activity and to further characterize their efficacy and toxicity in in vitro and in vivo systems in the hopes of future chemotherapeutic developments. METHODS/STUDY POPULATION: We developed a novel, target-free fluorometric high-throughput screen (HTS) to identify small molecules with anti-leishmanial activity. Screening of 10,000 small molecules from the ChemBridge DIVERset-EXP library cassette #5 yielded 210 compounds that killed 80% of parasites. One hundred nine (109) molecular scaffolds were represented within the hit compounds, including the 1,4-diaryl-pyrazolo-pyridinone (1,4-DAPP). A total of 27 novel 1,4-DAPP compounds were synthesized and anti-leishmanial efficacy and host cell toxicity was determined using L. donovani mCherry expressing amastigotes and THP-1 macrophages. Additional pharmacokinetic analyses of a potent 1,4-DAPP compound were conducted. RESULTS/ANTICIPATED RESULTS: Four experimental compounds had IC50 values less than 5μM, providing similar anti-leishmanial activity to miltefosine. Compound 9279817 had a clearance almost twice the rate of normal hepatic blood flow and had a relatively high volume of distribution, indicating this compound is rapidly cleared and distributes into tissues. in vitro rat liver microsome assays suggest a rapid metabolism of 9279817, and MS/MS results suggest this metabolite is most likely formed via oxidation of the sulfur on the lower aromatic ring. DISCUSSION/SIGNIFICANCE OF IMPACT: This study revealed a novel structural class of compounds that have anti-leishmanial activity. in vitro experiments show compounds with similar efficacy as miltefosine while having significantly less toxicity, suggesting that this class could be further developed as a potential chemotherapeutic.

This Letter describes the chemical optimization of a new series of muscarinic acetylcholine receptor subtype 1 (M(1)) positive allosteric modulators (PAMs) based on novel tricyclic triazolo- and imidazopyridine lactam cores, devoid of M(1) agonism, e.g., no M(1) ago-PAM activity, in high expressing recombinant cell lines. While all the new tricyclic congeners afforded excellent rat pharmacokinetic (PK) properties (CL(p) < 8 mL/min/kg and t(1/2) > 5 h), regioisomeric triazolopyridine analogues were uniformly not CNS penetrant (K(p) < 0.05), despite a lack of hydrogen bond donors. However, removal of a single nitrogen atom to afford imidazopyridine derivatives proved to retain the excellent rat PK and provide high CNS penetration (K(p) > 2), despite inclusion of a basic nitrogen. Moreover, 24c was devoid of M(1) agonism in high expressing recombinant cell lines and did not induce cholinergic seizures in vivo in mice. Interestingly, all of the new M(1) PAMs across the diverse tricyclic heterocyclic cores possessed equivalent CNS MPO scores (>4.5), highlighting the value of both “medicinal chemist’s eye” and experimental data, e.g., not sole reliance (or decision bias) on in silico calculated properties, for parameters as complex as CNS penetration.

Metabotropic glutamate receptor 7 (mGlu7) is a member of the group III mGlu receptors (mGlus), encompassed by mGlu4, mGlu6, mGlu7, and mGlu8. mGlu7 is highly expressed in the presynaptic active zones of both excitatory and inhibitory synapses, and activation of the receptor regulates the release of both glutamate and GABA. mGlu7 is thought to be a relevant therapeutic target for a number of neurological and psychiatric disorders, and polymorphisms in the GRM7 gene have been linked to autism, depression, ADHD, and schizophrenia. Here we report two new pan-group III mGlu positive allosteric modulators, VU0155094 and VU0422288, which show differential activity at the various group III mGlus. Additionally, both compounds show probe dependence when assessed in the presence of distinct orthosteric agonists. By pairing studies of these nonselective compounds with a synapse in the hippocampus that expresses only mGlu7, we have validated activity of these compounds in a native tissue setting. These studies provide proof-of-concept evidence that mGlu7 activity can be modulated by positive allosteric modulation, paving the way for future therapeutics development.

Herein we report the discovery and SAR of an indole-based protease activated receptor-4 (PAR-4) antagonist scaffold derived from a similarity search of the Vanderbilt HTS collection, leading to MLPCN probe ML354 (VU0099704). Using a novel PAC-1 fluorescent αIIbβ3 activation assay this probe molecule antagonist was found to have an IC50 of 140 nM for PAR-4 with 71-fold selectivity versus PAR-1 (PAR-1IC50 = 10 μM).

Positive allosteric modulators (PAMs) of the M4 muscarinic acetylcholine receptor (mAChR) represent a novel approach for the treatment of psychotic symptoms associated with schizophrenia and other neuropsychiatric disorders. We recently reported that the selective M4 PAM VU0152100 produced an antipsychotic drug-like profile in rodents after amphetamine challenge. Previous studies suggest that enhanced cholinergic activity may also improve cognitive function and reverse deficits observed with reduced signaling through the N-methyl-d-aspartate subtype of the glutamate receptor (NMDAR) in the central nervous system. Prior to this study, the M1 mAChR subtype was viewed as the primary candidate for these actions relative to the other mAChR subtypes. Here we describe the discovery of a novel M4 PAM, VU0467154, with enhanced in vitro potency and improved pharmacokinetic properties relative to other M4 PAMs, enabling a more extensive characterization of M4 actions in rodent models. We used VU0467154 to test the hypothesis that selective potentiation of M4 receptor signaling could ameliorate the behavioral, cognitive, and neurochemical impairments induced by the noncompetitive NMDAR antagonist MK-801. VU0467154 produced a robust dose-dependent reversal of MK-801-induced hyperlocomotion and deficits in preclinical models of associative learning and memory functions, including the touchscreen pairwise visual discrimination task in wild-type mice, but failed to reverse these stimulant-induced deficits in M4 KO mice. VU0467154 also enhanced the acquisition of both contextual and cue-mediated fear conditioning when administered alone in wild-type mice. These novel findings suggest that M4 PAMs may provide a strategy for addressing the more complex affective and cognitive disruptions associated with schizophrenia and other neuropsychiatric disorders.

Herein, we report the structure-activity relationships within a series of potent, selective, and orally bioavailable muscarinic acetylcholine receptor 4 (M

Herein we report a next generation muscarinic receptor 4 (M(4)) positive allosteric modulator (PAM), ML253 which exhibits nanomolar activity at both the human (EC(50)=56 nM) and rat (EC(50)=176 nM) receptors and excellent efficacy by the left-ward shift of the ACh concentration response curve (fold shift, human=106; rat=50). In addition, ML253 is selective against the four other muscarinic subtypes, displays excellent CNS exposure and is active in an amphetamine-induced hyperlocomotion assay.

This Letter describes the chemical optimization of a novel series of M4 positive allosteric modulators (PAMs) based on a 5,6-dimethyl-4-(piperidin-1-yl)thieno[2,3-d]pyrimidine core, identified from an MLPCN functional high-throughput screen. The HTS hit was potent and selective, but not CNS penetrant. Potency was maintained, while CNS penetration was improved (rat brain:plasma Kp = 0.74), within the original core after several rounds of optimization; however, the thieno[2,3-d]pyrimidine core was subject to extensive oxidative metabolism. Ultimately, we identified a 6-fluoroquinazoline core replacement that afforded good M4 PAM potency, muscarinic receptor subtype selectivity and CNS penetration (rat brain:plasma Kp > 10). Moreover, this campaign provided fundamentally distinct M4 PAM chemotypes, greatly expanding the available structural diversity for this exciting CNS target.

This Letter describes the continued optimization of an MLPCN probe molecule (ML012) through an iterative parallel synthesis approach. After exploring extensive modifications throughout the parent structure, we arrived at a more highly M(1)-selective antagonist, compound 13l (VU0415248). Muscarinic subtype selectivity across all five human and rat receptors for 13l, along with rat selectivity for the lead compound (ML012), is presented.

Despite the availability of >30 effective drugs for managing HIV-1 infection, no current therapy is curative, and long-term management is challenging owing to the emergence and spread of drug-resistant mutants. Identification of drugs against novel HIV-1 targets would expand the current treatment options and help to control resistance. The highly conserved HIV-1 capsid protein represents an attractive target because of its multiple roles in replication of the virus. However, the low antiviral potencies of the reported HIV-1 capsid–targeting inhibitors render them unattractive for therapeutic development. To facilitate the identification of more-potent HIV-1 capsid inhibitors, we developed a scintillation proximity assay to screen for small molecules that target a biologically active and specific intersubunit interface in the HIV-1 capsid. The assay, which is based on competitive displacement of a known capsid-binding small-molecule inhibitor, exhibited a signal-to-noise ratio of >9 and a Z factor of >0.8. In a pilot screen of a chemical library containing 2,400 druglike compounds, we obtained a hit rate of 1.8%. This assay has properties that are suitable for screening large compound libraries to identify novel HIV-1 capsid ligands with antiviral activity.

Schizophrenia patients exhibit deficits in signaling of the M1 subtype of muscarinic acetylcholine receptor (mAChR) in the prefrontal cortex (PFC) and also display impaired cortical long-term depression (LTD). We report that selective activation of the M1 mAChR subtype induces LTD in PFC and that this response is completely lost after repeated administration of phencyclidine (PCP), a mouse model of schizophrenia. Furthermore, discovery of a novel, systemically active M1 positive allosteric modulator (PAM), VU0453595, allowed us to evaluate the impact of selective potentiation of M1 on induction of LTD and behavioral deficits in PCP-treated mice. Interestingly, VU0453595 fully restored impaired LTD as well as deficits in cognitive function and social interaction in these mice. These results provide critical new insights into synaptic changes that may contribute to behavioral deficits in this mouse model and support a role for selective M1 PAMs as a novel approach for the treatment of schizophrenia.

Accumulating evidence suggests that selective M4 muscarinic acetylcholine receptor (mAChR) activators may offer a novel strategy for the treatment of psychosis. However, previous efforts to develop selective M4 activators were unsuccessful because of the lack of M4 mAChR subtype specificity and off-target muscarinic adverse effects. We recently developed VU0152100, a highly selective M4 positive allosteric modulator (PAM) that exerts central effects after systemic administration. We now report that VU0152100 dose-dependently reverses amphetamine-induced hyperlocomotion in rats and wild-type mice, but not in M4 KO mice. VU0152100 also blocks amphetamine-induced disruption of the acquisition of contextual fear conditioning and prepulse inhibition of the acoustic startle reflex. These effects were observed at doses that do not produce catalepsy or peripheral adverse effects associated with non-selective mAChR agonists. To further understand the effects of selective potentiation of M4 on region-specific brain activation, VU0152100 alone and in combination with amphetamine were evaluated using pharmacologic magnetic resonance imaging (phMRI). Key neural substrates of M4-mediated modulation of the amphetamine response included the nucleus accumbens (NAS), caudate-putamen (CP), hippocampus, and medial thalamus. Functional connectivity analysis of phMRI data, specifically assessing correlations in activation between regions, revealed several brain networks involved in the M4 modulation of amphetamine-induced brain activation, including the NAS and retrosplenial cortex with motor cortex, hippocampus, and medial thalamus. Using in vivo microdialysis, we found that VU0152100 reversed amphetamine-induced increases in extracellular dopamine levels in NAS and CP. The present data are consistent with an antipsychotic drug-like profile of activity for VU0152100. Taken together, these data support the development of selective M4 PAMs as a new approach to the treatment of psychosis and cognitive impairments associated with psychiatric disorders such as schizophrenia.