5-HT2C Agonists Modulate Schizophrenia-Like Behaviors in Mice.

All FDA-approved antipsychotic drugs (APDs) target primarily dopamine D2 or serotonin (5-HT2A) receptors, or both; however, these medications are not universally effective, they may produce undesirable side effects, and provide only partial amelioration of negative and cognitive symptoms. The heterogeneity of pharmacological responses in schizophrenic patients suggests that additional drug targets may be effective in improving aspects of this syndrome. Recent evidence suggests that 5-HT2C receptors may be a promising target for schizophrenia since their activation reduces mesolimbic nigrostriatal dopamine release (which conveys antipsychotic action), they are expressed almost exclusively in CNS, and have weight-loss-promoting capabilities. A difficulty in developing 5-HT2C agonists is that most ligands also possess 5-HT2B and/or 5-HT2A activities. We have developed selective 5-HT2C ligands and herein describe their preclinical effectiveness for treating schizophrenia-like behaviors. JJ-3-45, JJ-3-42, and JJ-5-34 reduced amphetamine-stimulated hyperlocomotion, restored amphetamine-disrupted prepulse inhibition, improved social behavior, and novel object recognition memory in NMDA receptor hypofunctioning NR1-knockdown mice, and were essentially devoid of catalepsy. However, they decreased motivation in a breakpoint assay and did not promote reversal learning in MK-801-treated mice. Somewhat similar effects were observed with lorcaserin, a 5-HT2C agonist with potent 5-HT2B and 5-HT2A agonist activities, which is approved for treating obesity. Microdialysis studies revealed that both JJ-3-42 and lorcaserin reduced dopamine efflux in the infralimbic cortex, while only JJ-3-42 decreased it in striatum. Collectively, these results provide additional evidence that 5-HT2C receptors are suitable drug targets with fewer side effects, greater therapeutic selectivity, and enhanced efficacy for treating schizophrenia and related disorders than current APDs.

Further Advances in Optimizing (2-Phenylcyclopropyl)methylamines as Novel Serotonin 2C Agonists: Effects on Hyperlocomotion, Prepulse Inhibition, and Cognition Models.

A series of novel compounds with two halogen substituents have been designed and synthesized to further optimize the 2-phenylcyclopropylmethylamine scaffold in the quest for drug-like 5-HT2C agonists. Compound (+)-22a was identified as a potent 5-HT2C receptor agonist, with good selectivity against the 5-HT2B and the 5-HT2A receptors. ADMET assays showed that compound (+)-22a possessed desirable properties in terms of its microsomal stability, and CYP and hERG inhibition, along with an excellent brain penetration profile. Evaluation of (+)-22a in animal models of schizophrenia-related behaviors revealed that it had a desirable activity profile, as it reduced d-amphetamine-stimulated hyperlocomotion in the open field test, it restored d-amphetamine-disrupted prepulse inhibition, it induced cognitive improvements in the novel object recognition memory test in NR1-KD animals, and it produced very little catalepsy relative to haloperidol. These data support the further development of (+)-22a as a drug candidate for the treatment of schizophrenia.

Effects of ß-Arrestin-Biased Dopamine D2 Receptor Ligands on Schizophrenia-Like Behavior in Hypoglutamatergic Mice.

Current antipsychotic drugs (APDs) show efficacy with positive symptoms, but are limited in treating negative or cognitive features of schizophrenia. Whereas all currently FDA-approved medications target primarily the dopamine D2 receptor (D2R) to inhibit G(i/o)-mediated adenylyl cyclase, a recent study has shown that many APDs affect not only G(i/o)- but they can also influence ß-arrestin- (ßArr)-mediated signaling. The ability of ligands to differentially affect signaling through these pathways is termed functional selectivity. We have developed ligands that are devoid of D2R-mediated G(i/o) protein signaling, but are simultaneously partial agonists for D2R/ßArr interactions. The purpose of this study was to test the effectiveness of UNC9975 or UNC9994 on schizophrenia-like behaviors in phencyclidine-treated or NR1-knockdown hypoglutamatergic mice. We have found the UNC compounds reduce hyperlocomotion in the open field, restore PPI, improve novel object recognition memory, partially normalize social behavior, decrease conditioned avoidance responding, and elicit a much lower level of catalepsy than haloperidol. These preclinical results suggest that exploitation of functional selectivity may provide unique opportunities to develop drugs with fewer side effects, greater therapeutic selectivity, and enhanced efficacy for treating schizophrenia and related conditions than medications that are currently available.

Data-independent MS/MS quantification of neuropeptides for determination of putative feeding-related neurohormones in microdialysate.

Food consumption is an important behavior that is regulated by an intricate array of neuropeptides (NPs). Although many feeding-related NPs have been identified in mammals, precise mechanisms are unclear and difficult to study in mammals, as current methods are not highly multiplexed and require extensive a priori knowledge about analytes. New advances in data-independent acquisition (DIA) MS/MS and the open-source quantification software Skyline have opened up the possibility to identify hundreds of compounds and quantify them from a single DIA MS/MS run. An untargeted DIA MS(E) quantification method using Skyline software for multiplexed, discovery-driven quantification was developed and found to produce linear calibration curves for peptides at physiologically relevant concentrations using a protein digest as internal standard. By using this method, preliminary relative quantification of the crab Cancer borealis neuropeptidome (<2 kDa, 137 peptides from 18 families) was possible in microdialysates from 8 replicate feeding experiments. Of these NPs, 55 were detected with an average mass error below 10 ppm. The time-resolved profiles of relative concentration changes for 6 are shown, and there is great potential for the use of this method in future experiments to aid in correlation of NP changes with behavior. This work presents an unbiased approach to winnowing candidate NPs related to a behavior of interest in a functionally relevant manner, and demonstrates the success of such a UPLC-MS(E) quantification method using the open source software Skyline.

Optimization of 2-phenylcyclopropylmethylamines as selective serotonin 2C receptor agonists and their evaluation as potential antipsychotic agents.

The discovery of a new series of compounds that are potent, selective 5-HT2C receptor agonists is described herein as we continue our efforts to optimize the 2-phenylcyclopropylmethylamine scaffold. Modifications focused on the alkoxyl substituent present on the aromatic ring led to the identification of improved ligands with better potency at the 5-HT2C receptor and excellent selectivity against the 5-HT2A and 5-HT2B receptors. ADMET studies coupled with a behavioral test using the amphetamine-induced hyperactivity model identified four compounds possessing drug-like profiles and having antipsychotic properties. Compound (+)-16b, which displayed an EC50 of 4.2 nM at 5-HT2C, no activity at 5-HT2B, and an 89-fold selectivity against 5-HT2A, is one of the most potent and selective 5-HT2C agonists reported to date. The likely binding mode of this series of compounds to the 5-HT2C receptor was also investigated in a modeling study, using optimized models incorporating the structures of ß2-adrenergic receptor and 5-HT2B receptor.

Mass spectrometric measurement of neuropeptide secretion in the crab, Cancer borealis, by in vivo microdialysis.

Neuropeptides (NPs), a unique and highly important class of signaling molecules across the animal kingdom, have been extensively characterized in the neuronal tissues of various crustaceans. Because many NPs are released into circulating fluid (hemolymph) and travel to distant sites in order to exhibit physiological effects, it is important to measure the secretion of these NPs from living animals. In this study, we report on extensive characterization of NPs released in the crab Cancer borealis by utilizing in vivo microdialysis to sample NPs from the hemolymph. We determined the necessary duration for collection of microdialysis samples, enabling more comprehensive identification of NP content while maintaining the temporal resolution of sampling. Analysis of in vivo microdialysates using a hybrid quadrupole-Orbitrap™ Q-Exactive mass spectrometer revealed that more than 50 neuropeptides from 9 peptide families-including the allatostatin, RFamide, orcokinin, tachykinin-related peptide and RYamide families - were released into the circulatory system. The presence of these peptides both in neuronal tissues as well as in hemolymph indicates their putative hormonal roles, a finding that merits further investigation. Preliminary quantitative measurement of these identified NPs suggested several potential candidates that maybe associated with the circadian rhythm in Cancer borealis.

Function-driven discovery of neuropeptides with mass spectrometry-based tools.

A number of unique challenges are inherent to the study of neuropeptides (NPs), both in determining their molecular structure and their function. Traditional studies follow a model in which novel NPs are discovered and identified, then investigated for function. These studies frequently use biochemical techniques that can be imprecise and cumbersome. Mass spectrometry (MS)-based tools are becoming important not only in precisely determining the identity of a NP or quantifying a compound with a known sequence, but also in studies where identity and putative function can be determined simultaneously. Tools based on MS and tandem MS (MS/MS) have been developed, both with isotope labeling strategies and label-free methods, that allow accurate quantitation of NP changes associated with behavior or physiological manipulation, concurrent with identification of sequence. MS and MS/MS have also been implemented with sampling methods that incorporate temporal or spatial information while determining functional role of a NP, such as microdialysis (MD) and imaging mass spectrometry (IMS). These advances in MS and sampling techniques allow investigation of a particular biological phenomenon to guide studies aimed to identify and characterize NPs. Permitting function to drive identification of relevant compounds allows for a broader understanding of the molecular underpinnings of these events. The NPs thus identified can then be validated with more conventional techniques, and successive iterations of identification and function determination will provide rich information about these compounds. This function-driven discovery of NPs using MS-based techniques is an important new approach for their study.

Mass spectrometric detection of neuropeptides using affinity-enhanced microdialysis with antibody-coated magnetic nanoparticles.

Microdialysis (MD) is a useful sampling tool for many applications due to its ability to permit sampling from an animal concurrent with normal activity. MD is of particular importance in the field of neuroscience, in which it is used to sample neurotransmitters (NTs) while the animal is behaving in order to correlate dynamic changes in NTs with behavior. One important class of signaling molecules, the neuropeptides (NPs), however, presented significant challenges when studied with MD, due to the low relative recovery (RR) of NPs by this technique. Affinity-enhanced microdialysis (AE-MD) has previously been used to improve recovery of NPs and similar molecules. For AE-MD, an affinity agent (AA), such as an antibody-coated particle or free antibody, is added to the liquid perfusing the MD probe. This AA provides an additional mass transport driving force for analyte to pass through the dialysis membrane and thus increases the RR. In this work, a variety of AAs have been investigated for AE-MD of NPs in vitro and in vivo, including particles with C18 surface functionality and antibody-coated particles. Antibody-coated magnetic nanoparticles (AbMnP) provided the best RR enhancement in vitro, with statistically significant (p < 0.05) enhancements for 4 out of 6 NP standards tested, and RR increases up to 41-fold. These particles were then used for in vivo MD in the Jonah crab, Cancer borealis, during a feeding study, with mass spectrometric (MS) detection. 31 NPs were detected in a 30 min collection sample, compared to 17 when no AA was used. The use of AbMnP also increased the temporal resolution from 4 to 18 h in previous studies to just 30 min in this study. The levels of NPs detected were also sufficient for reliable quantitation with the MS system in use, permitting quantitative analysis of the concentration changes for 7 identified NPs on a 30 min time course during feeding.