Synthesis and Behavioral Studies of Chiral Cyclopropanes as Selective α4ß2-Nicotinic Acetylcholine Receptor Partial Agonists Exhibiting an Antidepressant Profile. Part III.

We report the synthesis and biological characterization of novel derivatives of 3-[(1-methyl-2(S)-pyrrolidinyl)methoxy]-5-cyclopropylpyridine (4a-f and 5) as potent and highly selective α4ß2-nicotinic acetylcholine receptor (nAChR) full or partial agonists. A systematic structure-activity study was carried out on the previously described compound 3b, particularly concerning its (2-methoxyethyl)cyclopropyl side-chain, in an effort to improve its metabolic stability while maintaining receptor selectivity. Compound 4d exhibited very similar subnanomolar binding affinity for α4ß2- and α4ß2*-nAChRs compared to 3b, and it showed excellent potency in activating high-sensitivity (HS) α4ß2-nAChRs with an EC50 value of 8.2 nM. Testing of 4d in the SmartCube assay revealed that the compound has a combined antidepressant plus antipsychotic signature. In the forced swim test at a dose of 30 mg/kg given intraperitoneally, 4d was found to be as efficacious as sertraline, thus providing evidence of the potential use of the compound as an antidepressant. Additional promise for use of 4d in humans comes from pharmacokinetic studies in mice indicating brain penetration, and additional assays show compound stability in the presence of human microsomes and hepatocytes. Thus, 4d has a very favorable preclinical drug profile.

Synthesis and biological evaluation of novel hybrids of highly potent and selective α4ß2-Nicotinic acetylcholine receptor (nAChR) partial agonists.

We previously reported the cyclopropylpyridine and isoxazolylpyridine ether scaffolds to be versatile building blocks for creating potent α4ß2 nicotinic acetylcholine receptor (nAChR) partial agonists with excellent selectivity over the α3ß4 subtype. In our continued efforts to develop therapeutic nicotinic ligands, seven novel hybrid compounds were rationally designed, synthesized, and evaluated in [3H]epibatidine binding competition studies. Incorporation of a cyclopropane- or isoxazole-containing side chain onto the 5-position of 1-(pyridin-3-yl)-1,4-diazepane or 2-(pyridin-3-yl)-2,5-diazabicyclo[2.2.1]heptane led to highly potent and selective α4ß2* nAChR partial agonists with Ki values of 0.5-51.4 nM for α4ß2 and negligible affinities for α3ß4 and α7. Moreover, compounds 21, 25, and 30 maintained the functional profiles (EC50 and IC50 values of 15-50 nM) of the parent azetidine-containing compounds 3 and 4 in the 86Rb+ ion flux assays. In vivo efficacy of the most promising compound 21 was confirmed in the mouse SmartCube® platform and classical forced swim tests, supporting the potential use of α4ß2 partial agonists for treatment of depression.

Large-scale phenome analysis defines a behavioral signature for Huntington's disease genotype in mice.

Rapid technological advances for the frequent monitoring of health parameters have raised the intriguing possibility that an individual's genotype could be predicted from phenotypic data alone. Here we used a machine learning approach to analyze the phenotypic effects of polymorphic mutations in a mouse model of Huntington's disease that determine disease presentation and age of onset. The resulting model correlated variation across 3,086 behavioral traits with seven different CAG-repeat lengths in the huntingtin gene (Htt). We selected behavioral signatures for age and CAG-repeat length that most robustly distinguished between mouse lines and validated the model by correctly predicting the repeat length of a blinded mouse line. Sufficient discriminatory power to accurately predict genotype required combined analysis of >200 phenotypic features. Our results suggest that autosomal dominant disease-causing mutations could be predicted through the use of subtle behavioral signatures that emerge in large-scale, combinatorial analyses. Our work provides an open data platform that we now share with the research community to aid efforts focused on understanding the pathways that link behavioral consequences to genetic variation in Huntington's disease.

High-throughput analysis of behavior for drug discovery.

Drug testing with traditional behavioral assays constitutes a major bottleneck in the development of novel therapies. PsychoGenics developed three comprehensive high-throughput systems, SmartCube(®), NeuroCube(®) and PhenoCube(®) systems, to increase the efficiency of the drug screening and phenotyping in rodents. These three systems capture different domains of behavior, namely, cognitive, motor, circadian, social, anxiety-like, gait and others, using custom-built computer vision software and machine learning algorithms for analysis. This review exemplifies the use of the three systems and explains how they can advance drug screening with their applications to phenotyping of disease models, drug screening, selection of lead candidates, behavior-driven lead optimization, and drug repurposing.

Reprint of: Highthroughtput analysis of behavior for drug discovery.

Drug testing with traditional behavioral assays constitutes a major bottleneck in the development of novel therapies. PsychoGenics developed three comprehensive highthroughtput systems, SmartCube(®), NeuroCube(®) and PhenoCube(®) systems, to increase the efficiency of the drug screening and phenotyping in rodents. These three systems capture different domains of behavior, namely, cognitive, motor, circadian, social, anxiety-like, gait and others, using custom-built computer vision software and machine learning algorithms for analysis. This review exemplifies the use of the three systems and explains how they can advance drug screening with their applications to phenotyping of disease models, drug screening, selection of lead candidates, behavior-driven lead optimization, and drug repurposing.

In need of high-throughput behavioral systems.

One of the current major bottlenecks in drug discovery is in vivo testing of candidate drugs in behavioral paradigms in normal or genetically altered mice. This testing is essential in discovering gene function and predicting potential efficacy of CNS drugs in humans. New efforts in the biotech community aim to alleviate this bottleneck by developing higher-throughput systems of behavioral, neurological and physiological analyses. Together with large pharmacological databases, equipped with state-of-the-art bioinformatic and/or data-mining algorithms, these systems will provide rapid and accurate indices of the therapeutic potential of novel drugs. By providing a substantial increase in the speed of behavioral testing, new high-throughput systems will facilitate current behavioral research with faster, more reliable approaches. Furthermore, screening whole drug-libraries and comparing the profiles of novel compounds to those of known compounds will facilitate the discovery of novel drugs. Target validation will also become more efficient with the fast characterization of novel mutant mice.

Juvenile 5HT(1B) receptor knockout mice exhibit reduced pharmacological sensitivity to 5HT(1A) receptor activation.

Serotonin is an important modulator of anxiety and thus drugs that act on this system have frequently been shown to be either anxiogenic or anxiolytic. In addition serotonin has important trophic functions during early development and disruption of serotonin homeostasis is likely to have long-lasting repercussions in the adult. In the present study we examined the contribution of two serotonin receptor subtypes (5HT(1A) and 5HT(1B)) to the pathophysiology of anxiety during development. For this, we have studied homozygous knockout mice lacking the 5HT(1B) receptor and examined the effect of pharmacological manipulations of 5HT(1A) and 5HT(1B) receptors on locomotor activity and emission of ultrasonic vocalization (USV) in 7-8 days old mice. As shown before, drug naïve 5HT(1B) knockout pups showed reduced USV and were hyperactive, in comparison to wild type controls. The administration of RU24969 (a 5HT(1A/1B) agonist) showed a dose-dependent decrease in USV in the wild type and a biphasic effect in the mutants and resulted in dose-dependent increase in activity in the wild type and, to a lesser extent, in the knockouts. The selective 5HT(1A) agonist, 8OH-DPAT, dose-dependently blocked vocalization in both genotypes and also increased locomotion. To differentially activate 5HT(1B) receptors we first blocked 5HT(1A) receptors with WAY100315 and then treated with RU24969. At a high testing temperature, pretreatment with WAY100315 resulted in an anxiogenic effect in wild type pups but not in the knockouts. In agreement with our findings that 5HT(1B) knockout mice were in general less sensitive to 5HT(1A) activation, 5HT(1A) receptor binding was reduced in the knockouts in comparison to controls. Finally, treatment with diazepam dose-dependently decreased USVs in both group with the knockouts showing enhanced sensitivity to this drug. Our results show that important adaptations to a disturbance of serotonin homeostasis occur during the first week of life within the serotonergic system. The observed decreased in sensitivity of 5HT(1B) knockout mice to 5HT(1A) and increased to GABA(A) manipulations are discussed within the context of serotonergic plasticity during development and the implication for clinical treatment of anxiety in genetically predisposed individuals.

Enantiopure Cyclopropane-Bearing Pyridyldiazabicyclo[3.3.0]octanes as Selective α4ß2-nAChR Ligands.

We report the synthesis and characterization of a series of enantiopure 5-cyclopropane-bearing pyridyldiazabicyclo[3.3.0]octanes that display low nanomolar binding affinities and act as functional agonists at α4ß2-nicotinic acetylcholine receptor (nAChR) subtype. Structure-activity relationship studies revealed that incorporation of a cyclopropane-containing side chain at the 5-position of the pyridine ring provides ligands with improved subtype selectivity for nAChR ß2 subunit-containing nAChR subtypes (ß2*-nAChRs) over ß4*-nAChRs compared to the parent compound 4. Compound 15 exhibited subnanomolar binding affinity for α4ß2- and α4ß2*-nAChRs with negligible interaction. Functional assays confirm selectivity for α4ß2-nAChRs. Furthermore, using the SmartCube assay system, this ligand showed antidepressant, anxiolytic, and antipsychotic features, while mouse forced-swim assay further confirm the antidepressant-like property of 15.

Cognitive deficits in transgenic and knock-in HTT mice parallel those in Huntington's disease.

BACKGROUND: Huntington's disease (HD) is characterized not only by severe motor deficits but also by early cognitive dysfunction that significantly increases the burden of the disease for patients and caregivers. Considerable efforts have concentrated, therefore, on the assessment of cognitive deficits in some HD mouse models. However, many of these models that exhibit cognitive deficits also have contemporaneous serious motor deficits, confounding interpretation of cognitive decline. OBJECTIVE: The BACHD and zQ175 mouse models present a more slowly progressing disease phenotype in both motor and cognitive domains, and might therefore offer a better opportunity to measure cognitive decline over a longer timeframe; such models could be useful in screening therapeutic compounds. In order to better define the cognitive impairments evident in BACHD and zQ175 HD mice, both were tested in an instrumental touchscreen visual discrimination assay designed to assess discrimination learning and cognitive flexibility. METHODS: BACHD and zQ175 mice, as well as their WT controls were tested for their ability to discriminate two complex visual stimuli. Following this discrimination phase, the reinforcement contingencies were reversed and the previously incorrect stimulus became the correct stimulus. In a final, third phase of testing, two novel stimuli were introduced and mice were required to undergo a second round of discrimination testing with these stimuli. RESULTS: Our results show that learning during the discrimination phase was similar between the WT and BACHD mice. In contrast, the zQ175 at 26 weeks of age showed decreased accuracy over the last 10 days of discrimination, compared to WT controls. During subsequent reversal and novel stimuli phases, both BACHD and zQ175 mice exhibited significant deficits compared to WT controls. CONCLUSIONS: Our results suggest that the BACHD, and for the first time, zQ175 HD models exhibit cognitive inflexibility and psychomotor slowing, a phenotype that is consistent with cognitive symptoms described in HD patients.

Scientific rigor and the art of motorcycle maintenance.

The reliability of scientific research is under scrutiny. A recently convened working group proposes cultural adjustments to incentivize better research practices.

The potent and selective α4ß2*/α6*-nicotinic acetylcholine receptor partial agonist 2-[5-[5-((S)Azetidin-2-ylmethoxy)-3-pyridinyl]-3-isoxazolyl]ethanol demonstrates antidepressive-like behavior in animal models and a favorable ADME-tox profile.

Preclinical and clinical studies demonstrated that the inhibition of cholinergic supersensitivity through nicotinic antagonists and partial agonists can be used successfully to treat depressed patients, especially those who are poor responders to selective serotonin reuptake inhibitors (SSRIs). In our effort to develop novel antidepressant drugs, LF-3-88 was identified as a potent nicotinic acetylcholine receptor (nAChR) partial agonist with subnanomolar to nanomolar affinities for ß2-containing nAChRs (α2ß2, α3ß2, α4ß2, and α4ß2*) and superior selectivity away from α3ß4 - (K i > 10(4) nmol/L) and α7-nAChRs (K i > 10(4) nmol/L) as well as 51 other central nervous system (CNS)-related neurotransmitter receptors and transporters. Functional activities at different nAChR subtypes were characterized utilizing (86)Rb(+) ion efflux assays, two-electrode voltage-clamp (TEVC) recording in oocytes, and whole-cell current recording measurements. In mouse models, administration of LF-3-88 resulted in antidepressive-like behavioral signatures 15 min post injection in the SmartCube® test (5 and 10 mg/kg, i.p.; about 45-min session), decreased immobility in the forced swim test (1-3 mg/kg, i.p.; 1-10 mg/kg, p.o.; 30 min pretreatment, 6-min trial), and decreased latency to approach food in the novelty-suppressed feeding test after 29 days chronic administration once daily (5 mg/kg but not 10 mg/kg, p.o.; 15-min trial). In addition, LF-3-88 exhibited a favorable profile in pharmacokinetic/ADME-Tox (absorption, distribution, metabolism, excretion, and toxicity) assays. This compound was also shown to cause no mortality in wild-type Balb/CJ mice when tested at 300 mg/kg. These results further support the potential of potent and selective nicotinic partial agonists for use in the treatment of depression.

Genetic deletion of transglutaminase 2 does not rescue the phenotypic deficits observed in R6/2 and zQ175 mouse models of Huntington's disease.

Huntington's disease (HD) is an autosomal dominant, progressive neurodegenerative disorder caused by expansion of CAG repeats in the huntingtin gene. Tissue transglutaminase 2 (TG2), a multi-functional enzyme, was found to be increased both in HD patients and in mouse models of the disease. Furthermore, beneficial effects have been reported from the genetic ablation of TG2 in R6/2 and R6/1 mouse lines. To further evaluate the validity of this target for the treatment of HD, we examined the effects of TG2 deletion in two genetic mouse models of HD: R6/2 CAG 240 and zQ175 knock in (KI). Contrary to previous reports, under rigorous experimental conditions we found that TG2 ablation had no effect on either motor or cognitive deficits, or on the weight loss. In addition, under optimal husbandry conditions, TG2 ablation did not extend R6/2 lifespan. Moreover, TG2 deletion did not change the huntingtin aggregate load in cortex or striatum and did not decrease the brain atrophy observed in either mouse line. Finally, no amelioration of the dysregulation of striatal and cortical gene markers was detected. We conclude that TG2 is not a valid therapeutic target for the treatment of HD.

Characterization of maleimide-based glycogen synthase kinase-3 (GSK-3) inhibitors as stimulators of steroidogenesis.

Inhibition of GSK-3ß has been well documented to account for the behavioral actions of the mood stabilizer lithium in various animal models of mood disorders. Recent studies have showed that genetic or pharmacological inhibition of GSK-3ß resulted in anxiolytic-like and pro-social behavior. In our ongoing efforts to develop GSK-3ß inhibitors for the treatment of mood disorders, SAR studies on maleimide-based compounds were undertaken. We present herein for the first time that some of these GSK-3ß inhibitors, in particular analogues 1 and 9, were able to stimulate progesterone production in the MA-10 mouse tumor Leydig cell model of steroidogenesis without any significant toxicity. These two compounds were tested in the SmartCube behavioral assay and showed anxiolytic-like signatures following daily dose administration (50 mg/kg, ip) for 13 days. Taken together, these results support the hypothesis that GSK-3ß inhibition could influence neuroactive steroid production thereby mediating the modulation of anxiety-like behavior in vivo.

Deficits in a Simple Visual Go/No-go Discrimination Task in Two Mouse Models of Huntington's Disease.

Huntington's disease (HD), a devastating neurodegenerative disorder caused by a CAG repeat expansion on the HTT gene located on chromosome 4, is associated with a characteristic pattern of progressive cognitive dysfunction known to involve early deficits in executive function. A modified Go/No-go successive discrimination task was designed to assess the type of online response control/executive function known to be disrupted in patients with HD. The present studies show that this simple discrimination assay revealed early and robust deficits in two mouse models of HD, the zQ175 KI mouse (deficits from 28 weeks of age) and the R6/2 mouse, carrying ~240 CAG repeats (deficits from 9 weeks of age). These deficits are not due to gross motor dysfunction in the test animals, but instead appear to measure some inability to inhibit responding in the HD mouse models, suggesting this assay may measure deficits in underlying attentional and/or behavioral inhibition processes. Accordingly, this assay may be well suited to evaluation of simple deficits in cognitive function in mouse HD models, providing a potential platform for preclinical screening.

Increased Body Weight of the BAC HD Transgenic Mouse Model of Huntington's Disease Accounts for Some but Not All of the Observed HD-like Motor Deficits.

The genome of the Bacterial Artificial Chromosome (BAC) transgenic mouse model of Huntington's Disease (BAC HD) contains the 170 kb human HTT locus modified by the addition of exon 1 with 97 mixed CAA-CAG repeats. BAC HD mice present robust behavioral deficits in both the open field and the accelerating rotarod tests, two standard behavioral assays of motor function. BAC HD mice, however, also typically present significantly increased body weights relative to wildtype littermate controls (WT) which potentially confounds the interpretation of any motor deficits associated directly with the effects of mutant huntingtin. In order to evaluate this possible confound of body weight, we directly compared the performance of BAC HD and WT female mice under food restricted versus free feeding conditions in both the open field and rotarod tasks to test the hypothesis that some of the motor deficits observed in this HTT-transgenic mouse line results solely from increased body weight. Our results suggest that the rotarod deficit exhibited by BAC HD mice is modulated by both body weight and non-body weight factors resulting from overexpression of full length mutant Htt. When body weights of WT and BAC HD transgenic mice were normalized using restricted feeding, the deficits exhibited by BAC HD mice on the rotarod task were less marked, but were still significant. Since the rotarod deficit between WT and BAC HD mice is attenuated when body weight is normalized by food restriction, utilization of this task in BAC HD mice during pre-clinical evaluation must be powered accordingly and results carefully considered as therapeutic benefit can result from decreased overall body weight and or motoric improvement that may not be related to body mass. Furthermore, after controlling for body weight differences, the hypoactive phenotype displayed by ad libitum fed BAC HD mice in the open field assay was not observed in the BAC HD mice undergoing food restriction. These findings suggest that assessment of spontaneous locomotor activity, as measured in the open field test, may not be the appropriate behavioral endpoint to evaluate the BAC HD mouse during preclinical evaluation since it appears that the apparent hypoactive phenotype in this model is driven primarily by body weight differences.

High-Throughput Automated Phenotyping of Two Genetic Mouse Models of Huntington's Disease.

Phenotyping with traditional behavioral assays constitutes a major bottleneck in the primary screening, characterization, and validation of genetic mouse models of disease, leading to downstream delays in drug discovery efforts. We present a novel and comprehensive one-stop approach to phenotyping, the PhenoCube™. This system simultaneously captures the cognitive performance, motor activity, and circadian patterns of group-housed mice by use of home-cage operant conditioning modules (IntelliCage) and custom-built computer vision software. We evaluated two different mouse models of Huntington's Disease (HD), the R6/2 and the BACHD in the PhenoCube™ system. Our results demonstrated that this system can efficiently capture and track alterations in both cognitive performance and locomotor activity patterns associated with these disease models. This work extends our prior demonstration that PhenoCube™ can characterize circadian dysfunction in BACHD mice and shows that this system, with the experimental protocols used, is a sensitive and efficient tool for a first pass high-throughput screening of mouse disease models in general and mouse models of neurodegeneration in particular.

SAHA enhances synaptic function and plasticity in vitro but has limited brain availability in vivo and does not impact cognition.

Suberoylanilide hydroxamic acid (SAHA) is an inhibitor of histone deacetylases (HDACs) used for the treatment of cutaneous T cell lymphoma (CTCL) and under consideration for other indications. In vivo studies suggest reducing HDAC function can enhance synaptic function and memory, raising the possibility that SAHA treatment could have neurological benefits. We first examined the impacts of SAHA on synaptic function in vitro using rat organotypic hippocampal brain slices. Following several days of SAHA treatment, basal excitatory but not inhibitory synaptic function was enhanced. Presynaptic release probability and intrinsic neuronal excitability were unaffected suggesting SAHA treatment selectively enhanced postsynaptic excitatory function. In addition, long-term potentiation (LTP) of excitatory synapses was augmented, while long-term depression (LTD) was impaired in SAHA treated slices. Despite the in vitro synaptic enhancements, in vivo SAHA treatment did not rescue memory deficits in the Tg2576 mouse model of Alzheimer's disease (AD). Along with the lack of behavioral impact, pharmacokinetic analysis indicated poor brain availability of SAHA. Broader assessment of in vivo SAHA treatment using high-content phenotypic characterization of C57Bl6 mice failed to demonstrate significant behavioral effects of up to 150 mg/kg SAHA following either acute or chronic injections. Potentially explaining the low brain exposure and lack of behavioral impacts, SAHA was found to be a substrate of the blood brain barrier (BBB) efflux transporters Pgp and Bcrp1. Thus while our in vitro data show that HDAC inhibition can enhance excitatory synaptic strength and potentiation, our in vivo data suggests limited brain availability may contribute to the lack of behavioral impact of SAHA following peripheral delivery. These results do not predict CNS effects of SAHA during clinical use and also emphasize the importance of analyzing brain drug levels when interpreting preclinical behavioral pharmacology.

Comparative psychology and the grand challenge of drug discovery in psychiatry and neurodegeneration.

Drug discovery for brain disorders is undergoing a period of upheaval. Faced with an empty drug pipeline and numerous failures of potential new drugs in clinical trials, many large pharmaceutical companies have been shrinking or even closing down their research divisions that focus on central nervous system (CNS) disorders. In this paper, we argue that many of the difficulties facing CNS drug discovery stem from a lack of robustness in pre-clinical (i.e., non-human animal) testing. There are two main sources for this lack of robustness. First, there is the lack of replicability of many results from the pre-clinical stage, which we argue is driven by a combination of publication bias and inappropriate selection of statistical and experimental designs. Second, there is the frequent failure to translate results in non-human animals to parallel results in humans in the clinic. This limitation can only be overcome by developing new behavioral tests for non-human animals that have predictive, construct, and etiological validity. Here, we present these translational difficulties as a "grand challenge" to researchers from comparative cognition, who are well positioned to provide new methods for testing behavior and cognition in non-human animals. These new experimental protocols will need to be both statistically robust and target behavioral and cognitive processes that allow for better connection with human CNS disorders. Our hope is that this downturn in industrial research may represent an opportunity to develop new protocols that will re-kindle the search for more effective and safer drugs for CNS disorders.

Effect of the rd1 mutation on motor performance in R6/2 and wild type mice.

Homozygosis for the rd1 mutation in the Pbe6b gene results in the loss of the rod beta-subunit of the cyclic GMP phosphodiesterase and, eventually, of all rod and cone photoreceptors. The R6/2 mouse line is a widely used model of Huntington's disease (HD). The original line was made available on a mixed background obtained by crossing, via ovarian transplant, female R6/2 (on a B6CBA mixed background) with male B6CBAF1/J mice. As the CBA/J strain used in the US is homozygous for the rd1 mutation and the breeding scheme does not ensure heterozygosis for the mutation, a significant percentage of the offspring on this mixed background is expected to be homozygous for the rd1 mutation. We investigate here the effect of rd1 homozygosis on motor function and examined the effects of the mutation on the R6/2 phenotype. Homozygosis for the rd1 mutation resulted in increased activity in the open field test and reduced rotarod test performance. In addition, rd1 mutation absence or heterozygosis reduced the differences between the R6/2 and the WT mice. Our recommendation for the neurodegeneration field, and for all mouse studies in general, is to carefully control homozygosis for retinal degeneration mutation, even when using tests of motor function.

Discovery of highly potent and selective α4ß2-nicotinic acetylcholine receptor (nAChR) partial agonists containing an isoxazolylpyridine ether scaffold that demonstrate antidepressant-like activity. Part II.

In our continued efforts to develop α4ß2-nicotinic acetylcholine receptor (nAChR) partial agonists as novel antidepressants having a unique mechanism of action, structure-activity relationship (SAR) exploration of certain isoxazolylpyridine ethers is presented. In particular, modifications to both the azetidine ring present in the starting structure 4 and its metabolically liable hydroxyl side chain substituent have been explored to improve compound druggability. The pharmacological characterization of all new compounds has been carried out using [(3)H]epibatidine binding studies together with functional assays based on (86)Rb(+) ion flux measurements. We found that the deletion of the metabolically liable hydroxyl group or its replacement by a fluoromethyl group not only maintained potency and selectivity but also resulted in compounds showing antidepressant-like properties in the mouse forced swim test. These isoxazolylpyridine ethers appear to represent promising lead candidates in the design of innovative chemical tools containing reporter groups for imaging purposes and of possible therapeutics.