Antinociceptive effects of α2/α3-subtype selective GABAA receptor positive allosteric modulators KRM-II-81 and NS16085 in rats: behavioral specificity.

Recent studies suggest that amongst the GABAA receptor subtype heterogeneity, α2/α3 subunits of GABAA receptors mediate pain processing. Therefore, α2/α3-subtype selective GABAA receptor positive allosteric modulators (PAMs) may be candidate analgesics. Antinociceptive effects of α2/α3-subtype selective GABAA receptor PAMs have been reported, but the behavioral effects of these compounds have not been systematically evaluated. This study examined the behavioral effects of two α2/α3 subtype-selective GABAA receptor PAMs, KRM-II-81 and NS16085, in male rats. The antinociceptive effects of KRM-II-81 and NS16085 were examined using rat models of inflammatory (complete Freund's adjuvant) and neuropathic pain (chronic constriction injury). The effect of KRM-II-81 on affective pain was measured using the place escape/avoidance paradigm (PEAP). Rate-response of food-maintained operant responding, horizontal wire test, and the spontaneous alternation T-maze, were assessed to study the side-effect profiles of KRM-II-81 and NS16085. The benzodiazepine midazolam was used as a comparator in these studies. KRM-II-81 and NS16085 attenuated mechanical allodynia but not thermal hyperalgesia in both pain states, and their effects were attenuated by the benzodiazepine receptor antagonist flumazenil. KRM-II-81 attenuated affective pain-related behavior in the PEAP test. In the operant responding procedure and horizontal wire test, only midazolam produced significant effects at the dose that produced maximal antinociception. In the T-maze assay, only midazolam significantly decreased the percentage of alternation at an antinociceptive dose. Thus, KRM-II-81 and NS16085 but not midazolam selectively produced antinociceptive effects. Collectively, these data suggest that α2/α3-subtype selective GABAA PAMs could be a novel class of analgesics and warrant further investigation. Significance Statement This study demonstrates that α2/α3-subtype selective GABAA PAMs KRM-II-81 and NS16085 produce selective antinociceptive effects devoid of sedation, myorelaxation, cognitive impairment in two rat models of persistent pain. Unlikely classical benzodiazepines, this study supports the development of α2/α3-subtype selective GABAA PAMs as safe and novel analgesics for pain management.

Transmission mode predicts coinfection patterns of insect-specific viruses in field populations of the Queensland fruit fly.

Insect-specific viruses (ISVs) can affect insect health and fitness, but can also interact with other insect-associated microorganisms. Despite this, ISVs are often studied in isolation from each other, in laboratory populations. Consequently, their diversity, prevalence and associations with other viruses in field populations are less known, yet these parameters are important to understanding virus epidemiology. To help address this knowledge gap, we assessed the diversity, prevalence and coinfections of three ISVs (horizontally transmitted cripavirus, biparentally transmitted sigmavirus and maternally transmitted iflavirus) in 29 field populations of Queensland fruit fly, Australia's most significant horticultural pest, in the context of their different transmission modes. We detected new virus variant diversity. In contrast to the very high virus prevalence in laboratory populations, 46.8% of 293 field flies carried one virus and 4.8% had two viruses. Cripavirus and sigmavirus occurred in all regions, while iflavirus was restricted to subtropical and tropical regions. Cripavirus was most prevalent (37.5%), followed by sigmavirus (13.7%) and iflavirus (4.4%). Cripavirus coinfected some flies with either one of the two vertically transmitted viruses. However, sigmavirus did not coinfect individuals with iflavirus. Three different modelling approaches detected negative association patterns between sigmavirus and iflavirus, consistent with the absence of such coinfections in laboratory populations. This may be linked with their maternal transmission and the ineffective paternal transmission of sigmavirus. Furthermore, we found that, unlike sigmavirus and iflavirus, cripavirus load was higher in laboratory than field flies. Laboratory and mass-rearing conditions may increase ISV prevalence and load due to increased transmission opportunities. We conclude that a combination of field and laboratory studies is needed to uncover ISV interactions and further our understanding of ISV epidemiology.

Vascular effects of midazolam, flumazenil, and a novel imidazobenzodiazepine MP-III-058 on isolated rat aorta.

Hypotensive influences of benzodiazepines and other GABAA receptor ligands, recognized in clinical practice, seem to stem from the existence of "vascular" GABAA receptors in peripheral blood vessels, besides any mechanisms in the central and peripheral nervous systems. We aimed to further elucidate the vasodilatatory effects of ligands acting through GABAA receptors. Using immunohistochemistry, the rat aortic smooth muscle layer was found to express GABAA γ2 and α1-5 subunit proteins. To confirm the role of "vascular" GABAA receptors, we investigated the vascular effects of standard benzodiazepines, midazolam, and flumazenil, as well as the novel compound MP-III-058. Using two-electrode voltage clamp electrophysiology and radioligand binding assays, MP-III-058 was found to have modest binding but substantial functional selectivity for α5ß3γ2 over other αxß3γ2 GABAA receptors. Tissue bath assays revealed comparable vasodilatory effects of MP-III-058 and midazolam, both of which at 100 µmol/L concentrations had efficacy similar to prazosin. Flumazenil exhibited weak vasoactivity per se, but significantly prevented the relaxant effects of midazolam and MP-III-058. These studies indicate the existence of functional GABAA receptors in the rat aorta, where ligands exert vasodilatory effects by positive modulation of the benzodiazepine binding site, suggesting the potential for further quest for leads with optimized pharmacokinetic properties as prospective adjuvant vasodilators.

Development and validation of an LC-MS/MS method for the determination of ARN14988, an acid ceramidase inhibitor, and its application to a pharmacokinetic study in a mouse model.

Despite aggressive treatment approaches, the overall survival of glioblastoma (GBM) patients remained poor with a strong need for more effective chemotherapeutic agents. A previous study has shown that ARN14988 is more cytotoxic to GBM cells compared to US Food and Drug Administration-approved temozolomide. This finding makes ARN14988 a desirable candidate for further pharmacological assessment. Therefore, an efficient analytical method is needed to quantify ARN14988. Herein, we have developed and validated sample preparation and LC-MS/MS triple quadrupole (QQQ) method for quantification of ARN14988 in mouse plasma. In this method, the liquid-liquid extraction of ARN14988 from mouse plasma was performed using 5% ethyl acetate in hexane. The chromatographic separation was achieved using a C18 -column with mobile phases of 10 mm ammonium acetate (pH 5) and 0.1% formic acid in methanol, within a runtime of 10 min. The monitored transitions were m/z 391.20 → m/z 147.00 for ARN14988, and m/z 455.30 → m/z 165.00 for verapamil (internal standard) in positive electrospray ionization. The developed method for ARN14988 showed linearity over the range of 10-5,000 ng/ml (r2 > 0.99). The selectivity, sensitivity, matrix effect, recovery, stability, inter-day and intraday accuracy and precision were determined using four quality control samples. This validated method was successfully applied to the pharmacokinetic study of ARN14988 in mice.

Bee pollination services and the burden of biogeography.

Native bees augment pollination services in the Northern Hemisphere, especially cultivated apple crops, yet Southern Hemisphere contexts are poorly known. We observed the foraging behaviour of 69 354 invertebrate flower visitors in Australian orchards (two regions, 3 years) to assess the efficacy of pollination service (Peff). Native stingless bees and introduced honey bees were the most abundant visitors and most efficacious pollinators (Tetragonula Peff = 6.16; Apis Peff = 13.02), with Tetragonula becoming important service providers above 22°C. However, visits by tree-nesting stingless bees decreased with distance from native forest (less than 200 m) and their tropical/subtropical distribution precludes pollination service in other major Australian apple-producing regions. More broadly distributed native allodapine and halictine bees transferred the most pollen per-visit, but their low abundances reduce efficacies (Exoneura Peff = 0.03; Lasioglossum Peff = 0.06), resulting in a general dependence on honey bees. This reliance is a burden of biogeography, since key Northern Hemisphere pollinators of apple (Andrena, Apis, Bombus, Osmia) do not naturally occur in Australasia-where there is only 15% generic overlap with Central Asian bees sympatric with wild apple distributions (cf. Palaearctic 66% and Nearctic 46% generic overlaps). The historical biogeography of bees therefore drives an extreme dependence on one introduced species for apple pollination in Australia.

Structural Analogs of the GABAkine KRM-II-81 Are Orally Bioavailable Anticonvulsants without Sedation.

To provide back-up compounds to support the development of the GABAA receptor (GABAAR) potentiator KRM-II-81, three novel analogs were designed: replacing the pyridinyl with 2'-Cl-phenyl (FR-II-60), changing the positions of the N and O atoms in the oxazole ring with addition of an ethyl group (KPP-III-34 and KPP-III-51), or substituting a Br atom for the ethynyl of KRM-II-81 (KPP-III-34). The compounds bound to brain GABAARs. Intraperitoneal administration of FR-II-60 and KPP-III-34 produced anticonvulsant activity in mice [maximal electroshock (MES)-induced seizures or 6 Hz-induced seizures], whereas KPP-III-51 did not. Although all compounds were orally bioavailable, structural changes reduced the plasma and brain (FR-II-60 and KPP-III-51) exposures relative to KRM-II-81. Oral administration of each compound produced dose-dependent increases in the latency for both clonic and tonic seizures and the lethality induced by pentylenetetrazol (PTZ) in mice. Since KPP-III-34 produced the highest brain area under the curve (AUC) exposures, it was selected for further profiling. Oral administration of KPP-III-34 suppressed seizures in corneal-kindled mice, hippocampal paroxysmal discharges in mesial temporal lobe epileptic mice, and PTZ-induced convulsions in rats. Only transient sensorimotor impairment was observed in mice, and doses of KPP-III-34 up to 500 mg/kg did not produce impairment in rats. Molecular docking studies demonstrated that all compounds displayed a reduced propensity for binding to α1His102 compared with the sedating compound alprazolam; the bromine-substituted KPP-III-34 achieved the least interaction. Overall, these findings document the oral bioavailability and anticonvulsant efficacy of three novel analogs of KRM-II-81 with reduced sedative effects. SIGNIFICANCE STATEMENT: A new non-sedating compound, KRM-II-81, with reduced propensity for tolerance is moving into clinical development. Three new analogs were orally bioavailable, produced anticonvulsant effects in rodents, and displayed low sensorimotor impairment. KPP-III-34 demonstrated efficacy in models of pharmacoresistant epilepsy. Docking studies demonstrated a low propensity for compound binding to the α1His102 residue implicated in sedation. Thus, three additional structures have been added to the list of non-sedating imidazodiazepine anticonvulsants that could serve as backups in the clinical development of KRM-II-81.

Microbial diversity in stingless bee gut is linked to host wing size and influenced by the environment.

Stingless bees are important social corbiculate bees, fulfilling critical pollination roles in many ecosystems. However, their gut microbiota, particularly the fungal communities associated with them, remains inadequately characterised. This knowledge gap hinders our understanding of bee gut microbiomes and their impacts on the host fitness. We collected 121 samples from two species, Tetragonula carbonaria and Austroplebeia australis across 1200 km of eastern Australia. We characterised their gut microbiomes and investigated potential correlations between bee gut microbiomes and various geographical and morphological factors. We found their core microbiomes consisted of the abundant bacterial taxa Snodgrassella, Lactobacillus and Acetobacteraceae, and the fungal taxa Didymellaceae, Monocilium mucidum and Aureobasidium pullulans, but variances of their abundances among samples were large. Furthermore, gut bacterial richness of T. carbonaria was positively correlated to host forewing length, an established correlate to body size and fitness indicator in insects relating to flight capacity. This result indicates that larger body size/longer foraging distance of bees could associate with greater microbial diversity in gut. Additionally, both host species identity and management approach significantly influenced gut microbial diversity and composition, and similarity between colonies for both species decreased as the geographic distance between them increased. We also quantified the total bacterial and fungal abundance of the samples using qPCR analyses and found that bacterial abundance was higher in T. carbonaria compared to A. australis, and fungi were either lowly abundant or below the threshold of detection for both species. Overall, our study provides novel understanding of stingless bee gut microbiomes over a large geographic span and reveals that gut fungal communities likely not play an important role in host functions due to their low abundances.

Heat stress survival and thermal tolerance of Australian stingless bees.

Stingless bees (Meliponini) are important pollinators throughout the world's tropical and subtropical regions. Understanding their thermal tolerance is key to predicting their resilience to changing climates and increasingly frequent extreme heat events. We examined critical thermal maxima (CTmax), survival during 1-8 h heat periods, chill coma recovery and thermal preference for Australian meliponine species that occupy different climates across their ranges: Tetragonula carbonaria (tropical to temperate regions), T. hockingsi (tropical and subtropical regions only) and Austroplebeia australis (widely distributed including arid regions). We found interspecific differences in thermal tolerance consistent with differences in the climate variability observed in each species' range. Foragers of A. australis had a faster chill coma recovery (288 s) than foragers of T. hockingsi (1059 s) and T. carbonaria (872 s). Austroplebeia australis also had the highest CTmax of 44.5 °C, while the CTmax of the two Tetragonula species was ∼43.1 °C. After a 1-h heat exposure, T. carbonaria foragers experienced 95% mortality at 42 °C, and 100% at 45 °C. Surprisingly, larvae and pupae of both Tetragonula species were more resistant to heat exposure than foragers. Within an enclosed temperature gradient apparatus (17-38 °C), no clear preference was found for foragers; however, they were most frequently observed at ∼18 °C. Results indicate that in some regions of Australia, meliponines already experience periodic heat events exceeding their thermal maxima. Employing effective management strategies (such as nest site insulation and habitat preservation) may be crucial to colony survival under continued climate change.

Comparative anticonvulsant activity of the GABAkine KRM-II-81 and a deuterated analog.

A series of imidazodiazepines has been developed that possess reduced sedative liabilities but retain efficacy in anticonvulsant screening models. The latest of these compounds, (5-(8-ethynyl-6-(pyridin-2-yl)-4H-benzo[f]imidazole[1,5-α][1,4]diazepin-3-yl) oxazole known as KRM-II-81) is currently awaiting advancement into the clinic. A deuterated structural analog (D5-KRM-II-81) was made as a potential backup compound and studied here in comparison to KRM-II-81. In the present study, both compounds significantly prevented seizures in mice induced by 6 Hz (44 mA) electrical stimulation without significantly altering motoric function on a rotarod after intraperitoneal administration. Both compounds also significantly prevented clonic seizures, tonic seizures, and lethality induced by pentylenetetrazol in mice when given orally. D5-KRM-II-81 had a slightly longer duration of action against clonic and tonic seizures than KRM-II-81. Oral administration of 100 mg/kg of either KRM-II-81 or D5-KRM-II-81 was significantly less disruptive of sensorimotor function in mice than diazepam (5 mg/kg, p.o.). The present report documents that D5-KRM-II-81 represents another in this series of imidazodiazepines with anticonvulsant activity at doses that do not impair sensorimotor function.

GL-II-73, a Positive Allosteric Modulator of α5GABAA Receptors, Reverses Dopamine System Dysfunction Associated with Pilocarpine-Induced Temporal Lobe Epilepsy.

Although seizures are a hallmark feature of temporal lobe epilepsy (TLE), psychiatric comorbidities, including psychosis, are frequently associated with TLE and contribute to decreased quality of life. Currently, there are no defined therapeutic protocols to manage psychosis in TLE patients, as antipsychotic agents may induce epileptic seizures and are associated with severe side effects and pharmacokinetic and pharmacodynamic interactions with antiepileptic drugs. Thus, novel treatment strategies are necessary. Several lines of evidence suggest that hippocampal hyperactivity is central to the pathology of both TLE and psychosis; therefore, restoring hippocampal activity back to normal levels may be a novel therapeutic approach for treating psychosis in TLE. In rodent models, increased activity in the ventral hippocampus (vHipp) results in aberrant dopamine system function, which is thought to underlie symptoms of psychosis. Indeed, we have previously demonstrated that targeting α5-containing γ-aminobutyric acid receptors (α5GABAARs), an inhibitory receptor abundant in the hippocampus, with positive allosteric modulators (PAMs), can restore dopamine system function in rodent models displaying hippocampal hyperactivity. Thus, we posited that α5-PAMs may be beneficial in a model used to study TLE. Here, we demonstrate that pilocarpine-induced TLE is associated with increased VTA dopamine neuron activity, an effect that was completely reversed by intra-vHipp administration of GL-II-73, a selective α5-PAM. Further, pilocarpine did not alter the hippocampal α5GABAAR expression or synaptic localization that may affect the efficacy of α5-PAMs. Taken together, these results suggest augmenting α5GABAAR function as a novel therapeutic modality for the treatment of psychosis in TLE.

α5-GABAA Receptor Modulation Reverses Behavioral and Neurophysiological Correlates of Psychosis in Rats with Ventral Hippocampal Alzheimer's Disease-like Pathology.

Of the 35 million people in the world suffering from Alzheimer's Disease (AD), up to half experience comorbid psychosis. Antipsychotics, used to treat psychosis, are contraindicated in elderly patients because they increase the risk of premature death. Reports indicate that the hippocampus is hyperactive in patients with psychosis and those with AD. Preclinical studies have demonstrated that the ventral hippocampus (vHipp) can regulate dopamine system function, which is thought to underlie symptoms of psychosis. A viral-mediated approach was used to express mutated human genes known to contribute to AD pathology: the Swedish (K670N, M671L), Florida (I716V), and London (V717I) mutations of amyloid precursor protein and two mutations (M146L and L286V) of presenilin 1 specifically in the vHipp, to investigate the selective contribution of AD-like pathology in this region. We observed a significant increase in dopamine neuron population activity and behavioral deficits in this AD-AAV model that mimics observations in rodent models with psychosis-like symptomatologies. Further, systemic administration of MP-III-022 (α5-GABAA receptor selective positive allosteric modulator) was able to reverse aberrant dopamine system function in AD-AAV rats. This study provides evidence for the development of drugs that target α5-GABAA receptors for patients with AD and comorbid psychosis.

Synthesis and Receptor Binding Studies of α5 GABAAR Selective Novel Imidazodiazepines Targeted for Psychiatric and Cognitive Disorders.

GABA mediates inhibitory actions through various GABAA receptor subtypes, including 19 subunits in human GABAAR. Dysregulation of GABAergic neurotransmission is associated with several psychiatric disorders, including depression, anxiety, and schizophrenia. Selective targeting of α2/3 GABAARs can treat mood and anxiety, while α5 GABAA-Rs can treat anxiety, depression, and cognitive performance. GL-II-73 and MP-III-022, α5-positive allosteric modulators have shown promising results in animal models of chronic stress, aging, and cognitive disorders, including MDD, schizophrenia, autism, and Alzheimer's disease. Described in this article is how small changes in the structure of imidazodiazepine substituents can greatly impact the subtype selectivity of benzodiazepine GABAAR. To investigate alternate and potentially more effective therapeutic compounds, modifications were made to the structure of imidazodiazepine 1 to synthesize different amide analogs. The novel ligands were screened at the NIMH PDSP against a panel of 47 receptors, ion channels, including hERG, and transporters to identify on- and off-target interactions. Any ligands with significant inhibition in primary binding were subjected to secondary binding assays to determine their Ki values. The newly synthesized imidazodiazepines were found to have variable affinities for the benzodiazepine site and negligible or no binding to any off-target profile receptors that could cause other physiological problems.

Common endosymbionts affect host fitness and sex allocation via egg size provisioning.

It is hard to overemphasize the importance of endosymbionts in arthropod biology, ecology and evolution. Some endosymbionts can complement host metabolic function or provide defence against pathogens; others, such as ubiquitous Wolbachia and Cardinium, have evolved strategies to manipulate host reproduction. A common reproductive manipulation strategy is cytoplasmic incompatibility (CI) between differently infected individuals which can result in female mortality or male development of fertilized eggs in haplodiploid hosts. Recently, an additional role of endosymbionts has been recognized in the modification of sex allocation in sexually reproducing haplodiploids. This was theoretically expected due to the maternal inheritance of endosymbionts and natural selection for them to increase infected female production, yet the underlying mechanism remained unknown. Here, we tested whether and how Cardinium and Wolbachia causing different CI types interact to increase female production in a haplodiploid thrips species where sex allocation depends on both maternal condition and egg size provisioning. We found that Cardinium augmented female production by increasing maternal fitness and egg size, thereby boosting fertilization rate and offspring fitness. Wolbachia, in contrast, reduced the beneficial effects of Cardinium. Our results demonstrate different invasion strategies and antagonistic effects of endosymbiotic bacteria on host fitness and evolution of sex allocation.

Pathogen to commensal? Longitudinal within-host population dynamics, evolution, and adaptation during a chronic >16-year Burkholderia pseudomallei infection.

Although acute melioidosis is the most common outcome of Burkholderia pseudomallei infection, we have documented a case, P314, where disease severity lessened with time, and the pathogen evolved towards a commensal relationship with the host. In the current study, we used whole-genome sequencing to monitor this long-term symbiotic relationship to better understand B. pseudomallei persistence in P314's sputum despite intensive initial therapeutic regimens. We collected and sequenced 118 B. pseudomallei isolates from P314's airways over a >16-year period, and also sampled the patient's home environment, recovering six closely related B. pseudomallei isolates from the household water system. Using comparative genomics, we identified 126 SNPs in the core genome of the 124 isolates or 162 SNPs/indels when the accessory genome was included. The core SNPs were used to construct a phylogenetic tree, which demonstrated a close relationship between environmental and clinical isolates and detailed within-host evolutionary patterns. The phylogeny had little homoplasy, consistent with a strictly clonal mode of genetic inheritance. Repeated sampling revealed evidence of genetic diversification, but frequent extinctions left only one successful lineage through the first four years and two lineages after that. Overall, the evolution of this population is nonadaptive and best explained by genetic drift. However, some genetic and phenotypic changes are consistent with in situ adaptation. Using a mouse model, P314 isolates caused greatly reduced morbidity and mortality compared to the environmental isolates. Additionally, potentially adaptive phenotypes emerged and included differences in the O-antigen, capsular polysaccharide, motility, and colony morphology. The >13-year co-existence of two long-lived lineages presents interesting hypotheses that can be tested in future studies to provide additional insights into selective pressures, niche differentiation, and microbial adaptation. This unusual melioidosis case presents a rare example of the evolutionary progression towards commensalism by a highly virulent pathogen within a single human host.

Positive Allosteric Modulation of α5-GABAA Receptors Reverses Stress-Induced Alterations in Dopamine System Function and Prepulse Inhibition of Startle.

BACKGROUND: Up to 64% of patients diagnosed with posttraumatic stress disorder (PTSD) experience psychosis, likely attributable to aberrant dopamine neuron activity. We have previously demonstrated that positive allosteric modulators of α5-GABAARs can selectively decrease hippocampal activity and reverse psychosis-like physiological and behavioral alterations in a rodent model used to study schizophrenia; however, whether this approach translates to a PTSD model remains to be elucidated. METHODS: We utilized a 2-day inescapable foot shock (IS) procedure to induce stress-related pathophysiology in male Sprague-Dawley rats. We evaluated the effects of intra-ventral hippocampus (vHipp) administration GL-II-73, an α5-GABAAR, or viral overexpression of the α5 subunit, using in vivo electrophysiology and behavioral measures in control and IS-treated rats. RESULTS: IS significantly increased ventral tegmental area dopamine neuron population activity, or the number of dopamine neurons firing spontaneously (n = 6; P = .016), consistent with observation in multiple rodent models used to study psychosis. IS also induced deficits in sensorimotor gating, as measured by reduced prepulse inhibition of startle (n = 12; P = .039). Interestingly, intra-vHipp administration of GL-II-73 completely reversed IS-induced increases in dopamine neuron population activity (n = 6; P = .024) and deficits in prepulse inhibition (n = 8; P = .025), whereas viral overexpression of the α5 subunit in the vHipp was not effective. CONCLUSIONS: Our results demonstrate that pharmacological intervention augmenting α5-GABAAR function, but not α5 overexpression in itself, can reverse stress-induced deficits related to PTSD in a rodent model, providing a potential site of therapeutic intervention to treat comorbid psychosis in PTSD.

Rationalizing the binding and α subtype selectivity of synthesized imidazodiazepines and benzodiazepines at GABAA receptors by using molecular docking studies.

The pharmacological actions exerted by benzodiazepines are dependent on the discrete α protein subunits of the γ-aminobutyric acid type A receptor (GABAA R). Recent developments via a cryo-EM structure of the α1ß3γ2L GABAA R ion channel provide crucial insights into ligand efficacy and binding affinity at this subtype. We investigated the molecular interactions of diazepam and alprazolam bound GABAA R structures (6HUP and 6HUO) to determine key binding interaction domains. A halogen bond between the chlorine atoms of diazepam and alprazolam with the group on the backbone of the α1 histidine amino acid 102 is important to the positive allosteric modulatory actions of diazepam and alprazolam in the α1ß3γ2L GABAA R ion channel. In order to gain insight into α subtype selectivity we designed and synthesized close structural analogs of diazepam and alprazolam. These compounds were then docked into the recently publish cryo-EM structures of GABAA Rs (6HUP and 6HUO). This modeling along with radio-ligand binding data resulted in the conclusion that the non-classical bioisosteric replacement of the chlorine atom at C7 with an ethinyl group (compound 5) resulted in an 11-fold gain in α5 binding selectivity over the α1 subtype. Moreover, the potency of compound 5 resulted in a ligand with less sedation than diazepam, while still maintaining the same anxiolytic potency. These modeling data extend our understanding of the structural requirements for α-subtype-selective compounds that can be utilized to achieve improved medical treatments. It is clear that the ethinyl group in place of a halogen atom decreases the affinity and efficacy of benzodiazepines and imidazodiazepines at α1 subtypes, which results in less sedation and ataxia.

Reversal of Age-Related Neuronal Atrophy by α5-GABAA Receptor Positive Allosteric Modulation.

Aging is associated with reduced brain volume, altered neural activity, and neuronal atrophy in cortical-like structures, comprising the frontal cortex and hippocampus, together contributing to cognitive impairments. Therapeutic efforts aimed at reversing these deficits have focused on excitatory or neurotrophic mechanisms, although recent findings show that reduced dendritic inhibition mediated by α5-subunit containing GABA-A receptors (α5-GABAA-Rs) occurs during aging and contributes to cognitive impairment. Here, we aimed to confirm the beneficial effect on working memory of augmenting α5-GABAA-R activity in old mice and tested its potential at reversing age-related neuronal atrophy. We show that GL-II-73, a novel ligand with positive allosteric modulatory activity at α5-GABAA-R (α5-PAM), increases dendritic branching complexity and spine numbers of cortical neurons in vitro. Using old mice, we confirm that α5-PAM reverses age-related working memory deficits and show that chronic treatment (3 months) significantly reverses age-related dendritic shrinkage and spine loss in frontal cortex and hippocampus. A subsequent 1-week treatment cessation (separate cohort) resulted in loss of efficacy on working memory but maintained morphological neurotrophic effects. Together, the results demonstrate the beneficial effect on working memory and neurotrophic efficacy of augmenting α5-GABAA-R function in old mice, suggesting symptomatic and disease-modifying potential in age-related brain disorders.

Metabolism, pharmacokinetics, and anticonvulsant activity of a deuterated analog of the α2/3-selective GABAkine KRM-II-81.

The imidazodiazepine, (5-(8-ethynyl-6-(pyridin-2-yl)-4H-benzo [f]imidazole[1,5-α][1,4]diazepin-3-yl) oxazole or KRM-II-81) is a new α2/3-selective GABAkine (gamma aminobutyric acid A receptor potentiator) with anticonvulsant, anxiolytic, and antinociceptive activity in preclinical models. Reducing metabolism was utilized as a means of potentially extending the half-life of KRM-II-81. In vitro and in vivo studies were conducted to evaluate metabolic liabilities. Incubation of KRM-II-81 in hepatocytes revealed sites of potential metabolism on the oxazole and the diazepine rings. These sites were targeted in the design of a deuterated analog (D5-KRM-II-81) that could be evaluated as a potentially longer-acting analog. In contrast to computer predictions, peak plasma concentrations of D5-KRM-II-81 in rats were not significantly greater than those produced by KRM-II-81 after oral administration. Furthermore, brain disposition of KRM-II-81 was higher than that of D5-KRM-II-81. The half-life of the two compounds in either plasma or brain did not statistically differ from one another but the tmax for D5-KRM-II-81 occurred slightly earlier than for KRM-II-81. Non-metabolic considerations might be relevant to the lack of increases in exposure by D5-KRM-II-81. Alternative sites of metabolism on KRM-II-81, not targeted by the current deuteration process, are also possible. Despite its lack of augmented exposure, D5-KRM-II-81, like KRM-II-81, significantly prevented seizures induced by pentylenetetrazol when given orally. The present findings introduce a new orally active anticonvulsant GABAkine, D5-KRM-II-81.

Comparative pharmacodynamic and pharmacokinetic study of MIDD0301 and its (S) enantiomer.

MIDD0301 is being developed as an oral drug to relax airway smooth muscle (ASM) and reduce lung inflammation in asthma. We report a comparative study of MIDD0301 and its S isomer (MIDD0301S), and found that the compounds have equivalent affinity for γ-aminobutyric acid type A receptor (GABAA R) expressed in rat brain, with half maximal inhibitory concentration values of 25.1 and 26.3 nM for the S and R enantiomers, respectively. Both compounds relaxed substance P contracted ASM within 30 min and neither enantiomer revealed affinity to 48 receptors in an off-target screen. Both enantiomers reduced airway hyperresponsiveness (AHR) with nebulized and oral dosing in two mouse models of bronchoconstriction. In A/J mice, which are very sensitive to methacholine-induced bronchoconstriction, we observed reduction of AHR at 10.8 mg/kg MIDD0301 and 15 mg/kg MIDD0301S. Using oral administration, 100 mg/kg/day for 3 days of either enantiomer was sufficient to reduce AHR. In a model of severe airway inflammation induced by interferon-γ and lipopolysaccharide (LPS), we observed reduction of AHR at 7.2 mg/kg for both enantiomers using nebulized administration, and at 100 mg/kg for oral administration. MIDD0301 and MIDD0301S did not undergo Phase I metabolism. Glucuronidation was observed for both compounds, whereas only MIDD0301 formed the corresponding glucoside in the presence of kidney microsomes. Pharmacokinetic analysis identified glucuronides as the major metabolite with concentrations up to 20-fold more than the parent compound. MIDD0301 glucuronide and MIDD0301 taurine bind GABAA Rs, although 10-fold weaker than MIDD0301. In mouse blood, the taurine adduct was only observed for MIDD0301. Overall, both compounds exhibited similar receptor binding and pharmacodynamic properties with subtle differences in metabolism and greater oral availability and blood concentrations of MIDD0301S.

Completion of the Total Synthesis of Several Bioactive Sarpagine/Macroline Alkaloids including the Important NF-κB Inhibitor N4-Methyltalpinine.

The unification of the general synthetic strategy regarding the important and emerging group of C-19 methyl-substituted sarpagine/macroline alkaloids has culminated in the completion of the total synthesis of several bioactive alkaloids. Key transformations include an ACE-Cl mediated late-stage N(4)-demethylation and an anhydrous acid-mediated intramolecular quaternary hemiaminal formation between a tertiary amine and an aldehyde function to allow efficient access to several biologically important alkaloids from this group. Herein, the enantiospecific total synthesis of the first known sarpagine/macroline alkaloid with NF-κB inhibitory activity, N(4)-methyltalpinine (as a chloride salt), as well as the anticancer alkaloids talpinine, O-acetyltalpinine, and macrocarpines F-G, are described.