The α2,3-selective potentiator of GABAA receptors, KRM-II-81, reduces nociceptive-associated behaviors induced by formalin and spinal nerve ligation in rats.

Clinical evidence indicates that positive allosteric modulators (PAMs) of GABAA receptors have analgesic benefit in addition to efficacy in anxiety disorders. However, the utility of GABAA receptor PAMs as analgesics is compromised by the central nervous system side effects of non-selective potentiators. A selective potentiator of GABAA receptors associated with α2/3 subunits, KRM-II-81(5-(8-ethynyl-6-(pyridin-2-yl)-4H-benzo[f]imidazo[1,5-a][1,4]diazepin-3-yl)oxazole), has demonstrated anxiolytic, anticonvulsant, and antinociceptive effects in rodents with reduced motoric side effects. The present study evaluated the potential of KRM-II-81 as a novel analgesic. Oral administration of KRM-II-81 attenuated formalin-induced flinching; in contrast, diazepam was not active. KRM-II-81 attenuated nociceptive-associated behaviors engendered by chronic spinal nerve ligation (L5/L6). Diazepam decreased locomotion of rats at the dose tested in the formalin assay (10 mg/kg) whereas KRM-II-81 produced small decreases that were not dose-dependent (10-100 mg/kg). Plasma and brain levels of KRM-II-81 were used to demonstrate selectivity for α2/3- over α1-associated GABAA receptors and to define the degree of engagement of these receptors. Plasma and brain concentrations of KRM-II-81 were positively-associated with analgesic efficacy. GABA currents from isolated rat dorsal-root ganglion cultures were potentiated by KRM-II-81 with an ED50 of 32 nM. Measures of respiratory depression were reduced by alprazolam whereas KRM-II-81 was either inactive or produced effects with lower potency and efficacy. These findings add to the growing body of data supporting the idea that α2/3-selective GABAA receptor PAMs will have efficacy and tolerability as pain medications including those for neuropathic pain. Given their predicted anxiolytic effects, α2/3-selective GABAA receptor PAMs offer an additional inroad into the management of pain.

Bioisosteres of ethyl 8-ethynyl-6-(pyridin-2-yl)-4H-benzo[f]imidazo [1,5-a][1,4]diazepine-3-carboxylate (HZ-166) as novel alpha 2,3 selective potentiators of GABAA receptors: Improved bioavailability enhances anticonvulsant efficacy.

HZ-166 has previously been characterized as an α2,3-selective GABAA receptor modulator with anticonvulsant, anxiolytic, and anti-nociceptive properties but reduced motor effects. We discovered a series of ester bioisosteres with reduced metabolic liabilities, leading to improved efficacy as anxiolytic-like compounds in rats. In the present study, we evaluated the anticonvulsant effects KRM-II-81 across several rodent models. In some models we also evaluated key structural analogs. KRM-II-81 suppressed hyper-excitation in a network of cultured cortical neurons without affecting the basal neuronal activity. KRM-II-81 was active against electroshock-induced convulsions in mice, pentylenetetrazole (PTZ)-induced convulsions in rats, elevations in PTZ-seizure thresholds, and amygdala-kindled seizures in rats with efficacies greater than that of diazepam. KRM-II-81 was also active in the 6 Hz seizure model in mice. Structural analogs of KRM-II-81 but not the ester, HZ-166, were active in all models in which they were evaluated. We further evaluated KRM-II-81 in human cortical epileptic tissue where it was found to significantly-attenuate picrotoxin- and AP-4-induced increases in firing rate across an electrode array. These molecules generally had a wider margin of separation in potencies to produce anticonvulsant effects vs. motor impairment on an inverted screen test than did diazepam. Ester bioisosters of HZ-166 are thus presented as novel agents for the potential treatment of epilepsy acting via selective positive allosteric amplification of GABAA signaling through α2/α3-containing GABA receptors. The in vivo data from the present study can serve as a guide to dosing parameters that predict engagement of central GABAA receptors.

An antidepressant-related pharmacological signature for positive allosteric modulators of α2/3-containing GABAA receptors.

Data from transgenic animals and novel pharmacological agents has realigned scientific scrutiny on the therapeutic potential of positive allosteric modulators (PAMs) of α2/3-containing GABAA receptors. Evidence for analgesic, anticonvulsant, and anxiolytic activity of α2/3-selective PAMs has been presented along with the clinical potential for a milder motor-impacting profile compared to non-selective GABAA receptor PAMs. A new series of α2/3-selective PAMs was recently introduced which has anxiolytic and anticonvulsant activity in rodent models. These molecules also produce efficacy against pain in multiple animal models. Additionally, co-morbid states of depression are prevalent among patients with pain and patients with anxiety. Compounds were shown to be selective for α2 and α3 constructs over α1 (except KRM-II-82), α4, α5, and α6 proteins in electrophysiological assays in transfected HEK-293T cells. Utilizing the forced-swim assay in mice that detects conventional and novel antidepressant drugs, we demonstrate for the first time that α2/3-selective PAMs are active in the forced-swim assay at anxiolytic-producing doses. In contrast, activity in a related model, the tail-suspension test, was not observed. Diazepam was not active in the forced-swim assay when given alone but produced an antidepressant-like effect in mice when given in conjunction with the α1-preferring antagonist, ß-CCT, that attenuated the motor-impairing effects of diazepam. We conclude that these α2/3-selective PAMs deserve further scrutiny for their potential treatment of major depressive disorder. If effective, such a mechanism could add a beneficial antidepressant component to the anxiolytic, analgesic, and anticonvulsant spectrum of effects of these compounds.

Further evaluation of the potential anxiolytic activity of imidazo[1,5-a][1,4]diazepin agents selective for α2/3-containing GABAA receptors.

Positive allosteric modulators of GABAA receptors transduce a host of beneficial effects including anxiolytic actions. We have recently shown that bioavailability and anxiolytic-like activity can be improved by eliminating the ester functionality in imidazo[1,5-a][1,4]diazepines. In the present series of experiments, we further substantiate the value of heterocyle replacement of the ester for potential treatment of anxiety. None of three esters was active in a Vogel conflict test in rats that detects anxiolytic drugs like diazepam. Compounds 7 and 8, ester bioisosters, were selective for alpha 2 and 3 over alpha 1-containing GABAA receptors but also had modest efficacy at GABAA alpha 5-containing receptors. Compound 7 was efficacious and potent in this anxiolytic-detecting assay without affecting non-punished responding. The efficacies of the esters and of compound 7 were predicted from their efficacies as anticonvulsants against the GABAA antagonist pentylenetetrazole (PTZ). In contrast, the related structural analog, compound 8, did not produce anxiolytic-like effects in rats despite anticonvulsant efficacy. These data thus support the following conclusions: 1) ancillary pharmacological actions of compound 8 might be responsible for its lack of anxiolytic-like efficacy despite its efficacy as an anticonvulsant 2) esters of imidazo[1,5-a][1,4]diazepines do not demonstrate anxiolytic-like effects in rats due to their low bioavailability and 3) replacement of the ester function with suitable heterocycles markedly improves bioavailability and engenders molecules with the opportunity to have potent and efficacious effects in vivo that correspond to human anxiolytic actions.

Novel inhibitors of Erm methyltransferases from NMR and parallel synthesis.

The Erm family of methyltransferases confers resistance to the macrolide-lincosamide-streptogramin type B (MLS) antibiotics through the methylation of 23S ribosomal RNA. Upon the methylation of RNA, the MLS antibiotics lose their ability to bind to the ribosome and exhibit their antibiotic activity. Using an NMR-based screen, we identified a series of triazine-containing compounds that bind weakly to ErmAM. These initial lead compounds were optimized by the parallel synthesis of a large number of analogues, resulting in compounds which inhibit the Erm-mediated methylation of rRNA in the low micromolar range. NMR and X-ray structures of enzyme/inhibitor complexes reveal that the inhibitors bind to the S-adenosylmethionine binding site on the Erm protein. These compounds represent novel methyltransferase inhibitors that serve as new leads for the reversal of Erm-mediated MLS antibiotic resistance.

NMR structure and mutagenesis of the N-terminal Dbl homology domain of the nucleotide exchange factor Trio.

Guanine nucleotide exchange factors for the Rho family of GTPases contain a Dbl homology (DH) domain responsible for catalysis and a pleckstrin homology (PH) domain whose function is unknown. Here we describe the solution structure of the N-terminal DH domain of Trio that catalyzes nucleotide exchange for Rac1. The all-alpha-helical protein has a very different structure compared to other exchange factors. Based on site-directed mutagenesis, functionally important residues of the DH domain were identified. They are all highly conserved and reside in close proximity on two a helices. In addition, we have discovered a unique capability of the PH domain to enhance nucleotide exchange in DH domain-containing proteins.