Leslie J. Street

Sedative but not anxiolytic properties of benzodiazepines are mediated by the GABA(A) receptor alpha1 subtype.

Inhibitory neurotransmission in the brain is largely mediated by GABAA receptors. Potentiation of GABA receptor activation through an allosteric benzodiazepine (BZ) site produces the sedative, anxiolytic, muscle relaxant, anticonvulsant and cognition-impairing effects of clinically used BZs such as diazepam. We created genetically modified mice (α1 H101R) with a diazepam-insensitive α1 subtype and a selective BZ site ligand, L-838,417, to explore GABAA receptor subtypes mediating specific physiological effects. These two complimentary approaches revealed that the α1 subtype mediated the sedative, but not the anxiolytic effects of benzodiazepines. This finding suggests ways to improve anxiolytics and to develop drugs for other neurological disorders based on their specificity for GABAA receptor subtypes in distinct neuronal circuits.

Evidence for a Significant Role of α3-Containing GABAA Receptors in Mediating the Anxiolytic Effects of Benzodiazepines

The GABAA receptor subtypes responsible for the anxiolytic effects of nonselective benzodiazepines (BZs) such as chlordiazepoxide (CDP) and diazepam remain controversial. Hence, molecular genetic data suggest that α2-rather than α3-containing GABAA receptors are responsible for the anxiolytic effects of diazepam, whereas the anxiogenic effects of an α3-selective inverse agonist suggest that an agonist selective for this subtype should be anxiolytic. We have extended this latter pharmacological approach to identify a compound, 4,2′-difluoro-5′-[8-fluoro-7-(1-hydroxy-1-methylethyl)imidazo[1,2-á]pyridin-3-yl]biphenyl-2-carbonitrile (TP003), that is an α3 subtype selective agonist that produced a robust anxiolytic-like effect in both rodent and non-human primate behavioral models of anxiety. Moreover, in mice containing a point mutation that renders α2-containing receptors BZ insensitive (α2H101R mice), TP003 as well as the nonselective agonist CDP retained efficacy in a stress-induced hyperthermia model. Together, these data show that potentiation of α3-containing GABAA receptors is sufficient to produce the anxiolytic effects of BZs and that α2 potentiation may not be necessary.

Synthesis of cyclic ethers from copper carbenoids by formation and rearrangement of oxonium ylides

Abstract Copper carbenoids undergo efficient intramolecular insertion into allyl ethers, and the resulting ylide-type species rearrange to furnish cyclic ethers (ring sizes 6–8) in high yield. Copper(II) hexafluoroacetylacetonate is an extremely efficient catalyst for this reaction, and use of this complex minimises competing C-H insertion.

L‐694,247: a potent 5‐HT1D receptor agonist

1 The 5‐hydroxytryptamine (5‐HT) receptor binding selectivity profile of a novel, potent 5‐HT1D receptor agonist, L‐694,247 (2‐[5‐[3‐(4‐methylsulphonylamino)benzyl‐1,2,4‐oxadiazol‐5‐yl]‐1H‐indole‐3‐yl]ethylamine) was assessed and compared with that of the 5‐HT1‐like receptor agonist, sumatriptan. 2 L‐694,247 had an affinity (pIC50) of 10.03 at the 5‐HT1D binding site and 9.08 at the 5‐HT1B binding site (sumatriptan: pIC50 values 8.22 and 5.94 respectively). L‐694,247 retained good selectivity with respect to the 5‐HT1A binding site (pIC50 = 8.64), the 5‐HT1C binding site (6.42), the 5‐HT2 binding site (6.50) and the 5‐HT1E binding site (5.66). The pIC50 values for sumatriptan at these radioligand binding sites were 6.14, 5.0, < 5.0 and 5.64 respectively. Both L‐694,247 and sumatriptan were essentially inactive at the 5‐HT3 recognition site. 3 L‐694,247, like sumatriptan, displayed a similar efficacy to 5‐HT in inhibiting forskolin‐stimulated adenylyl cyclase in guinea‐pig substantia nigra although L‐694,247 (pEC50 = 9.1) was more potent than sumatriptan (6.2) in this 5‐HT1D receptor mediated functional response. L‐694,247 (pEC50 = 9.4) was also more potent than sumatriptan (6.5) in a second 5‐HT1D receptor mediated functional response, the inhibition of K+‐evoked [3H]‐5‐HT release from guinea‐pig frontal cortex slices. 4 The excellent agreement observed for L‐694,247 between the 5‐HT1D radioligand binding affinity and the functional potency confirm that the two functional models (the inhibition of forskolin‐stimulated adenylyl cyclase in guinea‐pig substantia nigra and the inhibition of K+‐evoked [3H]‐5‐HT release from guinea‐pig frontal cortex) do indeed reflect 5‐HT1D‐mediated events. 5 L‐694,247 is a novel, highly potent 5‐HT1D/5‐HT1B receptor ligand which should prove useful for the exploration of the physiological role of these receptors in animals.

The synthesis and preclinical evaluation in rhesus monkey of [18F]MK-6577 and [11C]CMPyPB glycine transporter 1 positron emission tomography radiotracers

Two positron emission tomography radiotracers for the glycine transporter 1 (GlyT1) are reported here. Each radiotracer is a propylsulfonamide‐containing benzamide and was labeled with either carbon‐11 or fluorine‐18. [11C]CMPyPB was synthesized by the alkylation of a 3‐hydroxypyridine precursor using [11C]MeI, and [18F]MK‐6577 was synthesized by a nucleophilic aromatic substitution reaction using a 2‐chloropyridine precursor. Each tracer shows good uptake into rhesus monkey brain with the expected distribution of highest uptake in the pons, thalamus, and cerebellum and lower uptake in the striatum and gray matter of the frontal cortex. In vivo blockade and chase studies of [18F]MK‐6577 showed a large specific signal and reversible binding. In vitro autoradiographic studies with [18F]MK‐6577 showed a large specific signal in both rhesus monkey and human brain slices and a distribution consistent with the in vivo results and those reported in the literature. In vivo metabolism studies in rhesus monkeys demonstrated that only more‐polar metabolites are formed for each tracer. Of these two tracers, [18F]MK‐6577 was more extensively characterized and is a promising clinical positron emission tomography tracer for imaging GlyT1 and for measuring GlyT1 occupancy of therapeutic compounds. Synapse, 2011.

Subtype-selective GABAergic drugs facilitate extinction of mouse operant behaviour

Several recent studies have shown that reducing gamma-aminobutyric acid (GABA)-mediated neurotransmission retards extinction of aversive conditioning. However, relatively little is known about the effect of GABA on extinction of appetitively motivated tasks. We examined the effect of chlordiazepoxide (CDP), a classical benzodiazepine (BZ) and two novel subtype-selective BZs when administered to male C57Bl/6 mice during extinction following training on a discrete-trial fixed-ratio 5 (FR5) food reinforced lever-press procedure. Initially CDP had no effect, but after several extinction sessions CDP significantly facilitated extinction, i.e. slowed responding, compared with vehicle-treated mice. This effect was not due to drug accumulation because mice switched from vehicle treatment to CDP late in extinction showed facilitation immediately. Likewise, this effect could not be attributed to sedation because the dose of CDP used (15 mg/kg i.p.) did not suppress locomotor activity. The two novel subtype-selective BZ partial agonists, L-838417 and TP13, selectively facilitated extinction in similar fashion to CDP. The non-GABAergic anxiolytic buspirone was also tested and found to have similar effects when administered at a non-sedating dose. These studies demonstrate that GABA-mediated processes are important during extinction of an appetitively motivated task, but only after the animals have experienced several extinction sessions.

Antimalarial efficacy of MMV390048, an inhibitor of Plasmodium phosphatidylinositol 4-kinase

MMV390048, a member of a new class of inhibitors of the Plasmodium phosphatidylinositol 4-kinase, shows potential for both treatment and prophylaxis. A new antimalarial in the armamentarium Paquet et al. screened a small-molecule library against the human malaria parasite, Plasmodium falciparum, and identified the 2-aminopyridine chemical class with potent activity. The optimized compound from this class, MMV390048, was active against multiple parasite life cycle stages, in both the mammalian host and the mosquito vector, and also killed drug-resistant parasites. MMV390048 killed the malaria parasite by blocking the parasite’s phosphatidylinositol 4-kinase (PI4K) and was able to protect monkeys from malaria infection. MMV390048 has potential as a new antimalarial drug that may contribute to global malaria eradication efforts. As part of the global effort toward malaria eradication, phenotypic whole-cell screening revealed the 2-aminopyridine class of small molecules as a good starting point to develop new antimalarial drugs. Stemming from this series, we found that the derivative, MMV390048, lacked cross-resistance with current drugs used to treat malaria. This compound was efficacious against all Plasmodium life cycle stages, apart from late hypnozoites in the liver. Efficacy was shown in the humanized Plasmodium falciparum mouse model, and modest reductions in mouse-to-mouse transmission were achieved in the Plasmodium berghei mouse model. Experiments in monkeys revealed the ability of MMV390048 to be used for full chemoprotection. Although MMV390048 was not able to eliminate liver hypnozoites, it delayed relapse in a Plasmodium cynomolgi monkey model. Both genomic and chemoproteomic studies identified a kinase of the Plasmodium parasite, phosphatidylinositol 4-kinase, as the molecular target of MMV390048. The ability of MMV390048 to block all life cycle stages of the malaria parasite suggests that this compound should be further developed and may contribute to malaria control and eradication as part of a single-dose combination treatment.

Fast in vitro methods to determine the speed of action and the stage-specificity of anti-malarials in Plasmodium falciparum

BackgroundRecent whole cell in vitro screening campaigns identified thousands of compounds that are active against asexual blood stages of Plasmodium falciparum at submicromolar concentrations. These hits have been made available to the public, providing many novel chemical starting points for anti-malarial drug discovery programmes. Knowing which of these hits are fast-acting compounds is of great interest. Firstly, a fast action will ensure rapid relief of symptoms for the patient. Secondly, by rapidly reducing the parasitaemia, this could minimize the occurrence of mutations leading to new drug resistance mechanisms.An in vitro assay that provides information about the speed of action of test compounds has been developed by researchers at GlaxoSmithKline (GSK) in Spain. This assay also provides an in vitro measure for the ratio between parasitaemia at the onset of drug treatment and after one intra-erythrocytic cycle (parasite reduction ratio, PRR). Both parameters are needed to determine in vitro killing rates of anti-malarial compounds. A drawback of the killing rate assay is that it takes a month to obtain first results.MethodsThe approach described in the present study is focused only on the speed of action of anti-malarials. This has the advantage that initial results can be achieved within 4–7 working days, which helps to distinguish between fast and slow-acting compounds relatively quickly. It is expected that this new assay can be used as a filter in the early drug discovery phase, which will reduce the number of compounds progressing to secondary, more time-consuming assays like the killing rate assay.ResultsThe speed of action of a selection of seven anti-malarial compounds was measured with two independent experimental procedures using modifications of the standard [3H]hypoxanthine incorporation assay. Depending on the outcome of both assays, the tested compounds were classified as either fast or non-fast-acting.ConclusionThe results obtained for the anti-malarials chloroquine, artesunate, atovaquone, and pyrimethamine are consistent with previous observations, suggesting the methodology is a valid way to rapidly identify fast-acting anti-malarial compounds. Another advantage of the approach is its ability to discriminate between static or cidal compound effects.

Structure&#8211;Activity Relationship Studies of Orally Active Antimalarial 3,5-Substituted 2-Aminopyridines

In an effort to address potential cardiotoxicity liabilities identified with earlier frontrunner compounds, a number of new 3,5-diaryl-2-aminopyridine derivatives were synthesized. Several compounds exhibited potent antiplasmodial activity against both the multidrug resistant (K1) and sensitive (NF54) strains in the low nanomolar range. Some compounds displayed a significant reduction in potency in the hERG channel inhibition assay compared to previously reported frontrunner analogues. Several of these new analogues demonstrated promising in vivo efficacy in the Plasmodium berghei mouse model and will be further evaluated as potential clinical candidates. The SAR for in vitro antiplasmodial and hERG activity was delineated.

Total synthesis of (±)-β-bulnesene, (±)-cryptofauronol, (±)-fauronyl acetate, and (±)-valeranone

A versatile method for the preparation of perhydroazulenes and cis-fused 1-decalones is described and exemplified by the synthesis of the title compounds.