Potential of 3-octanone as a lure and kill agent for control of the Brown garden snail.

The brown garden snail (Cornu aspersum) is a major agricultural pest, causing damage to a wide range of economically important crops. Withdrawal or restricted use of pollutant molluscicides like metaldehyde has prompted a search for more benign control products. This study investigated the response of snails to 3-octanone; a volatile organic compound (VOCs) produced by the insect pathogenic fungus Metarhizium brunneum. Concentrations of 1 - 1000 ppm of 3-octanone were first assessed in laboratory choice assays to determine behavioural response. Repellent activity was found at 1000 ppm whereas attractance was found for the lower concentrations of 1, 10 and 100 ppm. These three concentrations of 3-octanone were carried forward in field evaluations to assess potential for use in "lure and kill" strategies. The highest concentration (100 ppm) was the most attractive to the snails but also the most lethal. Even at the lowest concentration this compound proved toxic effects making 3-octanone an excellent candidate for the development as a snail attractant and molluscicide.

Pharmacological Profile of the Novel Antiepileptic Drug Candidate Padsevonil: Interactions with Synaptic Vesicle 2 Proteins and the GABAA Receptor.

Padsevonil is an antiepileptic drug (AED) candidate synthesized in a medicinal chemistry program initiated to rationally design compounds with high affinity for synaptic vesicle 2 (SV2) proteins and low-to-moderate affinity for the benzodiazepine binding site on GABAA receptors. The pharmacological profile of padsevonil was characterized in binding and electrophysiological experiments. At recombinant SV2 proteins, padsevonil's affinity for SV2A was greater than that of levetiracetam and brivaracetam (pKi 8.5, 5.2, and 6.6, respectively). Unlike the latter AEDs, both selective SV2A ligands, padsevonil also displayed high affinity for the SV2B and SV2C isoforms (pKi 7.9 and 8.5, respectively). Padsevonil's interaction with SV2A differed from that of levetiracetam and brivaracetam; it exhibited slower binding kinetics: dissociation t 1/2 30 minutes from the human protein at 37°C compared with <0.5 minute for levetiracetam and brivaracetam. In addition, its binding was not potentiated by the allosteric modulator UCB1244283. At recombinant GABAA receptors, padsevonil displayed low to moderate affinity (pIC50≤6.1) for the benzodiazepine site, and in electrophysiological studies, its relative efficacy compared with zolpidem (full-agonist reference drug) was 40%, indicating partial agonist properties. In in vivo (mice) receptor occupancy studies, padsevonil exhibited SV2A occupancy at low ED50 (0.2 mg/kg) and benzodiazepine site occupancy at higher doses (ED50 36 mg/kg), supporting in vitro results. Padsevonil's selectivity for its intended targets was confirmed in profiling studies, where it lacked significant effects on a wide variety of ion channels, receptors, transporters, and enzymes. Padsevonil is a first-in-class AED candidate with a unique target profile allowing for presynaptic and postsynaptic activity. SIGNIFICANCE STATEMENT: Padsevonil is an antiepileptic drug candidate developed as a single molecular entity interacting with both presynaptic and postsynaptic targets. Results of in vitro and in vivo radioligand binding assays confirmed this target profile: padsevonil displayed nanomolar affinity for the three synaptic vesicle 2 protein isoforms (SV2A, B, and C) and micromolar affinity for the benzodiazepine binding site on GABAA receptors. Furthermore, padsevonil showed greater affinity for and slower binding kinetics at SV2A than the selective SV2A ligands, levetiracetam, and brivaracetam.

Functional characterization of the antiepileptic drug candidate, padsevonil, on GABAA receptors.

OBJECTIVE: The antiepileptic drug candidate, padsevonil, is the first in a novel class of drugs designed to interact with both presynaptic and postsynaptic therapeutic targets: synaptic vesicle 2 proteins and γ-aminobutyric acid type A receptors (GABAA Rs), respectively. Functional aspects of padsevonil at the postsynaptic target, GABAA Rs, were characterized in experiments reported here. METHODS: The effect of padsevonil on GABA-mediated Cl- currents was determined by patch clamp on recombinant human GABAA Rs (α1ß2γ2) stably expressed in a CHO-K1 cell line and on native GABAA Rs in cultured rat primary cortical neurons. Padsevonil selectivity for GABAA R subtypes was evaluated using a two-electrode voltage clamp on recombinant human GABAA Rs (α1-5/ß2/γ2) in Xenopus oocytes. RESULTS: In recombinant GABAA Rs, padsevonil did not evoke Cl- currents in the absence of the agonist GABA. However, when co-administered with GABA at effective concentration (EC)20 , padsevonil potentiated GABA responses by 167% (EC50 138 nmol/L) and demonstrated a relative efficacy of 41% compared with zolpidem, a reference benzodiazepine site agonist. Similarly, padsevonil demonstrated GABA-potentiating activity at native GABAA Rs (EC50 208 nmol/L) in cultured rat cortical neurons. Padsevonil also potentiated GABA (EC20 ) responses in GABAA Rs expressed in oocytes, with higher potency at α1- and α5-containing receptors (EC50 295 and 281 nmol/L) than at α2- and α3-containing receptors (EC50 1737 and 2089 nmol/L). Compared with chlordiazepoxide-a nonselective, full GABAA R agonist-the relative efficacy of padsevonil was 60% for α1ß2γ2, 26% for α2ß2γ2, 56% for α3ß2γ2, and 41% for α5ß2γ2; no activity was observed at benzodiazepine-insensitive α4ß2γ2 receptors. SIGNIFICANCE: Results of functional investigations on recombinant and native neuronal GABAA Rs show that padsevonil acts as a positive allosteric modulator of these receptors, with a partial agonist profile at the benzodiazepine site. These properties may confer better tolerability and lower potential for tolerance development compared with classic benzodiazepines currently used in the clinic.

Crystal structure of the adenosine A2A receptor bound to an antagonist reveals a potential allosteric pocket.

The adenosine A2A receptor (A2AR) has long been implicated in cardiovascular disorders. As more selective A2AR ligands are being identified, its roles in other disorders, such as Parkinson's disease, are starting to emerge, and A2AR antagonists are important drug candidates for nondopaminergic anti-Parkinson treatment. Here we report the crystal structure of A2A receptor bound to compound 1 (Cmpd-1), a novel A2AR/N-methyl d-aspartate receptor subtype 2B (NR2B) dual antagonist and potential anti-Parkinson candidate compound, at 3.5 Å resolution. The A2A receptor with a cytochrome b562-RIL (BRIL) fusion (A2AR-BRIL) in the intracellular loop 3 (ICL3) was crystallized in detergent micelles using vapor-phase diffusion. Whereas A2AR-BRIL bound to the antagonist ZM241385 has previously been crystallized in lipidic cubic phase (LCP), structural differences in the Cmpd-1-bound A2AR-BRIL prevented formation of the lattice observed with the ZM241385-bound receptor. The crystals grew with a type II crystal lattice in contrast to the typical type I packing seen from membrane protein structures crystallized in LCP. Cmpd-1 binds in a position that overlaps with the native ligand adenosine, but its methoxyphenyl group extends to an exosite not previously observed in other A2AR structures. Structural analysis revealed that Cmpd-1 binding results in the unique conformations of two tyrosine residues, Tyr91.35 and Tyr2717.36, which are critical for the formation of the exosite. The structure reveals insights into antagonist binding that are not observed in other A2AR structures, highlighting flexibility in the binding pocket that may facilitate the development of A2AR-selective compounds for the treatment of Parkinson's disease.

Dried blood spot and mini-tube blood sample collection kits for postal HIV testing services: a comparative review of successes in a real-world setting.

OBJECTIVES: This is a comparative review between using dried blood spot (DBS) and mini-tube (MT) HIV sampling kits as part of an online sexually transmitted infection (STI) postal testing service. England has recently seen increases in internet-based and postal (eHealth) STI services. Expanding accessibility and testing for patients, cost implications and narrowing the HIV undiagnosed margin are drivers for this. METHODS: In 2017, data were reviewed from an online postal STI kit requesting service at a time of transitioning from MT to DBS. We compared the STI postal kit and HIV blood sample return rates, and the successful processing/analysis rates of the DBS and MT kits. Descriptive statistics were applied to participant characteristics, with Pearson's χ2 or Fisher exact test used to demonstrate statistical differences. We also describe and calculate a 'request-to-result ratio' (RRR) for both kit types. The RRR is defined as the number of online kit requests required to produce one successfully analysed result. RESULTS: 550 STI postal kit requests from a North-West of England region were reviewed from 13 June 2017 to 22 September 2017 (275 MT, 275 DBS). Baseline characteristics between the two groups were comparable (63% woman, 90% white British and 86% heterosexual with a median age of 26 years). The successful processing rate for the DBS was 98.8% c.f. 55.7% for the MT (p<0.001). The RRR for MT was 2.96, c.f. 1.70 for DBS. There was a 5.4% false positive HIV rate in the MT c.f. none in the DBS. CONCLUSIONS: This comparative analysis suggests that in this community setting, the use of postal HIV DBS kits resulted in a significantly improved RRR compared with MT. The biggest factor was the large number of MT samples not analysed due to inadequate blood volumes. The unexpected level of false positive results in the MT samples needs confirming in larger studies.

Further evidence for a differential interaction of brivaracetam and levetiracetam with the synaptic vesicle 2A protein.

Brivaracetam (BRV) and levetiracetam (LEV) are effective antiepileptic drugs that bind selectively to the synaptic vesicle 2A (SV2A) protein. BRV differs from LEV in preclinical studies in that it exhibits a more potent and complete seizure protection across animal models. We reported previously that an allosteric modulator of the SV2A protein had differential effects on BRV compared with LEV, suggesting that they act at different sites or with different conformations of the SV2A protein. If this is the case, then we hypothesized that mutations of specific amino acids in the SV2A protein may have differential effects on BRV and LEV binding by the modulator. Mutation of some amino acids identified previously in the binding site of racetams to the SV2A protein had marked effects on binding of both [3 H]BRV and [3 H]LEV (eg, W300F, F277A, G303A, F658A, Y462A, W666A, I663A, D670A, and V661A). However, 3 amino acids were identified (K694, I273, and S294) in which mutation lost the effect of the modulator on [3 H]LEV binding with no effect on the modulation of [3 H]BRV binding. These results confirm that BRV and LEV bind to the human synaptic vesicle 2A protein at closely related sites but interact with these sites in a different way.

Evidence for a differential interaction of brivaracetam and levetiracetam with the synaptic vesicle 2A protein.

OBJECTIVE: Brivaracetam (BRV) and levetiracetam (LEV) are effective antiepileptic drugs that bind selectively to the synaptic vesicle 2A (SV2A) protein. However, BRV differs from LEV in that it exhibits more potent and complete seizure suppression in animal models including in amygdala-kindled mice, where BRV afforded nearly complete seizure suppression. This raises the possibility that aside from potency differences, BRV and LEV may interact differently with the SV2A protein, which is not apparent in radioligand-binding competition studies. In this study, we used a recently identified SV2A allosteric modulator, UCB1244283, that appears to induce conformational changes in SV2A, to probe the binding properties of labeled BRV and LEV. METHODS: Radioligand binding studies were carried out using [3 H]BRV and [3 H]LEV. Studies were performed in membranes from both recombinant cells expressing human SV2A protein and human brain tissue. RESULTS: The modulator increased the binding of both radioligands but by different mechanisms. For [3 H]BRV, the increase was driven mainly by an increase in affinity, whereas for [3 H]LEV, the increase was due to an increase in the number of apparent binding sites. Kinetic studies confirmed this differential effect. SIGNIFICANCE: These studies suggest that LEV and BRV may act at different binding sites or interact with different conformational states of the SV2A protein. It is possible that some of the pharmacologic differences between BRV and LEV could be due to different interactions with the SV2A protein.

In Vitro and In Vivo Identification of Novel Positive Allosteric Modulators of the Human Dopamine D2 and D3 Receptor.

Agonists at dopamine D2 and D3 receptors are important therapeutic agents in the treatment of Parkinson's disease. Compared with the use of agonists, allosteric potentiators offer potential advantages such as temporal, regional, and phasic potentiation of natural signaling, and that of receptor subtype selectivity. We report the identification of a stereoselective interaction of a benzothiazol racemic compound that acts as a positive allosteric modulator (PAM) of the rat and human dopamine D2 and D3 receptors. The R isomer did not directly stimulate the dopamine D2 receptor but potentiated the effects of dopamine. In contrast the S isomer attenuated the effects of the PAM and the effects of dopamine. In radioligand binding studies, these compounds do not compete for binding of orthosteric ligands, but indeed the R isomer increased the number of high-affinity sites for [(3)H]-dopamine without affecting K(d). We went on to identify a more potent PAM for use in native receptor systems. This compound potentiated the effects of D2/D3 signaling in vitro in electrophysiologic studies on dissociated striatal neurons and in vivo on the effects of L-dopa in the 6OHDA (6-hydroxydopamine) contralateral turning model. These PAMs lacked activity at a wide variety of receptors, lacked PAM activity at related Gi-coupled G protein-coupled receptors, and lacked activity at D1 receptors. However, the PAMs did potentiate [(3)H]-dopamine binding at both D2 and D3 receptors. Together, these studies show that we have identified PAMs of the D2 and D3 receptors both in vitro and in vivo. Such compounds may have utility in the treatment of hypodopaminergic function.

Brivaracetam: Rationale for discovery and preclinical profile of a selective SV2A ligand for epilepsy treatment.

Despite availability of effective antiepileptic drugs (AEDs), many patients with epilepsy continue to experience refractory seizures and adverse events. Achievement of better seizure control and fewer side effects is key to improving quality of life. This review describes the rationale for the discovery and preclinical profile of brivaracetam (BRV), currently under regulatory review as adjunctive therapy for adults with partial-onset seizures. The discovery of BRV was triggered by the novel mechanism of action and atypical properties of levetiracetam (LEV) in preclinical seizure and epilepsy models. LEV is associated with several mechanisms that may contribute to its antiepileptic properties and adverse effect profile. Early findings observed a moderate affinity for a unique brain-specific LEV binding site (LBS) that correlated with anticonvulsant effects in animal models of epilepsy. This provided a promising molecular target and rationale for identifying selective, high-affinity ligands for LBS with potential for improved antiepileptic properties. The later discovery that synaptic vesicle protein 2A (SV2A) was the molecular correlate of LBS confirmed the novelty of the target. A drug discovery program resulted in the identification of anticonvulsants, comprising two distinct families of high-affinity SV2A ligands possessing different pharmacologic properties. Among these, BRV differed significantly from LEV by its selective, high affinity and differential interaction with SV2A as well as a higher lipophilicity, correlating with more potent and complete seizure suppression, as well as a more rapid brain penetration in preclinical models. Initial studies in animal models also revealed BRV had a greater antiepileptogenic potential than LEV. These properties of BRV highlight its promising potential as an AED that might provide broad-spectrum efficacy, associated with a promising tolerability profile and a fast onset of action. BRV represents the first selective SV2A ligand for epilepsy treatment and may add a significant contribution to the existing armamentarium of AEDs.

Development and laboratory validation of a plant-derived repellent blend, effective against Aedes aegypti [Diptera: Culicidae], Anopheles gambiae [Diptera: Culicidae] and Culex quinquefasciatus [Diptera: Culicidae].

Mosquitoes of the genera Aedes, Anopheles and Culex vector a wide range of pathogens seriously affecting humans and livestock on a global scale. Over-reliance on insecticides and repellents has driven research into alternative, naturally-derived compounds to fulfil the same objectives. Steam distilled extracts of four plants with strong, yet attractive, volatile profiles were initially assessed for repellency in a dual-port olfactometer using Aedes aegypti as the model species. Picea sitchensis was found to be the most repellent, proving comparable to leading products when applied at 100% (p = 1.000). Key components of conifer-derived volatile profiles were then screened via electroantennography before those components eliciting an electrophysiological response were assayed individually in the olfactometer; according to WHO protocol. The most promising 5 were selected for reductive analyses to produce an optimised semiochemical blend. This combination, and a further two variations of the blend, were then progressed to a multi-species analysis using the BG-test whereby bite-attempt frequency on hands was assessed under different repellent treatments; assays were compared between Aedes aegypti, Anopheles gambiae and Culex quinquefasciatus. Efficacy was found against all three species, although it was found that Ae. aegypti was the most susceptible to the repellent, with An. gambiae being the least. Here, a novel, naturally-derived blend is presented with weak spatial repellency, as confirmed in laboratory assays. Further work will be required to assess the full extent of the potential of the products, both in terms of field application and species screening; however, the success of the products developed demonstrate that plant metabolites have great capacity for use in the repellent sector; both to improve upon known compounds and to reduce the usage of toxic products currently on the market.

Comparative genomics of Metarhizium brunneum strains V275 and ARSEF 4556: unraveling intraspecies diversity.

Entomopathogenic fungi belonging to the Order Hypocreales are renowned for their ability to infect and kill insect hosts, while their endophytic mode of life and the beneficial rhizosphere effects on plant hosts have only been recently recognized. Understanding the molecular mechanisms underlying their different lifestyles could optimize their potential as both biocontrol and biofertilizer agents, as well as the wider appreciation of niche plasticity in fungal ecology. This study describes the comprehensive whole genome sequencing and analysis of one of the most effective entomopathogenic and endophytic EPF strains, Metarhizium brunneum V275 (commercially known as Lalguard Met52), achieved through Nanopore and Illumina reads. Comparative genomics for exploring intraspecies variability and analyses of key gene sets were conducted with a second effective EPF strain, M. brunneum ARSEF 4556. The search for strain- or species-specific genes was extended to M. brunneum strain ARSEF 3297 and other species of genus Metarhizium, to identify molecular mechanisms and putative key genome adaptations associated with mode of life differences. Genome size differed significantly, with M. brunneum V275 having the largest genome amongst M. brunneum strains sequenced to date. Genome analyses revealed an abundance of plant-degrading enzymes, plant colonization-associated genes, and intriguing intraspecies variations regarding their predicted secondary metabolic compounds and the number and localization of Transposable Elements. The potential significance of the differences found between closely related endophytic and entomopathogenic fungi, regarding plant growth-promoting and entomopathogenic abilities, are discussed, enhancing our understanding of their diverse functionalities and putative applications in agriculture and ecology.

Identification of the odorant binding proteins of Western Flower Thrips (Frankliniella occidentalis), characterization and binding analysis of FoccOBP3 with molecular modelling, molecular dynamics simulations and a confirmatory field trial.

Olfactory systems are indispensable for insects as they, including Western Flower Thrips (Frankliniella occidentalis), use olfactory cues for ovipositing and feeding. F. occidentalis use odorant binding proteins (OBPs) to transport semiochemicals to odorant receptors to induce a behavioural response from the sensillum lymph of the insect's antennae. This study identifies four OBPs of F. occidentalis and analyses their expression at three stages of growth: larvae, adult males and adult females. Further, it investigates the presence of conserved motifs and their phylogenetic relationship to other insect species. Moreover, FoccOBP3 was in silico characterized to analyse its structure along with molecular docking and molecular dynamics simulations to understand its binding with semiochemicals of F. occidentalis. Molecular docking revealed the interactions of methyl isonicotinate, p-anisaldehyde and (S)-(-)-verbenone with FoccOBP3. Moreover, molecular dynamics simulations showed bonding stability of these ligands with FoccOBP3, and field trials validated that Lurem TR (commercial product) and p-anisaldehyde had greater attraction as compared to (S)-(-)-verbenone, given the compound's binding with FoccOBP3. The current study helps in understanding the tertiary structure and interaction of FoccOBP3 with lures using computational and field data and will help in the identification of novel lures of insects in the future, given the importance of binding with OBPs.Communicated by Ramaswamy H. Sarma.

Evaluation of Metarhizium brunneum- and Metarhizium-Derived VOCs as Dual-Active Biostimulants and Pest Repellents in a Wireworm-Infested Potato Field.

Wireworm, the larval stages of click beetles, are a serious pest of tubers, brassicas and other important commercial crops throughout the northern hemisphere. No effective control agent has been developed specifically for them, and many of the pesticides marketed as having secondary application against them have been withdrawn from EU and Asian markets. Metarhizium brunneum, an effective entomopathogenic fungus, and its derived volatile metabolites are known to be effective plant biostimulants and plant protectants, although field efficacy has yet to be validated. Field validation of a combined M. brunneum and derived VOC treatments was conducted in Wales, UK, to assess the effects of each as a wireworm control agent and biostimulant. Plots were treated with Tri-Soil (Trichoderma atroviridae), M. brunneum, 1-octen-3-ol or 3-octanone, or combinations thereof. Treatments were applied subsurface during potato seeding (n = 52), and potatoes were harvested at the end of the growing season. Each potato was weighed individually and scored for levels of wireworm damage. Applications of both the VOCs and the M. brunneum individually were found to significantly decrease wireworm burden (p < 0.001). Combinations of M. brunneum and 3-octanone were also found to significantly decrease wireworm damage (p < 0.001), while no effect on yield was reported, resulting in an increased saleable mass over controls (p < 0.001). Herein, we present a novel 'stimulate and deter' wireworm control strategy that can be used to significantly enhance saleable potato yields and control wireworm populations, even under high pest pressure densities.

Stress-Mediated Responses of Aedes aegypti (Diptera: Culicidae) Larvae When Exposed to Metarhizium brunneum (Hypocreales: Clavicipitaceae) and Toxorhynchites brevipalpis (Diptera: Culicidae).

Aedes aegypti mosquitoes are capable of vectoring a wide range of diseases including dengue, yellow fever, and Zika viruses, with approximately half of the worlds' population at risk from such diseases. Development of combined predator-parasite treatments for the control of larvae consistently demonstrates increased efficacy over single-agent treatments, however, the mechanism behind the interaction remains unknown. Treatments using the natural predator Toxorhynchites brevipalpis and the entomopathogenic fungus Metarhizium brunneum were applied in the laboratory against Ae. aegypti larvae as both individual and combined treatments to determine the levels of interaction between control strategies. Parallel experiments involved the removal of larvae from test arenas at set intervals during the course of the trial to record whole body caspase and phenoloxidase activities. This was measured via luminometric assay to measure larval stress factors underlying the interactions. Combined Metarhizium and Toxorhynchites treatments were seen to drastically reduce lethal times as compared to individual treatments. This was accompanied by increased phenoloxidase and caspase activities in combination treatments after 18 h (p < 0.001). The sharp increases in caspase and phenoloxidase activities suggest that combined treatments act to increase stress factor responses in the larvae that result in rapid mortality above that of either control agent individually. This work concludes that the underlying mechanism for increased lethality in combined parasite-predator treatments may be related to additive stress factors induced within the target host larvae.

Metarhizium brunneum (Hypocreales: Clavicipitaceae) and Its Derived Volatile Organic Compounds as Biostimulants of Commercially Valuable Angiosperms and Gymnosperms.

Metarhizium brunneum is a highly effective entomopathogenic fungus that also functions as a plant biostimulant. It can act as both an endophyte and rhizosphere colonizer; however, the mechanisms driving biostimulation are multifactorial. In this work, oilseed rape (Brassica napus) seeds were grown in composts treated with different concentrations of M. brunneum strains ARSEF 4556 or V275, or the M. brunneum-derived volatile organic compounds 1-octen-3-ol and 3-octanone. Biostimulation efficacy was found to be strongly dose dependent. Concentrations of 1 × 106 conidia g-1 compost were found to be most effective for the M. brunneum, whereas dosages of 1 µL 100 g-1 compost were found to be efficacious for the volatiles. These optimized doses were assessed individually and in combined formulations with a hydrogel against oilseed rape (Brassica napus), sitka spruce (Picea sitchensis), maize (Zea mays) and strawberry (Fragaria annanassa). Both volatile compounds were highly effective biostimulants and were found to increase in biostimulatory efficiency when combined with M. brunneum conidia. Hydrogels were not found to interact with the growth process and may offer avenues for novel formulation technologies. This study demonstrates that Metarhizium-derived volatile organic compounds are actively involved in plant growth promotion and have potential for use in novel formulations to increase the growth of a wide range of commercially relevant crops.

Crystal structure of dopamine D1 receptor in complex with G protein and a non-catechol agonist.

Dopamine D1 receptor (D1R) is an important drug target implicated in many psychiatric and neurological disorders. Selective agonism of D1R are sought to be the therapeutic strategy for these disorders. Most selective D1R agonists share a dopamine-like catechol moiety in their molecular structure, and their therapeutic potential is therefore limited by poor pharmacological properties in vivo. Recently, a class of non-catechol D1R selective agonists with a distinct scaffold and pharmacological properties were reported. Here, we report the crystal structure of D1R in complex with stimulatory G protein (Gs) and a non-catechol agonist Compound 1 at 3.8 Å resolution. The structure reveals the ligand bound to D1R in an extended conformation, spanning from the orthosteric site to extracellular loop 2 (ECL2). Structural analysis reveals that the unique features of D1R ligand binding pocket explains the remarkable selectivity of this scaffold for D1R over other aminergic receptors, and sheds light on the mechanism for D1R activation by the non-catechol agonist.

Orthosteric and allosteric modes of interaction of novel selective agonists of the M1 muscarinic acetylcholine receptor.

Recent years have witnessed the discovery of novel selective agonists of the M(1) muscarinic acetylcholine (ACh) receptor (mAChR). One mechanism invoked to account for the selectivity of such agents is that they interact with allosteric sites. We investigated the molecular pharmacology of two such agonists, 1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone (77-LH-28-1) and 4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine hydrogen chloride (AC-42), at the wild-type M(1) mAChR and three mutant M(1) mAChRs. Both agonists inhibited the binding of the orthosteric antagonist [(3)H]N-methyl scopolamine ([(3)H]NMS) in a manner consistent with orthosteric competition or high negative cooperativity. Functional interaction studies between 77-LH-28-1 and ACh also indicated a competitive mechanism. Dissociation kinetics assays revealed that the agonists could bind allosterically when the orthosteric site was prelabeled with [(3)H]NMS and that 77-LH-28-1 competed with the prototypical allosteric modulator heptane-1,7-bis-[dimethyl-3'-phthalimidopropyl]-ammonium bromide under these conditions. Mutation of the key orthosteric site residues Y(381)A (transmembrane helix 6) and W(101)A (transmembrane helix 3) reduced the affinity of prototypical orthosteric agonists but increased the affinity of the novel agonists. Divergent effects were also noted on agonist signaling efficacies at these mutants. We identified a novel mutation, F(77)I (transmembrane helix 2), which selectively reduced the efficacy of the novel agonists in mediating intracellular Ca(2+) elevation and phosphorylation of extracellular signal regulated kinase 1/2. Molecular modeling suggested a possible "bitopic" binding mode, whereby the agonists extend down into the orthosteric site as well as up toward extracellular receptor regions associated with an allosteric site. It is possible that this bitopic mode may explain the pharmacology of other selective mAChR agonists.

The identification of a selective dopamine D2 partial agonist, D3 antagonist displaying high levels of brain exposure.

The identification of a highly selective D(2) partial agonist, D(3) antagonist tool molecule which demonstrates high levels of brain exposure and selectivity against an extensive range of dopamine, serotonin, adrenergic, histamine, and muscarinic receptors is described.

DNA Footprints: Using Parasites to Detect Elusive Animals, Proof of Principle in Hedgehogs.

The Western European Hedgehog (Erinaceous europaeus) is a nocturnal animal that is in decline in much of Europe, but the monitoring of this species is subjective, prone to error, and an inadequate basis for estimating population trends. Here, we report the use of Crenosoma striatum, a parasitic nematode specific to hedgehogs as definitive hosts, to detect hedgehog presence in the natural environment. This is achieved through collecting and sampling the parasites within their intermediate hosts, gastropoda, a group much simpler to locate and sample in both urban and rural habitats. C. striatum and Crenosoma vulpis were collected post-mortem from the lungs of hedgehogs and foxes, respectively. Slugs were collected in two sessions, during spring and autumn, from Skomer Island (n = 21), which is known to be free of hedgehogs (and foxes); and Pennard, Swansea (n = 42), known to have a healthy hedgehog population. The second internal transcribed spacer of parasite ribosomal DNA was used to develop a highly specific, novel, PCR based multiplex assay. Crenosoma striatum was found only at the site known to be inhabited by hedgehogs, at an average prevalence in gastropods of 10% in spring and autumn. The molecular test was highly specific: One mollusc was positive for both C. striatum and C. vulpis, and differentiation between the two nematode species was clear. This study demonstrates proof of principle for using detection of specific parasite DNA in easily sampled intermediate hosts to confirm the presence of an elusive nocturnal definitive host species. The approach has great potential as an adaptable, objective tool to supplement and support existing ecological survey methods.