Evaluating anthelmintic activity through Caenorhabditis elegans egg hatching assay.

The Caenorhabditis elegans egg hatching methodology is a valuable tool for assessing the anthelmintic activity of drugs and compounds and evaluating anthelmintic drug efficacy. Isolated eggs from gravid adults are exposed to different concentrations of selected drugs and the percentage of egg hatching is determined with respect to the control condition. The assay allows the construction of concentration-response curves and determination of EC50 or EC90 values for egg hatching inhibition. Also, it allows measurements of inhibition as a function of time of exposure. This approach addresses the urgent need for new anthelmintics, as resistance to current treatments poses a significant challenge in parasitic nematode infection. This resistance not only affects humans but also animals and plants, causing significant economic losses in livestock farming and agriculture. By using the free-living nematode C. elegans as a parasitic model organism, researchers can efficiently screen for potential treatments and assess drug combinations for synergistic effects. Importantly, this assay offers a cost-effective and accessible alternative to traditional methods, eliminating the need for specialized infrastructure, hosts, and trained animal maintenance personnel. Additionally, the methodology closely mimics natural conditions, providing insights into egg development and potential therapeutic targets. This method allows for evaluating the direct negative impact of drugs on egg hatching, which correlates with long-term anthelmintic effects, offering advantages in preventing or reducing the transmission and spread of worm infections by eggs. Overall, this approach represents a significant advancement for anthelmintic discovery, offering both practical applications and avenues for further scientific research. •The C. elegans egg hatching assay is a robust and effective method for assessing the anthelmintic potential of various drugs and compounds, allowing the generation of concentration-response curves.•By leveraging the free-living nematode C. elegans as a parasitic model organism, this method facilitates efficient screening of potential treatments and evaluation of drug combinations.•The method addresses the urgent need for new anthelmintics, offering a cost-effective and accessible alternative to traditional approaches.

Potent Anthelmintic Activity of Chalcones Synthesized by an Effective Green Approach.

There is currently an urgent need for new anthelmintic agents due to increasing resistance to the limited available drugs. The chalcone scaffold is a privileged structure for developing new drugs and has been shown to exhibit potential antiparasitic properties. We synthesized a series of chalcones via Claisen-Schmidt condensation, introducing a novel recoverable catalyst derived from biochar obtained from the pyrolysis of tree pruning waste. Employing microwave irradiation and a green solvent, this approach demonstrated significantly reduced reaction times and excellent compatibility with various functional groups. The result was the generation of a library of functionalized chalcones, exhibiting exclusive (E)-selectivity and high to excellent yields. The chalcone derivatives were evaluated on the free-living nematode Caenorhabditis elegans. The chalcone scaffold, along with two derivatives incorporating a methoxy substituent in either ring, caused a concentration-dependent decrease of worm motility, revealing potent anthelmintic activity and spastic paralysis not mediated by the nematode levamisole-sensitive nicotinic receptor. The combination of both methoxy groups in the chalcone scaffold resulted in a less potent compound causing worm hypermotility at the short term, indicating a distinct molecular mechanism. Through the identification of promising drug candidates, this work addresses the demand for new anthelmintic drugs while promoting sustainable chemistry.

New Multitarget Molecules Derived from Caffeine as Potentiators of the Cholinergic System.

Cholinergic deficit is a characteristic factor of several pathologies, such as myasthenia gravis, some types of congenital myasthenic syndromes, and Alzheimer's Disease. Two molecular targets for its treatment are acetylcholinesterase (AChE) and nicotinic acetylcholine receptor (nAChR). In previous studies, we found that caffeine behaves as a partial nAChR agonist and confirmed that it inhibits AChE. Here, we present new bifunctional caffeine derivatives consisting of a theophylline ring connected to amino groups by different linkers. All of them were more potent AChE inhibitors than caffeine. Furthermore, although some of them also activated muscle nAChR as partial agonists, not all of them stabilized nAChR in its desensitized conformation. To understand the molecular mechanism underlying these results, we performed docking studies on AChE and nAChR. The nAChR agonist behavior of the compounds depends on their accessory group, whereas their ability to stabilize the receptor in a desensitized state depends on the interactions of the linker at the binding site. Our results show that the new compounds can inhibit AChE and activate nAChR with greater potency than caffeine and provide further information on the modulation mechanisms of pharmacological targets for the design of novel therapeutic interventions in cholinergic deficit.

The diverse family of Cys-loop receptors in Caenorhabditis elegans: insights from electrophysiological studies.

Cys-loop receptors integrate a large family of pentameric ligand-gated ion channels that mediate fast ionotropic responses in vertebrates and invertebrates. Their vital role in converting neurotransmitter recognition into an electrical impulse makes these receptors essential for a great variety of physiological processes. In vertebrates, the Cys-loop receptor family includes the cation-selective channels, nicotinic acetylcholine and 5-hydroxytryptamine type 3 receptors, and the anion-selective channels, GABAA and glycine receptors, whereas in invertebrates, the repertoire is significantly larger. The free-living nematode Caenorhabditis elegans has the largest known Cys-loop receptor family as well as unique receptors that are absent in vertebrates and constitute attractive targets for anthelmintic drugs. Given the large number and variety of Cys-loop receptor subunits and the multiple possible ways of subunit assembly, C. elegans offers a large diversity of receptors although only a limited number of them have been characterized to date. C. elegans has emerged as a powerful model for the study of the nervous system and human diseases as well as a model for antiparasitic drug discovery. This nematode has also shown promise in the pharmaceutical industry search for new therapeutic compounds. C. elegans is therefore a powerful model organism to explore the biology and pharmacology of Cys-loop receptors and their potential as targets for novel therapeutic interventions. In this review, we provide a comprehensive overview of what is known about the function of C. elegans Cys-loop receptors from an electrophysiological perspective.

Side Groups Convert the α7 Nicotinic Receptor Agonist Ether Quinuclidine into a Type I Positive Allosteric Modulator.

The quinuclidine scaffold has been extensively used for the development of nicotinic acetylcholine receptor (nAChR) agonists, with hydrophobic substituents at position 3 of the quinuclidine framework providing selectivity for α7 nAChRs. In this study, six new ligands (4-9) containing a 3-(pyridin-3-yloxy)quinuclidine moiety (ether quinuclidine) were synthesized to gain a better understanding of the structural-functional properties of ether quinuclidines. To evaluate the pharmacological activity of these ligands, two-electrode voltage-clamp and single-channel recordings were performed. Only ligand 4 activated α7 nAChR. Ligands 5 and 7 had no effects on α7 nAChR, but ligands 6, 8, and 9 potentiated the currents evoked by ACh. Ligand 6 was the most potent and efficacious of the potentiating ligands, with an estimated EC50 for potentiation of 12.6 ± 3.32 µM and a maximal potentiation of EC20 ACh responses of 850 ± 120%. Ligand 6 increased the maximal ACh responses without changing the kinetics of the current responses. At the single-channel level, the potentiation exerted by ligand 6 was evidenced in the low micromolar concentration range by the appearance of prolonged bursts of channel openings. Furthermore, computational studies revealed the preference of ligand 6 for an intersubunit site in the transmembrane domain and highlighted some putative key interactions that explain the different profiles of the synthesized ligands. Notably, Met276 in the 15' position of the transmembrane domain 2 almost abolished the effects of ligand 6 when mutated to Leu. We conclude that ligand 6 is a novel type I positive allosteric modulator (PAM-I) of α7 nAChR.

Regulation of nicotinic acetylcholine receptors by post-translational modifications.

Nicotinic acetylcholine receptors (nAChRs) comprise a family of pentameric ligand-gated ion channels widely distributed in the central and peripheric nervous system and in non-neuronal cells. nAChRs are involved in chemical synapses and are key actors in vital physiological processes throughout the animal kingdom. They mediate skeletal muscle contraction, autonomic responses, contribute to cognitive processes, and regulate behaviors. Dysregulation of nAChRs is associated with neurological, neurodegenerative, inflammatory and motor disorders. In spite of the great advances in the elucidation of nAChR structure and function, our knowledge about the impact of post-translational modifications (PTMs) on nAChR functional activity and cholinergic signaling has lagged behind. PTMs occur at different steps of protein life cycle, modulating in time and space protein folding, localization, function, and protein-protein interactions, and allow fine-tuned responses to changes in the environment. A large body of evidence demonstrates that PTMs regulate all levels of nAChR life cycle, with key roles in receptor expression, membrane stability and function. However, our knowledge is still limited, restricted to a few PTMs, and many important aspects remain largely unknown. There is thus a long way to go to decipher the association of aberrant PTMs with disorders of cholinergic signaling and to target PTM regulation for novel therapeutic interventions. In this review we provide a comprehensive overview of what is known about how different PTMs regulate nAChR.

Cannabidiol as a modulator of α7 nicotinic receptors.

Cannabidiol (CBD), an important terpenoid compound from marijuana with no psychoactive effects, has become of great pharmaceutical interest for several health conditions. As CBD is a multitarget drug, there is a need to establish the molecular mechanisms by which CBD may exert therapeutic as well as adverse effects. The α7 nicotinic acetylcholine receptor (α7 nAChR) is a cation-permeable ACh-gated channel present in the nervous system and in non-neuronal cells. It is involved in different pathological conditions, including neurological and neurodegenerative disorders, inflammation, and cancer. By high-resolution single-channel recordings and confocal microscopy, we here reveal how CBD modulates α7 nAChR ionotropic and metabotropic functions. CBD leads to a profound concentration-dependent decrease of α7 nAChR single-channel activity with an IC50 in the sub-micromolar range. The inhibition of α7 nAChR activity, which takes place through a membrane pathway, is neither mediated by receptor phosphorylation nor overcome by positive allosteric modulators and is compatible with CBD stabilization of resting or desensitized α7 nAChR conformational states. CBD modulation is complex as it also leads to the later appearance of atypical, low-frequency α7 nAChR channel openings. At the cellular level, CBD inhibits the increase in intracellular calcium triggered by α7 nAChR activation, thus decreasing cell calcium responses. The modulation of α7 nAChR is of pharmacological relevance and should be considered in the evaluation of CBD potential therapeutic uses. Thus, our study provides novel molecular information of CBD multiple actions and targets, which is required to set the basis for prospective applications in human health.

The nematode serotonin-gated chloride channel MOD-1: A novel target for anthelmintic therapy.

Anthelmintics are used to treat human and veterinary parasitic diseases and to reduce crop and livestock production loss associated with parasitosis. The free-living nematode Caenorhabditis elegans, a model system for anthelmintic drug discovery, has a serotonin (5-HT)-gated chloride channel, MOD-1, which belongs to the Cys-loop receptor family and modulates locomotory and behavioral functions. Since MOD-1 is unique to nematodes, it is emerging as an attractive anthelmintic drug target, but details of MOD-1 function are unclear. Here, we revealed novel aspects of MOD-1 function from the molecular level to the organism level and identified compounds targeting this receptor, which may provide new directions for anthelmintic drug discovery. We used whole-cell current recordings from heterologously expressed MOD-1 to show that tryptamine (Tryp), a weak partial agonist of vertebrate serotonin type 3 (5-HT3) receptors, efficaciously activates MOD-1. A screen for modulators revealed that GABAergic ligands piperazine (PZE) and muscimol reduce 5-HT-elicited currents, thus identifying novel MOD-1 allosteric inhibitors. Next, we performed locomotor activity assays, and we found 5-HT and Tryp rapidly decrease worm motility, which is reversible only at low 5-HT concentrations. Mutants lacking MOD-1 are partially resistant to both drugs, demonstrating its role in locomotion. Acting as an antagonist of MOD-1, we showed PZE reduces the locomotor effects of exogenous 5-HT. Therefore, Tryp- and PZE-derived compounds, acting at MOD-1 through different molecular mechanisms, emerge as promising anthelmintic agents. This study enhances our knowledge of the function and drug selectivity of Cys-loop receptors and postulates MOD-1 as a potential target for anthelmintic therapy.

A Functional Interaction Between Y674-R685 Region of the SARS-CoV-2 Spike Protein and the Human α7 Nicotinic Receptor.

The α7 nicotinic acetylcholine receptor (nAChR) is present in neuronal and non-neuronal cells and has anti-inflammatory actions. Molecular dynamics simulations suggested that α7 nAChR interacts with a region of the SARS-CoV-2 spike protein (S), and a potential contribution of nAChRs to COVID-19 pathophysiology has been proposed. We applied whole-cell and single-channel recordings to determine whether a peptide corresponding to the Y674-R685 region of the S protein can directly affect α7 nAChR function. The S fragment exerts a dual effect on α7. It activates α7 nAChRs in the presence of positive allosteric modulators, in line with our previous molecular dynamics simulations showing favourable binding of this accessible region of the S protein to the nAChR agonist binding site. The S fragment also exerts a negative modulation of α7, which is evidenced by a profound concentration-dependent decrease in the durations of openings and activation episodes of potentiated channels and in the amplitude of macroscopic responses elicited by ACh. Our study identifies a potential functional interaction between α7 nAChR and a region of the S protein, thus providing molecular foundations for further exploring the involvement of nAChRs in COVID-19 pathophysiology.

Vigilancia epidemiológica de COVID-19 mediante testeos agrupados focalizada en trabajadores de salud asintomáticos en Bahía Blanca / Epidemiological surveillance of COVID-19 through group testing focused on asymptomatic health workers in Bahía Blanca

INTRODUCCIÓN: Uno de los desafíos más relevantes al comienzo de la pandemia consistió en implementar estrategias dirigidas a prevenir la transmisión del virus SARS-CoV-2 y mitigar el impacto de la COVID-19. El propósito de este estudio fue contribuir a reducir la transmisión comunitaria a través de una iniciativa interinstitucional, cuyos objetivos fueron validar un método de detección de ARN del SARS-CoV-2 e implementar y evaluar la vigilancia en trabajadores de salud asintomáticos de instituciones de salud pública de Bahía Blanca. MÉTODOS: Se validó una prueba de detección del gen E del coronavirus mediante RT-PCR a partir de ARN aislado de hisopados nasofaríngeos. Para aumentar la capacidad de testeo se validó la detección del ARN viral en muestras agrupadas (pooles). Se realizó un estudio de cohorte prospectiva entre el 15/09/20 y el 15/09/21. RESULTADOS: La sensibilidad y especificidad de la prueba en muestras individuales fue del 95% (IC 95%: 85%-100%). La sensibilidad de la detección en pooles fue del 73% (IC 95%: 46%-99%) y la especificidad, del 100%. A lo largo de la vigilancia se incluyeron 855 trabajadores y 1764 hisopados, con una incidencia acumulada anual de 2,3% (IC 95%: 1,2%-3,4%). Se detectaron 20 casos asintomáticos positivos. DISCUSIÓN: El tamizaje de trabajadores de salud asintomáticos en la pandemia contribuyó a reducir el riesgo de brotes hospitalarios. Asimismo, se generó un marco de trabajo interdisciplinario aplicable a otros problemas de salud.

COVID-19 epidemiological surveillance through pool testing focused on asymptomatic healthcare workers in the city of Bahía Blanca / Vigilancia epidemiológica de covid-19 mediante testeos agrupados focalizada en trabajadores de salud asintomáticos en bahía blanca

RESUMEN INTRODUCCIÓN: Uno de los desafíos más relevantes al comienzo de la pandemia consistió en implementar estrategias dirigidas a prevenir la transmisión del virus SARS-CoV-2 y mitigar el impacto de la COVID-19. El propósito de este estudio fue contribuir a reducir la transmisión comunitaria a través de una iniciativa interinstitucional, cuyos objetivos fueron validar un método de detección de ARN del SARS-CoV-2 e implementar y evaluar la vigilancia en trabajadores de salud asintomáticos de instituciones de salud pública de Bahía Blanca. MÉTODOS: Se validó una prueba de detección del gen E del coronavirus mediante RT-PCR a partir de ARN aislado de hisopados nasofaríngeos. Para aumentar la capacidad de testeo se validó la detección del ARN viral en muestras agrupadas (pooles). Se realizó un estudio de cohorte prospectiva entre el 15/09/20 y el 15/09/21. RESULTADOS: La sensibilidad y especificidad de la prueba en muestras individuales fue del 95% (IC 95%: 85%-100%). La sensibilidad de la detección en pooles fue del 73% (IC 95%: 46%-99%) y la especificidad, del 100%. A lo largo de la vigilancia se incluyeron 855 trabajadores y 1764 hisopados, con una incidencia acumulada anual de 2,3% (IC 95%: 1,2%-3,4%). Se detectaron 20 casos asintomáticos positivos. DISCUSIÓN: El tamizaje de trabajadores de salud asintomáticos en la pandemia contribuyó a reducir el riesgo de brotes hospitalarios. Asimismo, se generó un marco de trabajo interdisciplinario aplicable a otros problemas de salud.

ABSTRACT INTRODUCTION: One of the most important challenges at the beginning of the pandemic was to implement strategies to prevent SARS-CoV-2 virus transmission and reduce the impact of COVID-19. The purpose of this study was to contribute to the reduction of community transmission through an interinstitutional initiative, aimed at validating a SARS-CoV-2 RNA detection method, and at implementing and assessing the surveillance of asymptomatic infected healthcare workers (HCWs) at public health institutions in the city of Bahía Blanca. METHODS: To validate a coronavirus RNA detection method, RNA was extracted from nasopharyngeal swabs and identification of the viral E gene was done by RT-PCR. Validation of sample pooling was performed to increase the testing capacity. A prospective cohort study was conducted from 15 September 2020 to 15 September 2021. RESULTS: The sensitivity and specificity of the test in individual samples was 95% (CI 95%: 85%-100%). The sensitivity of the pooling strategy was 73% (CI 95%: 46%-99%) and the specificity was 100%. A total of the 855 HCWs were included in the surveillance and 1764 swabs were performed, with an annual cumulative incidence of 2.3% (CI 95%: 1,2%-3,4%), and 20 positive asymptomatic cases were detected. DISCUSSION: The screening of asymptomatic HCWs during the pandemic contributed to reduce the risk of outbreaks in hospital settings. Moreover, an interdisciplinary team framework applicable to other health problems was generated.

Tyrosine phosphorylation differentially fine-tunes ionotropic and metabotropic responses of human α7 nicotinic acetylcholine receptor.

The α7 nicotinic acetylcholine receptor is involved in neurological, neurodegenerative, and inflammatory disorders. It operates both as a ligand-gated cationic channel and as a metabotropic receptor in neuronal and non-neuronal cells. As protein phosphorylation is an important cell function regulatory mechanism, deciphering how tyrosine phosphorylation modulates α7 dual ionotropic/metabotropic molecular function is required for understanding its integral role in physiological and pathological processes. α7 single-channel activity elicited by ACh appears as brief isolated openings and less often as episodes of few openings in quick succession. The reduction of phosphorylation by tyrosine kinase inhibition increases the duration and frequency of activation episodes, whereas the inhibition of phosphatases has the opposite effect. Removal of two tyrosine residues at the α7 intracellular domain recapitulates the effects mediated by tyrosine kinase inhibition. The tyrosine-free mutant receptor shows longer duration-activation episodes, reduced desensitization rate and significantly faster recovery from desensitization, indicating that phosphorylation decreases α7 channel activity by favoring the desensitized state. However, the mutant receptor is incapable of triggering ERK1/2 phosphorylation in response to the α7-agonist. Thus, while tyrosine phosphorylation is absolutely required for α7-triggered ERK pathway, it negatively modulates α7 ionotropic activity. Overall, phosphorylation/dephosphorylation events fine-tune the integrated cell response mediated by α7 activation, thus having a broad impact on α7 cholinergic signaling.

Gender inequality in Latin American Neuroscience community.

Gender bias in Science, Technology, Engineering, and Mathematics (STEM) has been identified since a long time ago. However, gender imbalance in neuroscience has not yet been adequately explored worldwide. Here we report the first study on the development of the careers of men and women neuroscientists in Latin America in relation to family life and their perceptions of obstacles to success. Apart from revealing gender inequality in the neuroscience field, distinctive Latin American traits have become evident, thus providing novel insights into the global comprehension of gender imbalance in the region, which is required for guiding future actions, including the design of public policies in the region.

Loss of Choline Agonism in the Inner Ear Hair Cell Nicotinic Acetylcholine Receptor Linked to the α10 Subunit.

The α9α10 nicotinic acetylcholine receptor (nAChR) plays a fundamental role in inner ear physiology. It mediates synaptic transmission between efferent olivocochlear fibers that descend from the brainstem and hair cells of the auditory sensory epithelium. The α9 and α10 subunits have undergone a distinct evolutionary history within the family of nAChRs. Predominantly in mammalian vertebrates, the α9α10 receptor has accumulated changes at the protein level that may ultimately relate to the evolutionary history of the mammalian hearing organ. In the present work, we investigated the responses of α9α10 nAChRs to choline, the metabolite of acetylcholine degradation at the synaptic cleft. Whereas choline is a full agonist of chicken α9α10 receptors it is a partial agonist of the rat receptor. Making use of the expression of α9α10 heterologous receptors, encompassing wild-type, heteromeric, homomeric, mutant, chimeric, and hybrid receptors, and in silico molecular docking, we establish that the mammalian (rat) α10 nAChR subunit underscores the reduced efficacy of choline. Moreover, we show that whereas the complementary face of the α10 subunit does not play an important role in the activation of the receptor by ACh, it is strictly required for choline responses. Thus, we propose that the evolutionary changes acquired in the mammalian α9α10 nAChR resulted in the loss of choline acting as a full agonist at the efferent synapse, without affecting the triggering of ACh responses. This may have accompanied the fine-tuning of hair cell post-synaptic responses to the high-frequency activity of efferent medial olivocochlear fibers that modulate the cochlear amplifier.

A conserved arginine with non-conserved function is a key determinant of agonist selectivity in α7 nicotinic ACh receptors.

BACKGROUND AND PURPOSE: The α7 and α4ß2* ("*" denotes possibly assembly with another subunit) nicotinic acetylcholine receptors (nAChRs) are the most abundant nAChRs in the mammalian brain. These receptors are the most targeted nAChRs in drug discovery programmes for brain disorders. However, the development of subtype-specific agonists remains challenging due to the high degree of sequence homology and conservation of function in nAChRs. We have developed C(10) variants of cytisine, a partial agonist of α4ß2 nAChR that has been used for smoking cessation. The C(10) methyl analogue used in this study displays negligible affinity for α7 nAChR, while retaining high affinity for α4ß2 nAChR. EXPERIMENTAL APPROACH: The structural underpinning of the selectivity of 10-methylcytisine for α7 and α4ß2 nAChRs was investigated using molecular dynamic simulations, mutagenesis and whole-cell and single-channel current recordings. KEY RESULTS: We identified a conserved arginine in the ß3 strand that exhibits a non-conserved function in nAChRs. In α4ß2 nAChR, the arginine forms a salt bridge with an aspartate residue in loop B that is necessary for receptor expression, whereas in α7 nAChR, this residue is not stabilised by electrostatic interactions, making its side chain highly mobile. This lack of constrain produces steric clashes with agonists and affects the dynamics of residues involved in agonist binding and the coupling network. CONCLUSION AND IMPLICATIONS: We conclude that the high mobility of the ß3-strand arginine in the α7 nAChR influences agonist binding and possibly gating network and desensitisation. The findings have implications for rational design of subtype-selective nAChR agents.

A New Antagonist of Caenorhabditis elegans Glutamate-Activated Chloride Channels With Anthelmintic Activity.

Nematode parasitosis causes significant mortality and morbidity in humans and considerable losses in livestock and domestic animals. The acquisition of resistance to current anthelmintic drugs has prompted the search for new compounds for which the free-living nematode Caenorhabditis elegans has emerged as a valuable platform. We have previously synthetized a small library of oxygenated tricyclic compounds and determined that dibenzo[b,e]oxepin-11(6H)-one (doxepinone) inhibits C. elegans motility. Because doxepinone shows potential anthelmintic activity, we explored its behavioral effects and deciphered its target site and mechanism of action on C. elegans. Doxepinone reduces swimming rate, induces paralysis, and decreases the rate of pharyngeal pumping required for feeding, indicating a marked anthelmintic activity. To identify the main drug targets, we performed an in vivo screening of selected strains carrying mutations in Cys-loop receptors involved in worm locomotion for determining resistance to doxepinone effects. A mutant strain that lacks subunit genes of the invertebrate glutamate-gated chloride channels (GluCl), which are targets of the widely used antiparasitic ivermectin (IVM), is resistant to doxepinone effects. To unravel the molecular mechanism, we measured whole-cell currents from GluClα1/ß receptors expressed in mammalian cells. Glutamate elicits macroscopic currents whereas no responses are elicited by doxepinone, indicating that it is not an agonist of GluCls. Preincubation of the cell with doxepinone produces a statistically significant decrease of the decay time constant and net charge of glutamate-elicited currents, indicating that it inhibits GluCls, which contrasts to IVM molecular actions. Thus, we identify doxepinone as an attractive scaffold with promising anthelmintic activity and propose the inhibition of GluCls as a potential anthelmintic mechanism of action.

Orthosteric and Allosteric Activation of Human 5-HT3A Receptors.

The serotonin type 3 receptor (5-HT3) is a ligand-gated ion channel that converts the binding of the neurotransmitter serotonin (5-HT) into a transient cation current that mediates fast excitatory responses in peripheral and central nervous systems. Information regarding the activation and modulation of the human 5-HT3 type A receptor has been based only on macroscopic current measurements because of its low ion conductance. By constructing a high-conductance human 5-HT3A receptor, we here revealed mechanistic information regarding the orthosteric activation by 5-HT and by the partial agonist tryptamine, and the allosteric activation by the terpenoids, carvacrol, and thymol. Terpenoids potentiated macroscopic currents elicited by the orthosteric agonist and directly elicited currents with slow-rising phases and submaximal amplitudes. At the single-channel level, activation by orthosteric and allosteric agonists appeared as openings in quick succession (bursts) that showed no ligand concentration dependence. Bursts were grouped into long-duration clusters in the presence of 5-HT and even longer in the presence of terpenoids, whereas they remained isolated in the presence of tryptamine. Kinetic analysis revealed that allosteric and orthosteric activation mechanisms can be described by the same scheme that includes transitions of the agonist-bound receptor to closed intermediate states before opening (priming). Reduced priming explained the partial agonism of tryptamine; however, equilibrium constants for gating and priming were similar for 5-HT and terpenoid activation. Thus, our kinetic analysis revealed that terpenoids are efficacious agonists for 5-HT3A receptors. These findings not only extend our knowledge about the human 5-HT3A molecular function but also provide novel insights into the mechanisms of action of allosteric ligands, which are of increasing interest as therapeutic drugs in all the superfamily.

Design, Synthesis, and Functional Evaluation of a Novel Series of Phosphonate-Functionalized 1,2,3-Triazoles as Positive Allosteric Modulators of α7 Nicotinic Acetylcholine Receptors.

The α7 nicotinic acetylcholine receptor is a pentameric ligand-gated ion channel widely distributed in the central nervous system, mainly in the hippocampus and cortex. The enhancement of its activity by positive allosteric modulators (PAMs) is a promising therapeutic strategy for cognitive deficits and neurodegenerative disorders. With the aim of developing novel scaffolds with PAM activity, we designed and synthesized a series of phosphonate-functionalized 1,4-disubstituted 1,2,3-triazoles using supported copper nanoparticles as the cycloaddition reaction catalyst and evaluated their activity on α7 receptors by single-channel and whole-cell recordings. We identified several triazole derivatives that displayed PAM activity, with the compound functionalized with the methyl phosphonate group being the most efficacious one. At the macroscopic level, α7 potentiation was evidenced as an increase of the maximal currents elicited by acetylcholine with minimal effects on desensitization, recapitulating the actions of type I PAMs. At the single-channel level, the active compounds did not affect channel amplitude but significantly increased the duration of channel openings and activation episodes. By using chimeric and mutant α7 receptors, we demonstrated that the new α7 PAMs share transmembrane structural determinants of potentiation with other chemically nonrelated PAMs. To gain further insight into the chemical basis of potentiation, we applied structure-activity relationship strategies involving modification of the chain length, inversion of substituent positions in the triazole ring, and changes in the aromatic nucleus. Our findings revealed that the phosphonate-functionalized 1,4-disubstituted 1,2,3-triazole is a novel pharmacophore for the development of therapeutic agents for neurological and neurodegenerative disorders associated with cholinergic dysfunction.

Mechanism of calcium potentiation of the α7 nicotinic acetylcholine receptor.

The α7 nicotinic acetylcholine receptor (nAChR) is among the most abundant types of nAChR in the brain, yet the ability of nerve-released ACh to activate α7 remains enigmatic. In particular, a major population of α7 resides in extra-synaptic regions where the ACh concentration is reduced, owing to dilution and enzymatic hydrolysis, yet ACh shows low potency in activating α7. Using high-resolution single-channel recording techniques, we show that extracellular calcium is a powerful potentiator of α7 activated by low concentrations of ACh. Potentiation manifests as robust increases in the frequency of channel opening and the average duration of the openings. Molecular dynamics simulations reveal that calcium binds to the periphery of the five ligand binding sites and is framed by a pair of anionic residues from the principal and complementary faces of each site. Mutation of residues identified by simulation prevents calcium from potentiating ACh-elicited channel opening. An anionic residue is conserved at each of the identified positions in all vertebrate species of α7. Thus, calcium associates with a novel structural motif on α7 and is an obligate cofactor in regions of limited ACh concentration.

Caenorhabditis elegans muscle Cys-loop receptors as novel targets of terpenoids with potential anthelmintic activity.

The anthelmintic treatment of nematode infections remains the pillar of worm control in both human and veterinary medicine. Since control is threatened by the appearance of drug resistant nematodes, there is a need to develop novel compounds, among which phytochemicals constitute potential anthelmintic agents. Caenorhabditis elegans has been pivotal in anthelmintic drug discovery and in revealing mechanisms of drug action and resistance. By using C. elegans, we here revealed the anthelmintic actions of three plant terpenoids -thymol, carvacrol and eugenol- at the behavioral level. Terpenoids produce a rapid paralysis of worms with a potency rank order carvacrol > thymol > eugenol. In addition to their paralyzing activity, they also inhibit egg hatching, which would, in turn, lead to a broader anthelmintic spectrum of activity. To identify drug targets, we performed an in vivo screening of selected strains carrying mutations in receptors involved in worm locomotion for determining resistance to the paralyzing effect of terpenoids. The assays revealed that two Cys-loop receptors with key roles in worm locomotion -Levamisole sensitive nicotinic receptor (L-AChR) and GABA(A) (UNC-49) receptor- are involved in the paralyzing effects of terpenoids. To decipher the mechanism by which terpenoids affect these receptors, we performed electrophysiological studies using a primary culture of C. elegans L1 muscle cells. Whole cell recordings from L1 cells demonstrated that terpenoids decrease macroscopic responses of L-AChR and UNC-49 receptor to their endogenous agonists, thus acting as inhibitors. Single-channel recordings from L-AChR revealed that terpenoids decrease the frequency of opening events, probably by acting as negative allosteric modulators. The fact that terpenoids act at different receptors may have important advantages regarding efficacy and development of resistance. Thus, our findings give support to the use of terpenoids as either an alternative or a complementary anthelmintic strategy to overcome the ever-increasing resistance of parasites to classical anthelmintic drugs.