Differential role of the menthol-binding residue Y745 in the antagonism of thermally gated TRPM8 channels

<p>Abstract</p> <p>Background</p> <p>TRPM8 is a non-selective cation channel that belongs to the melastatin subfamily of the transient receptor potential (TRP) ion channels. TRPM8 is activated by voltage, cold and cooling compounds such as menthol. Despite its essential role for cold temperature sensing in mammals, the pharmacology of TRPM8 is still in its infancy. Recently, tyrosine 745 (Y745) was identified as a critical residue for menthol sensitivity of the channel. In this report, we study the effect of mutating this residue on the action of several known TRPM8 antagonists: BCTC, capsazepine, SKF96365, and clotrimazole as well as two new inhibitor candidates, econazole and imidazole.</p> <p>Results</p> <p>We show that Y745 at the menthol binding site is critical for inhibition mediated by SKF96365 of cold- and voltage-activated TRPM8 currents. In contrast, the inhibition by other antagonists was unaffected by the mutation (BCTC) or only partially reduced (capsazepine, clotrimazole, econazole), suggesting that additional binding sites exist on the TRPM8 channel from where the inhibitors exert their negative modulation. Indeed, a molecular docking model implies that menthol and SKF96365 interact readily with Y745, while BCTC is unable to bind to this residue.</p> <p>Conclusion</p> <p>In summary, we identify structural elements on the TRPM8 channel that are critical for the action of channel antagonists, providing valuable information for the future design of new, specific modulator compounds.</p

Structural stabilization of botulinum neurotoxins by tyrosine phosphorylation

AbstractTyrosine phosphorylation of botulinum neurotoxins augments their proteolytic activity and thermal stability, suggesting a substantial modification of the global protein conformation. We used Fourier-transform infrared (FTIR) spectroscopy to study changes of secondary structure and thermostability of tyrosine phosphorylated botulinum neurotoxins A (BoNT A) and E (BoNT E). Changes in the conformationally-sensitive amide I band upon phosphorylation indicated an increase of the α-helical content with a concomitant decrease of less ordered structures such as turns and random coils, and without changes in β-sheet content. These changes in secondary structure were accompanied by an increase in the residual amide II absorbance band remaining upon H-D exchange, consistent with a tighter packing of the phosphorylated proteins. FTIR and differential scanning calorimetry (DSC) analyses of the denaturation process show that phosphorylated neurotoxins denature at temperatures higher than those required by non-phosphorylated species. These findings indicate that tyrosine phosphorylation induced a transition to higher order and that the more compact structure presumably imparts to the phosphorylated neurotoxins the higher catalytic activity and thermostability

DD04107-Derived neuronal exocytosis inhibitor peptides: Evidences for synaptotagmin-1 as a putative target

The analgesic peptide DD04107 (Pal-EEMQRR-NH2) and its acetylated analogue inhibit a-calcitonin gene-related peptide (a-CGRP) exocytotic release from primary sensory neurons. Examining the crystal structure of the SNARE-Synaptotagmin-1(Syt1) complex, we hypothesized that these peptides could inhibit neuronal exocytosis by binding to Syt1, hampering at least partially its interaction with the SNARE complex. To address this hypothesis, we first interrogate the role of individual side-chains on the inhibition of a-CGRP release, finding that E1, M3, Q4 and R6 residues were crucial for activity. CD and NMR conformational analysis showed that linear peptides have tendency to adopt a-helical conformations, but the results with cyclic analogues indicated that this secondary structure is not needed for activity. Isothermal titration calorimetry (ITC) measurements demonstrate a direct interaction of some of these peptides with Syt1-C2B domain, but not with Syt7-C2B region, indicating selectivity. As expected for a compound able to inhibit a-CGRP release, cyclic peptide derivative Pal-E-cyclo[EMQK]R-NH2 showed potent in vivo analgesic activity, in a model of inflammatory pain. Molecular dynamics simulations provided a model consistent with KD values for the interaction of peptides with Syt1-C2B domain, and with their biological activity. Altogether, these results identify Syt1 as a potential new analgesic target. © 202

Estudio piloto de metilfenidato y entrenamiento a padres en el tratamiento de niños con trastorno por déficit de atención hiperactividad [A pilot study of methylphenidate and parent training in the treatment of children with attention-deficit hiperactivity disorder]

Objetivo. Este estudio comunica la eficacia de un programa de entrenamiento a padres y un ensayo clínico con medicación estimulante (metilfenidato), en la disminución de síntomas de trastorno por déficit de atención-hiperactividad (TDAH) en una muestra de niños venezolanos.Pacientes y métodos. 24 niños, con edades entre 6 y 10 años, con diagnóstico de TDAH, identificados en unas jornadas de cribado de TDAH, se asignaron aleatoriamente a los dos grupos de tratamiento: entrenamiento a padres y medicación estimulante (metilfenidato).Resultados. Ambos grupos mostraron reducción de síntomas al finalizar la fase de intervención terapéutica en diferentes grados. Estas diferencias fueron estadísticamente significativas para las valoraciones de los padres en ambos grupos, en los síntomas de falta de atención, hiperactividad e impulsividad. Las valoraciones de los profesores mostraron disminución de síntomas significativos en el grupo de medicación para las medidas de hiperactividad e impulsividad, pero no para las de falta de atención, mientras que para el grupo de entrenamiento a padres sólo hubo diferencias significativas en el índice de TDAH. No hubo diferencia en cuanto a la efectividad de ambas intervenciones.Conclusiones. Al comparar ambos programas de tratamiento, se observa que tanto el entrenamiento a padres como la medicación psicoestimulante son tratamientos efectivos para la disminución de los síntomas del TDAH. A pesar de que no existen diferencias entre la efectividad de ambas intervenciones, se observa una tendencia a que la medicación sea más efectiva. - OBJECTIVE. This study gives information about a parent training program and a clinical trial with a stimulant drug (methylphenidate) to reduce the symptoms of attention deficit hiperactivity disorder (ADHD) in a group of children in Venezuela.PATIENTS AND METHODS. 24 children, aged between 6 and 10 years and diagnosed as having ADHD, identified in ADHD screening days, were randomly assigned to two groups of treatment: parent training and a stimulant drug (methylphenidate).RESULTS. Both groups showed an improvement in their symptoms, to different degrees, after treatment. The differences were statistically significant, as evaluated by their parents, regarding the symptoms of inattention, hyperactivity and impulsivity. The teachers observed a significant improvement for the medication group with respect to the symptoms of hyperactivity and impulsivity, but no change in the inattention, whilst in the parent training group there was only significant difference in the ADHD index. There was no difference in the effectiveness of the two types of treatment.CONCLUSIONS. When the two programs of treatment were compared it was observed that both parent training and psychostimulant medication were effective in reducing the symptoms of ADHD. Although there was no difference in the effectiveness of the two programs, there was a tendency for medication to be more effectiv

Smart biosensing device for tracking fish behaviour

Biosensor technology for tracking individual challenged fish behaviour has the potential to revolutionize aquaculture, allowing farmers and breeders to orientate selective breeding towards more robust and efficient fish or improve culture conditions for a more sustainable and ethical production. The proposed solution within the AQUAEXCEL2020 EU project is a stand-alone, small and light (1 g) device (AEFishBIT), based on a tri-axial accelerometer and a microprocessor. It is externally attached to the operculum to monitor physical activity by mapping accelerations in x- and y-axes, while operculum beats (z-axis) serve as a measurement of respiratory frequency. The conducted operculum attachment protocol does not show signs of tissue damage or growth impairment in active feeding gilthead sea bream. AEFishBIT offers a wide range of new information based on individual behaviour, allowing to point out the asynchrony of movements as an indirect measure of aging and adaptability to farming environment, as well as to discriminate different coping behaviour (proactive or reactive) of gilthead sea bream challenged with low water oxygen concentrations. AEFishBIT also provides reliable information of disease outcome in fish parasitized with an intestinal myxozoan, emerging as a powerful tool for sensing the quality of the environment and improving selective breeding protocols.The study has received funding from the European Union’s Horizon 2020 research and innovation programme, GA no 652831 (AQUAEXCEL2020)

β–Lactam TRPM8 Antagonist RGM8-51 Displays Antinociceptive Activity in Different Animal Models

Transient receptor potential melastatin subtype 8 (TRPM8) is a cation channel extensively expressed in sensory neurons and implicated in different painful states. However, the effectiveness of TRPM8 modulators for pain relief is still a matter of discussion, since structurally diverse modulators lead to different results, depending on the animal pain model. In this work, we described the antinociceptive activity of a β–lactam derivative, RGM8-51, showing good TRPM8 antagonist activity, and selectivity against related thermoTRP channels and other pain-mediating receptors. In primary cultures of rat dorsal root ganglion (DRG) neurons, RGM8-51 potently reduced menthol-evoked neuronal firing without affecting the major ion conductances responsible for action potential generation. This compound has in vivo antinociceptive activity in response to cold, in a mouse model of oxaliplatin-induced peripheral neuropathy. In addition, it reduces cold, mechanical and heat hypersensitivity in a rat model of neuropathic pain arising after chronic constriction of the sciatic nerve. Furthermore, RGM8-51 exhibits mechanical hypersensitivity-relieving activity, in a mouse model of NTG-induced hyperesthesia. Taken together, these preclinical results substantiate that this TRPM8 antagonist is a promising pharmacological tool to study TRPM8-related diseases

DD04107-Derived neuronal exocytosis inhibitor peptides: Evidences for synaptotagmin-1 as a putative target

15 pags, 8 figs, 3 tabs. -- Supplementary data to this article can be found online at https://doi.org/10.1016/j.bioorg.2021.105231.The analgesic peptide DD04107 (Pal-EEMQRR-NH2) and its acetylated analogue inhibit α-calcitonin gene-related peptide (α-CGRP) exocytotic release from primary sensory neurons. Examining the crystal structure of the SNARE-Synaptotagmin-1(Syt1) complex, we hypothesized that these peptides could inhibit neuronal exocytosis by binding to Syt1, hampering at least partially its interaction with the SNARE complex. To address this hypothesis, we first interrogate the role of individual side-chains on the inhibition of α-CGRP release, finding that E1, M3, Q4 and R6 residues were crucial for activity. CD and NMR conformational analysis showed that linear peptides have tendency to adopt α-helical conformations, but the results with cyclic analogues indicated that this secondary structure is not needed for activity. Isothermal titration calorimetry (ITC) measurements demonstrate a direct interaction of some of these peptides with Syt1-C2B domain, but not with Syt7-C2B region, indicating selectivity. As expected for a compound able to inhibit α-CGRP release, cyclic peptide derivative Pal-E-cyclo[EMQK]R-NH2 showed potent in vivo analgesic activity, in a model of inflammatory pain. Molecular dynamics simulations provided a model consistent with KD values for the interaction of peptides with Syt1-C2B domain, and with their biological activity. Altogether, these results identify Syt1 as a potential new analgesic target.This work was supported by the Spanish Ministerio de Economía y Competitividad (MINECO-FEDER), RTI2018-097189-C2 and CTQ2017-84371-P), and the Spanish National Research Council (CSIC, 201880E109, 201980E030). The NMR experiments were performed in the “Manuel Rico” NMR laboratory, LMR, CSIC, a node of the Spanish Large-Scale National Facility ICTS R-LRB. We thank Prof. Josep Rizo and R. Voleti (Dept. Biophysics, Biochemistry and Pharmacology, UT Southwestern Medical Center, Dallas, USA) for providing the clones required for expressing Syt1 and Syt7 proteins. SG-R and AB belong to the Instituto de Investigación Sanitaria del Principado de Asturias (ISPA).Peer reviewe

Botulinum Neurotoxin Devoid of Receptor Binding Domain Translocates Active Protease

Clostridium botulinum neurotoxin (BoNT) causes flaccid paralysis by disabling synaptic exocytosis. Intoxication requires the tri-modular protein to undergo conformational changes in response to pH and redox gradients across endosomes, leading to the formation of a protein-conducting channel. The ∼50 kDa light chain (LC) protease is translocated into the cytosol by the ∼100 kDa heavy chain (HC), which consists of two modules: the N-terminal translocation domain (TD) and the C-terminal Receptor Binding Domain (RBD). Here we exploited the BoNT modular design to identify the minimal requirements for channel activity and LC translocation in neurons. Using the combined detection of substrate proteolysis and single-channel currents, we showed that a di-modular protein consisting only of LC and TD was sufficient to translocate active protease into the cytosol of target cells. The RBD is dispensable for cell entry, channel activity, or LC translocation; however, it determined a pH threshold for channel formation. These findings indicate that, in addition to its individual functions, each module acts as a chaperone for the others, working in concert to achieve productive intoxication