Structure-activity relationship of potent photo-switchable neuromuscular inhibitors

Muscular nicotinic acetylcholine receptors (nAChR) are ligand-gated ion channels located in the plasma membrane of the post-junctional motor endplate in skeletal muscles. These receptors are responsible for triggering the electrical signal that leads to muscle contraction. Here, we have developed a family of photoisomerizable nAChR inhibitors, using the azobenzene scaffold. Biochemical assays showed that all these novel compounds have higher affinity for muscular nAChR (up to 60-fold) than for either ¿7 or ¿4ß2 nAChRs. Thus, we proceeded to characterize the action of these compounds on the activity of the embryonic muscular nAChR expressed in Xenopus oocytes. To do so, we recorded currents evoked by acetylcholine, using the Two-Electrode Voltage-Clamp technique. In doing so, we observed that all but one of the compounds were effective inhibitors of the receptor¿s activity. As reported elsewhere, azobenzene-based molecules can be as either cis- or trans-isomers, displaying remarkable difference in their pharmacology. To test whether the isomer had different inhibitory activity, we proceeded to irradiate the drugs with either near UV (335-365 nm) or blue light (400-450 nm) while recording currents. In all cases, we observed that the trans-isomers were the most potent form of these molecules. The main difference among the compounds of this family is the ¿molecular volume¿ of the functional group. The molecules were effective inhibitors of the receptor with nano- to micromolar activity, decreasing their potency as their size decrease. In fact, the trans-isomer of the smallest derivative reverting its behavior, acting as an agonist drug, with the cis-isomer being unable to activate the channels. In terms structure-activity relationship (SAR) parameters, these observations led us to infer the that decreasing the bulkiness of the molecule is a critical determinant of the character and potency of these novel azobenzene-based inhibitors.MINECO (SAF2015-64948-C2-1-R; CHA: FPU16/01704; FPUEST17/00233), CSIC (PIE201580E109), Nutting Foundation Grant (CAVG)

Optical control of muscular nicotinic channels with azocuroniums, photoswitchable azobenzenes bearing two N-methyl-N-carbocyclic quaternary ammonium groups

By linking two N-methyl-N-carbocyclic quaternary ammonium groups to an azobenzene scaffold in meta- or para-positions we generated a series of photoswitchable neuromuscular ligands for which we coined the term “azocuroniums”. These compounds switched between the (E)- and (Z)-isomers by light irradiation at 400–450 nm and 335–340 nm, respectively. Meta-azocuroniums were potent nicotinic ligands with a clear selectivity for the muscular nAChRs compared to neuronal α7 and α4β2 subtypes, showed good solubility in physiologic media, negligible cell toxicity, and would not reach the CNS. Electrophysiological studies in muscle-type nAChRs expressed in Xenopus laevis oocytes showed that (E)-isomers were more potent than (Z)-forms. All meta-azocuroniums were neuromuscular blockers, with the exception of the pyrrolidine derivative that was an agonist. These new meta-azocuroniums, which can be modulated ad libitum by light, could be employed as photoswitchable muscle relaxants with fewer side effects for surgical interventions and as tools to better understand the pharmacology of muscle-type nAChRs.The authors gratefully acknowledge the following financial supports: Spanish Ministry of Science, Innovation and Universities; Spanish Research Agency; and European Regional Development Funds (grants SAF2015-64948-C2-1-R and RTI2018-093955-B-C21 to MIRF), General Council for Research and Innovation of the Community of Madrid and European Structural Funds (grant S2017/ BMD-3827 e NRF24ADCM to MIRF) and Spanish National Research Council CSIC (grant PIE-202080E118). CH-A also thanks her PhD fellowships from Spanish Ministry of Education (MEC, PhD grant FPU16/01704 and mobility grant FPUEST17/00233)

New neurogenic inducers with combined activities in key targets related to Alzheimer¿s disease

Alzheimer¿s Disease (AD) is a complex multifactorial illness with no effective cure, characterized by the irreversible memory loss and global cognitive impairment. Given that the marketed drugs act only at one single target, being a symptomatic treatment, exploration of new molecules acting in different pathways is required. In this sense, we have developed a large family of multitarget directed ligands (MTDL), which interacts in different targets related to neurodegeneration, such as the nuclear factor (erythroid-derived 2)-like 2 (Nrf2), lipoxygenase-5 (LOX-5), melatonin receptors (MT1, MT2, and MT3), and monoamine oxidases (MAO-A and MAO-B). The affinity or inhibition constants of these compounds in such targets are in the nanomolar and micromolar ranges. In general, these molecules have potent antioxidant properties (ORAC assay) and are able to penetrate into the central nervous system (CNS) by passive diffusion (PAMPA-BBB assay). Moreover, some of them are able to promote neurogenesis in vitro by inducing the maturation of neural stem cells into a neuronal phenotype. A structure-activity relationship has been stablished, reaching an optimized structure as possible candidate to treat AD, able to cross the blood brain barrier, reaching the CNS where it would act in three targets involved in the regulation of oxidative stress, as Nrf2 inducer, MT3 ligand and selective MAO B inhibitor. Furthermore, this MTDL is an effective neuroprotective and neurogenic agent.Dirección General de Investigación e Innovación de la Comunidad de Madrid y Fondo Europeo de Desarrollo Regional (B2017/BMD-3827); Ministerio de Ciencia, Innovación y Universidades, Agencia Estatal de Investigación y FEDER (SAF2015-64948-C2-1-R and RTI2018-093955-B-C21). C.H.-A. thanks Spanish Ministry of Education for a FPU fellowship (grant FPU16/01704

Caracterización farmacológica de derivados de melatonina y resveratrol para el potencial tratamiento de la enfermedad de Alzheimer

This study has been developed within a multidisciplinary project designed to search for multitarget compounds with NRF2 inducing capacity, by inhibiting the Keap1-NRF2 interaction, for the treatment of Alzheimer¿s disease. We have focused on melatonin and resveratrol derivatives, which additionally induce NRF2 transcription factor, master regulator of oxidative stress. For this study, two melatonin (CH213 and CH507) and one resveratrol (PL119) derivatives, which were the ones that duplicated NRF2 expression at concentrations below 10 ¿M, were selected. We found that these compounds were not neurotoxic in the human neuroblastoma cell line SH-SY5Y at the concentration of 100 ¿M. Thereafter, neuroprotection in a tau hyperphosphorylation in vitro model induced by okadaic acid (OA) was studied; compounds CH507 and PLC119 presented significant neuroprotection, while CH213 did not. Nevertheless, neuroprotection of compound CH213 against OA was studied in primary neuronal cultures achieving significant results. Additionally, their potential anti neuroinflammatory effects were studied in primary rat glial cultures exposed to LPS; in this model, only compound CH213 reduced nitric oxide production and IL-1b. CH123 was further studied in an acute model of hippocampal slices, where toxicity was induced by incubation with OA for 6 h and in organotypic hippocampal cultures of 14 months old APPTau mice; in these models, CH213 was able to provide neuroprotection and reduce oxidative stress. Finally, since QR2, also known as MT3, is overexpressed in AD patients, we sought of interest to perform a molecular docking study to evaluate how the compounds could interact with this receptor. In conclusion, CH213 is a melatonin derivative that induces NRF2 with antineuroinflammatory, antioxidant and neuroprotective properties; however, future studies in in vivo AD models need to be conducted to validate the in vitro results here obtained

Identification of tetracyclic lactams as NMDA receptor antagonists with potential application in neurological disorders

N-Methyl-D-aspartate receptors (NMDARs) are crucial for the normal function of the central nervous system (CNS), and fundamental in memory and learning-related processes. The overactivation of these receptors is associated with numerous neurodegenerative and psychiatric disorders. Therefore, NMDAR is considered a relevant therapeutic target for many CNS disorders. Herein, we report the synthesis and pharmacological evaluation of a new scaffold with antagonistic activity for NMDAR. Specifically, a chemical library of eighteen 1-aminoindan-2-ol tetracyclic lactams was synthesized and screened as NMDAR antagonists. The compounds were obtained by chiral pool synthesis using enantiomerically pure 1-aminoindan-2-ols as chiral inductors, and their stereochemistry was proven by X-ray crystallographic analysis of two target compounds. Most compounds reveal NMDAR antagonism, and eleven compounds display IC values in a Ca entry-sensitive fluo-4 assay in the same order of magnitude of memantine, a clinically approved NMDAR antagonist. Docking studies suggest that the novel compounds can act as NMDAR channel blockers since there is a compatible conformation with MK-801 co-crystallized with NMDAR channel. In addition, we show that the tetracyclic 1-aminoindan-2-ol derivatives are brain permeable and non-toxic, and we identify promising hits for further optimization as modulators of the NMDAR function.This work was supported by FCT (Fundaç~ao para a Ci^encia e a Tecnologia, I.P.) through iMed.ULisboa (UID/DTP/04138/2019), Principal Researcher grant CEECIND/01772/2017 (M. M. M. Santos), and PhD fellowships SFRH/BD/117931/2016 (M. Espadinha) and SFRH/BD/121664/2016 (R. Lopes). Financial support from FCT and Portugal 2020 to the Portuguese Mass Spectrometry Network (Rede Nacional de Espectrometria de Massa e RNEM; LISBOA-01-0145- FEDER-402-022125) is also acknowledged. M.I.R.-F. thanks funding from the Spanish Ministry of Science, Innovation and Universities (grant RTI2018-093955-B-C21) and the technical assistance of Ms. Cristina Tortosa (European contract for young professionals). C.d.l.R. thanks funding from Instituto de Salud Carlos III, Madrid, Spain (grant PI16/01041 and PhD fellowship FI17/00079 for L. Viejo)