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Depto. de Bioquímica y Biología Molecular, Fac. de Farmacia, FALSE, pub

The transcription factor nuclear factor erythroid 2-related factor 2 (NRF2) is considered the master regulator of the phase II antioxidant response. It controls a plethora of cytoprotective genes related to oxidative stress, inflammation, and protein homeostasis, among other processes. Activation of these pathways has been described in numerous pathologies including cancer, cardiovascular, respiratory, renal, digestive, metabolic, autoimmune, and neurodegenerative diseases. Considering the increasing interest of discovering novel NRF2 activators due to its clinical application, initial efforts were devoted to the development of electrophilic drugs able to induce NRF2 nuclear accumulation by targeting its natural repressor protein Kelch-like ECH-associated protein 1 (KEAP1) through covalent modifications on cysteine residues. However, off-target effects of these drugs prompted the development of an innovative strategy, the search of KEAP1-NRF2 protein–protein interaction (PPI) inhibitors. These innovative activators are proposed to target NRF2 in a more selective way, leading to potentially improved drugs with the application for a variety of diseases that are currently under investigation. In this review, we summarize known KEAP1-NRF2 PPI inhibitors to date and the bases of their design highlighting the most important features of their respective interactions. We also discuss the preclinical pharmacological properties described for the most promising compounds.

Soluble epoxide hydrolase inhibitors (sEHI) are a new class of non-opioid analgesics, with a representative compound, EC5026, currently in clinical trials for the management of neuropathic pain [1]. Our group has recently designed, synthesized and pharmacologically evaluated novel series of potent benzohomoadamantane-based sEHI [2]. Herein, we report further medicinal chemistry around the abovementioned polycyclic scaffold to improve the potency and, particularly, the DMPK properties of previous hits. After an extensive in vitro screening cascade, molecular modeling, and in vivo pharmacokinetics studies, three candidates were selected for in vivo studies. Two compounds evaluated in a murine model of capsaicin-induced allodynia displayed potent anti-allodynic effect in a dose-dependent manner. Next, the most potent compound was evaluated in the cyclophosphamide-induced murine model of cystitis, a well-established model of visceral pain, presenting robust analgesic efficacy [3]. Finally, considering that chemotherapy-induced neuropathic pain (CINP), a severe side effect of several anticancer agents, is a largely unmet medical need [4], our third candidate was evaluated in a murine model of paclitaxel-induced neuropathic pain. CINP was performed by a daily injection of paclitaxel via i.p. (2 mg/kg), for 5 consecutive days. Mice developed neuropathic mechanical allodynia, which peaked on day 10 after the first paclitaxel administration ¿time when the acute effects of sEHI were tested. Subcutaneous administration of this candidate (2.5-5 mg/kg) completely reversed in a dose dependent manner the sensory hypersensitivity. Additionally, administration of the sEHI (5 mg/kg, s.c.) 30 min before each paclitaxel injection completely prevented the development of neuropathic allodynia. Collectively, these results suggest interstitial cystitis/pain bladder syndrome and CINP as possible new indications for sEHI. Acknowledgements: This work was funded by the Grant PID2020-118127RB-I

The soluble epoxide hydrolase (sEH) has been suggested as a pharmacological target for the treatment of several diseases, including pain-related disorders. Herein, we report further medicinal chemistry around new benzohomoadamantane-based sEH inhibitors (sEHI) in order to improve the drug metabolism and pharmacokinetics properties of a previous hit. After an extensive in vitro screening cascade, molecular modeling, and in vivo pharmacokinetics studies, two candidates were evaluated in vivo in a murine model of capsaicin-induced allodynia. The two compounds showed an anti-allodynic effect in a dose-dependent manner. Moreover, the most potent compound presented robust analgesic efficacy in the cyclophosphamide-induced murine model of cystitis, a well-established model of visceral pain. Overall, these results suggest painful bladder syndrome as a new possible indication for sEHI, opening a new range of applications for them in the visceral pain field.

The transcription factor nuclear factor erythroid 2‐related factor 2 (NRF2) is considered the master regulator of the phase II antioxidant response. It controls a plethora of cytoprotective genes related to oxidative stress, inflammation, and protein homeostasis, among other processes. Activation of these pathways has been described in numerous pathologies.

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 derivatives, which additionally induce the NRF2 transcription factor, master regulator of oxidative stress. NF1086 is a melatonin derivative which duplicates NRF2 expression at a concentration below 1 ¿M, has antioxidant properties and is predicted to cross the BBB through the PAMPA test. Further on, in vivo target validation was assessed by measuring NRF2 and some of its target genes in brain samples after peripheral administration. To test the potential effects of NF1086 in tauopathy, primary cortical neuronal cultures were treated with adeno-associated particles which express the human tau protein with P301L mutation (AAV-hTauP301L). In this in vitro model, phosphorylated Tau was significantly reduced in cultures treated with NF1086. We next evaluated if the compound could provide neuroprotective effects in vivo. Tauopathy in adult (3-4 months) C57bl/6 mice was generated by injecting stereotaxically AAVhTauP 301L in both hippocampi; mice were treated with 20 or 50 mg/Kg/day orally for 35 days, starting after induction of tauopathy. NF1086 treatment prevented cognitive decline promoted by tauopathy. Moreover, it reduced tau hyperphosphorylated protein in different brain regions and reduced neuroinflammation. Interestingly, similar results were achieved in aged mice 20 months old. In conclusion, NF1086 is a melatonin derivative that induces NRF2 with neuroprotective properties with potential therapeutic interest in tauopathy and related NDDs like Alzheimer¿s disesase; however, future studies need to be conducted to elucidate its mechanism of action.

The role of spinal glia in the development and maintenance of chronic pain has become over the last years a subject of increasing interest. In this regard, toll-like receptor 4 (TLR4) signaling has been proposed as a major trigger mechanism. Hence, in this study we explored the implications of TLR4 inhibition in the periphery and primarily in the CNS, focusing on the impact this inhibition renders in pain development and glia activation in the dorsal horn in two models of pain. Making use of a synthetic cluster of differentiation 14 (CD14)/TLR4 antagonist, the effect of TLR4 blockade on tactile allodynia and heat hyperalgesia was evaluated in osteoarthritic and postoperative rat models. An in vitro parallel artificial membrane permeation assay was performed to determine the proneness of the drug to permeate the blood-brain barrier prior to systemic and central admin- istration. Findings suggest a dominant role of peripheral TLR4 in the model of incisional pain, whilst both pe- ripheral and central TLR4 seem to be responsible for osteoarthritic pain. That is, central and peripheral TLR4 may be differently involved in the etiopathology of diverse types of pain what potentially seems a promising approach in the management of pain

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 derivatives, which additionally induce the NRF2 transcription factor, master regulator of oxidative stress. NF1086 is a melatonin derivative which duplicates NRF2 expression at a concentration below 1 ¿M, has antioxidant properties and is predicted to cross the BBB through the PAMPA test. Further on, in vivo target validation was assessed by measuring NRF2 and some of its target genes in brain samples after peripheral administration. To test the potential effects of NF1086 in tauopathy, primary cortical neuronal cultures were treated with adeno-associated particles which express the human tau protein with P301L mutation (AAV-hTauP301L). In this in vitro model, phosphorylated tau was signi¿¿cantly reduced in cultures treated with NF1086. We next evaluated if the compound could provide neuroprotective effects in vivo. Tauopathy in adult (3-4 months) C57BL/6J mice was generated by injecting stereotaxically AAV-hTauP301L in both hippocampi; mice were treated with 20 or 50 mg/Kg/day orally for 35 days, starting after the tauopathy induction. NF1086 treatment prevented cognitive decline promoted by tauopathy. Moreover, it reduced tau hyperphosphorylated protein in different brain regions and reduced neuroin¿¿ammation. Interestingly, similar results were achieved in aged 20 months old mice. In conclusion, NF1086 is a melatonin derivative that induces NRF2 with neuroprotective properties with potential therapeutic interest in tauopathy and related NDDs; however, future studies need to be conducted to elucidate its mechanism of action. Acknowledgments. The Comunidad Autónoma de Madrid (grant B2017/BMD-3827), the Universidad Autónoma de Madrid, the Ministerio de Economía y Competencia (RTI2018- 095793-B-I00) and the Ministerio d

A new series of 2-phenylbenzofuran derivatives were designed and synthesized to determine relevant structural features for the MAO inhibitory activity and selectivity. Methoxy substituents were introduced in the 2-phenyl ring, whereas the benzofuran moiety was not substituted or substituted at the positions 5 or 7 with a nitro group. Substitution patterns on both the phenyl ring and the benzofuran moiety determine the affinity for MAO-A or MAO-B. The 2-(3-methoxyphenyl)-5-nitrobenzofuran 9 was the most potent MAO-B inhibitor (IC = 0.024 µM) identified in this series, whereas 7-nitro-2-phenylbenzofuran 7 was the most potent MAO-A inhibitor (IC = 0.168 µM), both acting as reversible inhibitors. The number and position of the methoxyl groups on the 2-phenyl ring, have an important influence on the inhibitory activity. Molecular docking studies confirmed the experimental results and highlighted the importance of key residues in enzyme inhibition.

In vivo pharmacological inhibition of soluble epoxide hydrolase (sEH) reduces inflammatory diseases, including acute pancreatitis (AP). Adamantyl ureas are very potent sEH inhibitors, but the lipophilicity and metabolism of the adamantane group compromise their overall usefulness. Herein, we report that the replacement of a methylene unit of the adamantane group by an oxygen atom increases the solubility, permeability, and stability of three series of urea-based sEH inhibitors. Most of these oxa-analogues are nanomolar inhibitors of both the human and murine sEH. Molecular dynamics simulations rationalize the molecular basis for their activity and suggest that the presence of the oxygen atom on the adamantane scaffold results in active site rearrangements to establish a weak hydrogen bond. The 2-oxaadamantane 22, which has a good solubility, microsomal stability, and selectivity for sEH, was selected for further in vitro and in vivo studies in models of cerulein-induced AP. Both in prophylactic and treatment studies, 22 diminished the overexpression of inflammatory and endoplasmic reticulum stress markers induced by cerulein and reduced the pancreatic damage.

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.

New multi-target indole and naphthalene derivatives containing the oxadiazolone scaffold as a bioisostere of the melatonin acetamido group have been developed. The novel compounds were characterized at melatonin receptors MTR and MTR, quinone reductase 2 (QR2), lipoxygenase-5 (LOX-5), and monoamine oxidases (MAO-A and MAO-B), and also as radical scavengers. We found that selectivity within the oxadiazolone series can be modulated by modifying the side chain functionality and co-planarity with the indole or naphthalene ring. In phenotypic assays, several oxadiazolone-based derivatives induced signalling mediated by the transcription factor NRF2 and promoted the maturation of neural stem-cells into a neuronal phenotype. Activation of NRF2 could be due to the binding of indole derivatives to KEAP1, as deduced from surface plasmon resonance (SPR) experiments. Molecular modelling studies using the crystal structures of QR2 and the KEAP1 Kelch-domain, as well as the recently described X-ray free-electron laser (XFEL) structures of chimeric MTR and MTR, provided a rationale for the experimental data and afforded valuable insights for future drug design endeavours.

Alzheimer´s disease (AD) is a complex multifactorial illness with no effective treatment, characterized by irreversible global cognitive impairment. As only symptomatic treatment sare available with drugs acting at one single target, exploration of molecules active indifferent pathological targets is required. In this chapter, our objective was to develop new families of multitarget directed ligands (MTDLs) focused on some pathological pathways underlying AD, namely, oxidative stress and neuro inflammation. Thus, we design melatonin- and resveratrol-based MTDLs looking for activity in melatonin receptors (MT1-3Rs), monoaminoxidases (MAO-A/B), lipoxygenase-5 (LOX-5), and nuclear factor erythroid 2-related factor 2 (Nrf2). Considering the neuroprotective and neurogenic properties found in melatonin and resveratrol, we reasonably expected these activities in our MTDLs...

Alzheimer´s disease (AD) is a complex multifactorial illness with no effective treatment, characterized by irreversible global cognitive impairment. As only symptomatic treatment sare available with drugs acting at one single target, exploration of molecules active indifferent pathological targets is required. In this chapter, our objective was to develop new families of multitarget directed ligands (MTDLs) focused on some pathological pathways underlying AD, namely, oxidative stress and neuro inflammation. Thus, we design melatonin- and resveratrol-based MTDLs looking for activity in melatonin receptors (MT1-3Rs), monoaminoxidases (MAO-A/B), lipoxygenase-5 (LOX-5), and nuclear factor erythroid 2-related factor 2 (Nrf2). Considering the neuroprotective and neurogenic properties found in melatonin and resveratrol, we reasonably expected these activities in our MTDLs..., La enfermedad de Alzheimer (EA) es un desorden multifactorial sin cura efectiva,caracterizada por un deterioro cognitivo global. Los tratamientos disponibles son sólo sintomáticos con fármacos que actúan sobre una sola diana, siendo necesarias moléculas activas por diferentes vías. En este capítulo, nuestro objetivo es desarrollar nuevas familias de ligandos multidiana (MTDL) enfocados hacia vías patológicas de la EA, concretamente, el estrés oxidativo y neuroinflamación. Así, desarrollamos MTDLs basados en melatoninay resveratrol buscando actividad en receptores de melatonina (MT1-3Rs), monoaminooxidasas (MAO-A/B), lipoxigenasa-5 (LOX-5) y el factor nuclear (erythroid-derived 2)-like 2 (Nrf2). Considerando las propiedades neuroprotectoras y neurogénicas de la melatonina y el resveratrol, esperamos razonablemente estas actividades en nuestros MTDLs...

The design of multi-target directed ligands (MTDLs) is a valid approach for obtaining effective drugs for complex pathologies. MTDLs that combine neuro-repair properties and block the first steps of neurotoxic cascades could be the so long wanted remedies to treat neurodegenerative diseases (NDs). By linking two privileged scaffolds with well-known activities in ND-targets, the flavonoid and the N,N-dibenzyl(N-methyl)- amine (DBMA) fragments, new CNS-permeable flavonoid – DBMA hybrids (1–13) were obtained. They were subjected to biological evaluation in a battery of targets involved in Alzheimer’s disease (AD) and other NDs, namely human cholinesterases (hAChE/hBuChE), b-secretase (hBACE-1), monoamine oxidases (hMAO-A/B), lipoxygenase-5 (hLOX-5) and sigma receptors (r1R/r2R). After a funnel-type screening, 6,7- dimethoxychromone – DBMA (6) was highlighted due to its neurogenic properties and an interesting MTD-profile in hAChE, hLOX-5, hBACE-1 and r1R. Molecular dynamic simulations showed the most relevant drug-protein interactions of hybrid 6, which could synergistically contribute to neuronal regeneration and block neurodegeneration.

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.