The proof of concept of 2-{3-[N-(1-benzylpiperidin-4-yl)propyl]amino}-6-[N-methyl-N-(prop-2-yn-1-yl)amino]-4-phenylpyridine-3,5-dicarbonitrile for the therapy of neuropathic pain.

In the search for new small molecules for the therapy of neuropathic pain, we found that 2-{3-[N-(1-benzylpiperidin-4-yl)propyl]amino}-6-[N-methyl-N-(prop-2-yn-1-yl)amino]-4-phenylpyridine-3,5-dicarbonitrile (12) induced a robust antiallodynic effect in capsaicin-induced mechanical allodynia, a behavioural model of central sensitization, through σ1R antagonism. Furthermore, administration of compound 12 to neuropathic animals, fully reversed mechanical allodynia, increasing its mechanical threshold to levels that were not significantly different from those found in paclitaxel-vehicle treated mice or from basal levels before neuropathy was induced. Ligand 12 is thus a promising hit-compound for the therapy of neuropathic pain.

SK2 channels set a signalling hub bolstering CAF-triggered tumourigenic processes in pancreatic cancer.

OBJECTIVE: Intercellular communication within pancreatic ductal adenocarcinoma (PDAC) dramatically contributes to metastatic processes. The underlying mechanisms are poorly understood, resulting in a lack of targeted therapy to counteract stromal-induced cancer cell aggressiveness. Here, we investigated whether ion channels, which remain understudied in cancer biology, contribute to intercellular communication in PDAC. DESIGN: We evaluated the effects of conditioned media from patient-derived cancer-associated fibroblasts (CAFs) on electrical features of pancreatic cancer cells (PCC). The molecular mechanisms were deciphered using a combination of electrophysiology, bioinformatics, molecular and biochemistry techniques in cell lines and human samples. An orthotropic mouse model where CAF and PCC were co-injected was used to evaluate tumour growth and metastasis dissemination. Pharmacological studies were carried out in the Pdx1-Cre, Ink4afl/fl LSL-KrasG12D (KICpdx1) mouse model. RESULTS: We report that the K+ channel SK2 expressed in PCC is stimulated by CAF-secreted cues (8.84 vs 2.49 pA/pF) promoting the phosphorylation of the channel through an integrin-epidermal growth factor receptor (EGFR)-AKT (Protein kinase B) axis. SK2 stimulation sets a positive feedback on the signalling pathway, increasing invasiveness in vitro (threefold) and metastasis formation in vivo. The CAF-dependent formation of the signalling hub associating SK2 and AKT requires the sigma-1 receptor chaperone. The pharmacological targeting of Sig-1R abolished CAF-induced activation of SK2, reduced tumour progression and extended the overall survival in mice (11.7 weeks vs 9.5 weeks). CONCLUSION: We establish a new paradigm in which an ion channel shifts the activation level of a signalling pathway in response to stromal cues, opening a new therapeutic window targeting the formation of ion channel-dependent signalling hubs.

A Polyaminobiaryl-Based ß-secretase Modulator Alleviates Cognitive Impairments, Amyloid Load, Astrogliosis, and Neuroinflammation in APPSwe/PSEN1ΔE9 Mice Model of Amyloid Pathology.

The progress in Alzheimer's disease (AD) treatment suggests a combined therapeutic approach targeting the two lesional processes of AD, which include amyloid plaques made of toxic Aß species and neurofibrillary tangles formed of aggregates of abnormally modified Tau proteins. A pharmacophoric design, novel drug synthesis, and structure-activity relationship enabled the selection of a polyamino biaryl PEL24-199 compound. The pharmacologic activity consists of a non-competitive ß-secretase (BACE1) modulatory activity in cells. Curative treatment of the Thy-Tau22 model of Tau pathology restores short-term spatial memory, decreases neurofibrillary degeneration, and alleviates astrogliosis and neuroinflammatory reactions. Modulatory effects of PEL24-199 towards APP catalytic byproducts are described in vitro, but whether PEL24-199 can alleviate the Aß plaque load and associated inflammatory counterparts in vivo remains to be elucidated. We investigated short- and long-term spatial memory, Aß plaque load, and inflammatory processes in APPSwe/PSEN1ΔE9 PEL24-199 treated transgenic model of amyloid pathology to achieve this objective. PEL24-199 curative treatment induced the recovery of spatial memory and decreased the amyloid plaque load in association with decreased astrogliosis and neuroinflammation. The present results underline the synthesis and selection of a promising polyaminobiaryl-based drug that modulates both Tau and, in this case, APP pathology in vivo via a neuroinflammatory-dependent process.

Discovery of Compounds That Selectively Repress the Amyloidogenic Processing of the Amyloid Precursor Protein: Design, Synthesis and Pharmacological Evaluation of Diphenylpyrazoles.

The rationale to define the biological and molecular parameters derived from structure-activity relationships (SAR) is mandatory for the lead selection of small drug compounds. Several series of small molecules have been synthesized based on a computer-assisted pharmacophore design derived from two series of compounds whose scaffold originates from chloroquine or amodiaquine. All compounds share similar biological activities. In vivo, Alzheimer's disease-related pathological lesions are reduced, consisting of amyloid deposition and neurofibrillary degeneration, which restore and reduce cognitive-associated impairments and neuroinflammation, respectively. Screening election was performed using a cell-based assay to measure the repression of Aß1-x peptide production, the increased stability of APP metabolites, and modulation of the ratio of autophagy markers. These screening parameters enabled us to select compounds as potent non-competitive ß-secretase modulators, associated with various levels of lysosomotropic or autophagy modulatory activities. Structure-activity relationship analyses enabled us to define that (1) selectively reducing the production of Aß1-x, and (2) little Aßx-40/42 modification together with (3) a decreased ratio of p62/(LC3-I/LC3-II) enabled the selection of non-competitive ß-secretase modulators. Increased stability of CTFα and AICD precluded the selection of compounds with lysosomotropic activity whereas cell toxicity was associated with the sole p62 enhanced expression shown to be driven by the loss of nitrogen moieties. These SAR parameters are herein proposed with thresholds that enable the selection of potent anti-Alzheimer drugs for which further investigation is necessary to determine the basic mechanism underlying their mode of action.

Crosstalk between the Hippo Pathway and the Wnt Pathway in Huntington's Disease and Other Neurodegenerative Disorders.

The Hippo pathway consists of a cascade of kinases that controls the phosphorylation of the co-activators YAP/TAZ. When unphosphorylated, YAP and TAZ translocate into the nucleus, where they mainly bind to the TEAD transcription factor family and activate genes related to cell proliferation and survival. In this way, the inhibition of the Hippo pathway promotes cell survival, proliferation, and stemness fate. Another pathway can modulate these processes, namely the Wnt/ß-catenin pathway that is indeed involved in cellular functions such as proliferation and cell survival, as well as apoptosis, growth, and cell renewal. Wnt signaling can act in a canonical or noncanonical way, depending on whether ß-catenin is involved in the process. In this review, we will focus only on the canonical Wnt pathway. It has emerged that YAP/TAZ are components of the ß-catenin destruction complex and that there is a close relationship between the Hippo pathway and the canonical Wnt pathway. Furthermore, recent data have shown that both of these pathways may play a role in neurodegenerative diseases, such as Huntington's disease, Alzheimer's disease, or Amyotrophic Lateral Sclerosis. Thus, this review analyzes the Hippo pathway and the Wnt pathway, their crosstalk, and their involvement in Huntington's disease, as well as in other neurodegenerative disorders. Altogether, these data suggest possible therapeutic approaches targeting key players of these pathways.

Safe and Efficient Sigma1 Ligand: A Potential Drug Candidate for Multiple Sclerosis.

Multiple Sclerosis (MS) is an autoimmune demyelinating and neurodegenerative disease of the central nervous system (CNS). Current management strategies suppress or modulate immune function, all with consequences and known side effects. They demonstrate a high level of success in limiting new relapses. However, the neurodegenerative process still affects both grey and white matter in the central nervous system. The sigma1 (S1R) ligand-regulated chaperone is implicated in many biological processes in various CNS-targeted diseases, acting on neural plasticity, myelination and neuroinflammation. Among the proteins involved in MS, S1R has therefore emerged as a promising new target. Standard and robust methods have been adopted to analyze the adsorption, distribution, metabolism, excretion (ADME) properties, safety pharmacology and toxicology of a previously synthetized simple benzamide-derived compound with nanomolar affinity for S1R, high selectivity, no cytotoxicity and good metabolic stability. The compound was also characterized as an agonist based on well-validated assays prior to in vivo investigations. Interestingly, we found that the oral administration of this compound resulted in an overall significant reduction in clinical progression in an MS experimental model. This effect is mediated through S1R action. Our results further suggest the potential use of this compound in the treatment of MS.

Repositioning of FDA-Approved antifungal agents to interrogate Acyl-CoA synthetase long chain family member 4 (ACSL4) in ferroptosis.

Ferroptosis, first coined in 2012, is an iron-dependent regulated cell death (RCD) characterized by the accumulation of lipid peroxides to toxic levels. This mechanism is currently being evaluated as a target for a variety of diseases offering new opportunities for drug design and development. Recent reports uncovered acyl-CoA synthetase long-chain 4 (ACSL4) as a critical contributor to ferroptosis execution. Therefore, ACSL4 inhibitors are emerging as attractive anti-ferroptotic agents. Herein, we developed a robust screening cascade with orthogonal biophysical and biochemical techniques to identify original human ACSL4 inhibitors. By screening an FDA-approved drug library, we were able to identify and validate new inhibitors with micromolar-range activities against ACSL4. With an IC50 of 280 nM against hACSL4, antifungal agent sertaconazole is to our knowledge, the most potent ACSL4 inhibitor identified so far. In addition, sertaconazole significantly reduced lipid peroxidation and ferroptosis in human differentiated dopaminergic neurons (Lund human mesencephalic LUHMES cells), demonstrating that it is a valuable chemical tool for further investigating the role of ACSL4 in ferroptosis. This study highlights the phenethyl-imidazole scaffold as a novel and promising starting point for the development of anti-ferroptotic agents targeting ACSL4.

High ligand efficiency quinazoline compounds as novel A2A adenosine receptor antagonists.

The past fifty years have been marked by the surge of neurodegenerative diseases. Unfortunately, current treatments are only symptomatic. Hence, the search for new and innovative therapeutic targets for curative treatments becomes a major challenge. Among these targets, the adenosine A2A receptor (A2AAR) has been the subject of much research in recent years. In this paper, we report the design, synthesis and pharmacological analysis of quinazoline derivatives as A2AAR antagonists with high ligand efficiency. This class of molecules has been discovered by a virtual screening and bears no structural semblance with reference antagonist ZM-241385. More precisely, we identified a series of 2-aminoquinazoline as promising A2AAR antagonists. Among them, one compound showed a high affinity towards A2AAR (21a, Ki = 20 nM). We crystallized this ligand in complex with A2AAR, confirming one of our predicted docking poses and opening up possibilities for further optimization to derive selective ligands for specific adenosine receptor subtypes.

Progress with YAP/TAZ-TEAD inhibitors: a patent review (2018-present).

INTRODUCTION: The Hippo pathway represents a new opportunity for the treatment of cancer. Overexpression of Yes-associated protein (YAP) or transcriptional coactivator with PDZ-binding motif (TAZ) or TEAD has been demonstrated in cancers and YAP mediates resistance to cancer drugs. Since 2018, the potential of this pathway has been illustrated by numerous articles and patents and the first drugs entering in clinical trial phase 1. AREAS COVERED: This review is limited to published patent applications that have disclosed direct small-molecule inhibitors of the YAP/TAZ-TEAD interaction. EXPERT OPINION: The YAP/TAZ-TEAD transcriptional complex is a promising target for the treatment of cancer. Approximately 30 international patents (used database: Sci-finder, query: TEAD; documents: patents; period: from 2017-January 2022) that disclose TEAD transcriptional inhibitors have been filled since 2018. The mechanism of action is not always described in the patents, we can divide the drugs into three different categories: (i) external TEAD ligands; (ii) non-covalent TEAD ligands of the palmitate pocket; (iii) covalent TEAD ligands, which bind into the palmitate pocket. The first molecules in clinical trial phase 1 are non-covalent TEAD ligands. The selective TEAD ligand have also been patented, published and selectivity could be of great interest for personalized medicine.

Toward the Design of Ligands Selective for the C-Terminal Domain of TEADs.

The Hippo signaling pathway plays a fundamental role in the control of organ growth, cell proliferation, and stem cell characters. TEADs are the main transcriptional output regulators of the Hippo signaling pathway and bind to YAP and TAZ co-activators. TEAD1-4 are expressed differently, depending on the tissue and developmental level, and can be overexpressed in certain pathologies. TEAD ligands mainly target the internal pocket of the C-terminal domain of TEAD, and the first ligands selective for TEAD1 and TEAD3 have been recently reported. In this paper, we focus on the topographic homology of the TEAD C-terminal domain both externally and in the internal pocket to highlight the possibility of rationally designing ligands selective for one of the TEAD family members. We identified a novel TEAD2-specific pocket and reported its first ligand. Finally, AlphaFold2 models of full-length TEADs suggest TEAD autoregulation and emphasize the importance of the interface 2.

A Phosphosite Mutant Approach on LRRK2 Links Phosphorylation and Dephosphorylation to Protective and Deleterious Markers, Respectively.

The Leucine Rich Repeat Kinase 2 (LRRK2) gene is a major genetic determinant of Parkinson's disease (PD), encoding a homonymous multi-domain protein with two catalytic activities, GTPase and Kinase, involved in intracellular signaling and trafficking. LRRK2 is phosphorylated at multiple sites, including a cluster of autophosphorylation sites in the GTPase domain and a cluster of heterologous phosphorylation sites at residues 860 to 976. Phosphorylation at these latter sites is found to be modified in brains of PD patients, as well as for some disease mutant forms of LRRK2. The main aim of this study is to investigate the functional consequences of LRRK2 phosphorylation or dephosphorylation at LRRK2's heterologous phosphorylation sites. To this end, we generated LRRK2 phosphorylation site mutants and studied how these affected LRRK2 catalytic activity, neurite outgrowth and lysosomal physiology in cellular models. We show that phosphorylation of RAB8a and RAB10 substrates are reduced with phosphomimicking forms of LRRK2, while RAB29 induced activation of LRRK2 kinase activity is enhanced for phosphodead forms of LRRK2. Considering the hypothesis that PD pathology is associated to increased LRRK2 kinase activity, our results suggest that for its heterologous phosphorylation sites LRRK2 phosphorylation correlates to healthy phenotypes and LRRK2 dephosphorylation correlates to phenotypes associated to the PD pathological processes.

Synthesis and SAR Studies of Isoquinoline and Tetrahydroisoquinoline Derivatives as Melatonin Receptor Ligands.

In our constant search for new successors of agomelatine, we report herein a new series of compounds resulting from bioisosteric modulation of the naphthalene ring. The isoquinoline and tetrahydroisoquinoline derivatives were synthesized and pharmacologically evaluated. This isosteric replacement of the naphthalene group of agomelatine has led to potent agonist and partial agonist compounds with nanomolar melatonergic binding affinities. Overall, the presence of a nitrogen atom was accompanied with a decrease in the binding affinity toward both MT1 and MT2 and the loss of 5HT2C response, especially for tetrahydroisoquinoline in comparison with the parent compound. Interestingly, due to the presence of this nitrogen atom, a notable improvement in the pharmacokinetic properties was observed for all compounds.

The EGF Domains of MUC4 Oncomucin Mediate HER2 Binding Affinity and Promote Pancreatic Cancer Cell Tumorigenesis.

The HER2 receptor and its MUC4 mucin partner form an oncogenic complex via an extracellular region of MUC4 encompassing three EGF domains that promotes tumor progression of pancreatic cancer (PC) cells. However, the molecular mechanism of interaction remains poorly understood. Herein, we decipher at the molecular level the role and impact of the MUC4EGF domains in the mediation of the binding affinities with HER2 and the PC cell tumorigenicity. We used an integrative approach combining in vitro bioinformatic, biophysical, biochemical, and biological approaches, as well as an in vivo study on a xenograft model of PC. In this study, we specified the binding mode of MUC4EGF domains with HER2 and demonstrate their "growth factor-like" biological activities in PC cells leading to stimulation of several signaling proteins (mTOR pathway, Akt, and ß-catenin) contributing to PC progression. Molecular dynamics simulations of the MUC4EGF/HER2 complexes led to 3D homology models and identification of binding hotspots mediating binding affinity with HER2 and PC cell proliferation. These results will pave the way to the design of potential MUC4/HER2 inhibitors targeting the EGF domains of MUC4. This strategy will represent a new efficient alternative to treat cancers associated with MUC4/HER2 overexpression and HER2-targeted therapy failure as a new adapted treatment to patients.

Discovery of a cryptic site at the interface 2 of TEAD - Towards a new family of YAP/TAZ-TEAD inhibitors.

The Hippo pathway is involved in organ size control and tissue homeostasis by regulating cell growth, proliferation and apoptosis. It controls the phosphorylation of the transcription co-activator YAP (Yes associated protein) and TAZ (Transcriptional coactivator with PDZ-binding motif) in order to control their nuclear import and their interaction with TEAD (Transcriptional Enhanced Associated Domain). YAP, TAZ and TEADs are dysregulated in several cancers making YAP/TAZ-TEAD interaction a new emerging anti-cancer target. We report the synthesis of a set of trisubstituted pyrazoles which bind to hTEAD2 at the interface 2 revealing for the first time a cryptic pocket created by the movement of the phenol ring of Y382. Compound 6 disrupts YAP/TAZ-TEAD interaction in HEK293T cells and inhibits TEAD target genes and cell proliferation in MDA-MB-231 cells. Compound 6 is therefore the first inhibitor of YAP/TAZ-TEAD targeting interface 2. This molecule could serve with other pan-TEAD inhibitors such as interface 3 ligands, for the delineation of the relative importance of VGLL vs YAP/TAZ in a given cellular model.

A ß-Secretase Modulator Decreases Tau Pathology and Preserves Short-Term Memory in a Mouse Model of Neurofibrillary Degeneration.

Identifying which among several in cellulo pharmacological activities is necessary for the proper in vivo activity is essential for further drug development against Alzheimer's disease pathophysiological processes. An in-depth structure-activity relationship-based study has been carried out, and two molecules, named MAGS02-14 and PEL24-199, that share a ß-secretase modulatory effect associated or not to a lysosomotropic activity in cellulo have been identified. In terms of chemical formulas, MAGS02-14 and PEL24-199 only differ from each other by a single nitrogen atom. The study aimed to elucidate the in vivo pharmacological effects of lysosomotropic and/or the ß-secretase modulatory activity in a tau pathology mouse model. To address this question, the THY-Tau22 transgenic model of tauopathy was treated with both compounds for 6 weeks in a curative paradigm. Short-term memory, tau burden, and inflammatory processes were analyzed using orthogonal methods, and PEL24-199, but not MAGS02-14, was shown to restore the short-term memory and reduce the neurofibrillary degenerating process. These effects were associated with a reduced phosphorylation of tau, an increased phosphatase expression, and decreased astrogliosis. Our results, therefore, suggest that the lysosomotropic activity may be nonessential for the effect on tau pathology.

Design, Synthesis and Evaluation of a Series of 1,5-Diaryl-1,2,3-triazole-4-carbohydrazones as Inhibitors of the YAP-TAZ/TEAD Complex.

Starting from our previously reported hit, a series of 1,5-diaryl-1,2,3-triazole-4-carbohydrazones were synthesized and evaluated as inhibitors of the YAP/TAZ-TEAD complex. Their binding to hTEAD2 was confirmed by nanodifferential scanning fluorimetry, and some of the compounds were also found to moderately disrupt the YAP-TEAD interaction, as assessed by a fluorescence polarization assay. A TEAD luciferase gene reporter assay performed in HEK293T cells and RTqPCR measurements in MDA-MB231 cells showed that these compounds inhibit YAP/TAZ-TEAD activity to cells in the micromolar range. In spite of the cytotoxic effects displayed by some of the compounds of this series, they are still good starting points and can be suitably modified into an effective and viable YAP-TEAD disruptor in the future.

Publisher Correction: MUC4-ErbB2 Oncogenic Complex: Binding studies using Microscale Thermophoresis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

EGF-Containing Membrane-Bound Mucins: A Hidden ErbB2 Targeting Pathway?

Membrane-bound mucins belong to a heterogeneous family of large O-glycoproteins involved in numerous cancers and inflammatory diseases of the epithelium. Some of them are also involved in protein-protein interactions, with receptor tyrosine kinase ErbB2, and fundamental and clinical data showed that these complexes have a detrimental impact on cancer outcome, thus raising interest in therapeutic targeting. This paper aims to demonstrate that MUC3, MUC4, MUC12, MUC13, and MUC17 have a common evolutionary origin and share a common structural organization with EGF-like and SEA domains. Theoretical structure-function relationship analysis of the conserved domains indicated that the studied membrane-bound mucins share common biological properties along with potential specific functions. Finally, the potential druggability of these complexes is discussed, revealing ErbB2-related pathways of cell signaling to be targeted.

Quinazoline and phthalazine derivatives as novel melatonin receptor ligands analogues of agomelatine.

For further development of successors of Agomelatine through modulation of its pharmacokinetic properties, we report herein the design, synthesis and pharmacological results of a new family of melatonin receptor ligands. Issued from the introduction of quinazoline and phthalazine scaffolds carrying an ethyl amide lateral chain and a methoxy group as bioisosteric ligands analogues of previously developed Agomelatine. The biological activity of the prepared analogues was compared with that of Agomelatine. Quinazoline and phthalazine rings proved to be a versatile scaffold for easy feasible MT1 and MT2 ligands. Potent agonists with sub-micromolar binding affinity were obtained. However, the presence of two nitrogen atoms resulted in compounds with lower affinity for both MT1 and MT2, in comparison with the parent compound, balanced by the exhibition of good pharmacokinetic properties.

Synthesis of a novel series of (Z)-3,5-disubstituted thiazolidine-2,4-diones as promising anti-breast cancer agents.

A novel series of (Z)-3,5-disubstituted thiazolidine-2,4-diones 4-16 has been designed and synthesized. Preliminary screening of these compounds for their anti-breast cancer activity revealed that compounds 5, 7, and 9 possess the highest anti-cancer activities. The anti-tumor effects of compounds 5, 7, and 9 were evaluated against human breast cancer cell lines (MCF-7 and MDA-MB-231) and human breast cancer cells. They were also evaluated against normal non-cancerous breast cells, isolated from the same patients, to conclude about their use in a potential targeted therapy. Using MTT uptake method, these three compounds 5, 7, and 9 blunt the proliferation of these cancer cells in a dose-dependent manner with an IC50 of 1.27, 1.50 and 1.31 µM respectively. Interestingly, using flow cytometry analysis these three compounds significantly mediated apoptosis of human breast cancer cells without affecting the survival of normal non-cancerous breast cells that were isolated from the same patients. Mechanistically, these compounds blunt the proliferation of MCF-7 breast cancer cells by robustly decreasing the phosphorylation of AKT, mTOR and the expression of VEGF and HIF-1α. Most importantly, compounds 5, 7, and 9 without affecting the phosphorylation and expression of these crucial cellular factors in normal non-cancerous breast cells that were isolated from the same patients. Additionally, using Western blot analysis the three compounds significantly (P < 0.05) decreased the expression of the anti-apoptotic Bcl-2 members (Bcl-2, Bcl-XL and Mcl-1) and increased the expression of the pro-apoptotic Bcl-2 members (Bak, Bax and Bim) in MCF-7, MDA-MB-231 and human breast cancer cells making these breast cancer cells susceptible for apoptosis induction. Taken together, these data provide great evidences for the inhibitory activity of these compounds against breast cancer cells without affecting the normal breast cells.