Distinct binding hotspots for natural and synthetic agonists of FFA4 from in silico approaches.

FFA4 has gained interest in recent years since its deorphanization in 2005 and the characterization of the Free Fatty Acids receptors family for their therapeutic potential in metabolic disorders. The expression of FFA4 (also known as GPR120) in numerous organs throughout the human body makes this receptor a highly potent target, particularly in fat sensing and diet preference. This offers an attractive approach to tackle obesity and related metabolic diseases. Recent cryo-EM structures of the receptor have provided valuable information for a potential active state although the previous studies of FFA4 presented diverging information. We performed molecular docking and molecular dynamics simulations of four agonist ligands, TUG-891, Linoleic acid, α-Linolenic acid, and Oleic acid, based on a homology model. Our simulations, which accumulated a total of 2 µs of simulation, highlighted two binding hotspots at Arg992.64 and Lys293 (ECL3). The results indicate that the residues are located in separate areas of the binding pocket and interact with various types of ligands, implying different potential active states of FFA4 and a highly adaptable binding intra-receptor pocket. This article proposes additional structural characteristics and mechanisms for agonist binding that complement the experimental structures.

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.

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.

Melatonin drugs inhibit SARS-CoV-2 entry into the brain and virus-induced damage of cerebral small vessels.

COVID-19 is a complex disease with short- and long-term respiratory, inflammatory and neurological symptoms that are triggered by the infection with SARS-CoV-2. Invasion of the brain by SARS-CoV-2 has been observed in humans and is postulated to be involved in post-COVID state. Brain infection is particularly pronounced in the K18-hACE2 mouse model of COVID-19. Prevention of brain infection in the acute phase of the disease might thus be of therapeutic relevance to prevent long-lasting symptoms of COVID-19. We previously showed that melatonin or two prescribed structural analogs, agomelatine and ramelteon delay the onset of severe clinical symptoms and improve survival of SARS-CoV-2-infected K18-hACE2 mice. Here, we show that treatment of K18-hACE2 mice with melatonin and two melatonin-derived marketed drugs, agomelatine and ramelteon, prevents SARS-CoV-2 entry in the brain, thereby reducing virus-induced damage of small cerebral vessels, immune cell infiltration and brain inflammation. Molecular modeling analyses complemented by experimental studies in cells showed that SARS-CoV-2 entry in endothelial cells is prevented by melatonin binding to an allosteric-binding site on human angiotensin-converting enzyme 2 (ACE2), thus interfering with ACE2 function as an entry receptor for SARS-CoV-2. Our findings open new perspectives for the repurposing of melatonergic drugs and its clinically used analogs in the prevention of brain infection by SARS-CoV-2 and COVID-19-related long-term neurological symptoms.

Pyrazolones as inhibitors of immune checkpoint blocking the PD-1/PD-L1 interaction.

Immuno-therapy has become a leading strategy to fight cancer. Over the past few years, immuno-therapies using checkpoint inhibitor monoclonal antibodies (mAbs) against programmed death receptor 1 (PD-1) and programmed death ligand 1 (PD-L1) have demonstrated improved survival compared with chemotherapy. We describe the microwave-assisted synthesis and the characterization of an innovative series of synthetic compounds endowed with nanomolar activity against PD-L1. The properties of the compounds were characterized using several biophysical techniques including microscale thermophoresis (MST) and fluorescence resonance energy transfer (FRET) measurements. A few small molecules demonstrated a high affinity for human PD-L1, potently disrupted the PD-L1:PD-1 interaction and inhibited Src homology region 2 domain-containing phosphatase (SHP2) recruitment to PD-1. More than 30 molecules from the pyrazolone family have been synthesized and 5 highly potent "PD-L1 silencing compounds" have been identified, based on in vitro measurements. Structure-activity relationships have been defined and ADME properties were evaluated. The phenyl-pyrazolone unit offers novel perspectives to design PD-L1-targeting agents, potentially useful to combat cancer and other pathologies implicating the PD-1/PD-L1 checkpoint.

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.

SINAPs: A Software Tool for Analysis and Visualization of Interaction Networks of Molecular Dynamics Simulations.

As long as the structural study of molecular mechanisms requires multiple molecular dynamics reflecting contrasted bioactive states, the subsequent analysis of molecular interaction networks remains a bottleneck to be fairly treated and requires a user-friendly 3D view of key interactions. Structural Interaction Network Analysis Protocols (SINAPs) is a proprietary python tool developed to (i) quickly solve key interactions able to distinguish two protein states, either from two sets of molecular dynamics simulations or from two crystallographic structures, and (ii) render a user-friendly 3D view of these key interactions through a plugin of UCSF Chimera, one of the most popular open-source viewing software for biomolecular systems. Through two case studies, glucose transporter-1 (GLUT-1) and A2A adenosine receptor (A2AR), SINAPs easily pinpointed key interactions observed experimentally and relevant for their bioactivities. This very effective tool was thus applied to identify the amino acids involved in the molecular enzymatic mechanisms ruling the activation of an immunomodulator drug candidate, P28 glutathione-S-transferase (P28GST). SINAPs is freely available at https://github.com/ParImmune/SINAPs.

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.

A small-molecule P2RX7 activator promotes anti-tumor immune responses and sensitizes lung tumor to immunotherapy.

Only a subpopulation of non-small cell lung cancer (NSCLC) patients responds to immunotherapies, highlighting the urgent need to develop therapeutic strategies to improve patient outcome. We develop a chemical positive modulator (HEI3090) of the purinergic P2RX7 receptor that potentiates αPD-1 treatment to effectively control the growth of lung tumors in transplantable and oncogene-induced mouse models and triggers long lasting antitumor immune responses. Mechanistically, the molecule stimulates dendritic P2RX7-expressing cells to generate IL-18 which leads to the production of IFN-γ by Natural Killer and CD4+ T cells within tumors. Combined with immune checkpoint inhibitor, the molecule induces a complete tumor regression in 80% of LLC tumor-bearing mice. Cured mice are also protected against tumor re-challenge due to a CD8-dependent protective response. Hence, combination treatment of small-molecule P2RX7 activator followed by immune checkpoint inhibitor represents a strategy that may be active against NSCLC.

Pyroglutamide-Based P2X7 Receptor Antagonists Targeting Inflammatory Bowel Disease.

This report deals with the design, the synthesis, and the pharmacological evaluation of pyroglutamide-based P2X7 antagonists. A dozen were shown to possess improved properties, among which inhibition of YO-PRO-1/TO-PRO-3 uptake and IL1ß release upon BzATP activation of the receptor and dampening signs of DSS-induced colitis on mice, in comparison with reference antagonist GSK1370319A. Docking study and biological evaluation of synthesized compounds has highlighted new SAR, and low toxicity profiles of pyroglutamides herein described are clues for the finding of a usable h-P2X7 antagonist drug. Such a drug would raise the hope for a cure to many P2X7-dependent pathologies, including inflammatory, neurological, and immune diseases.

Benzo[d]thiazol-2(3H)-ones as new potent selective CB2 agonists with anti-inflammatory properties.

The high distribution of CB2 receptors in immune cells suggests their important role in the control of inflammation. Growing evidence offers this receptor as an attractive therapeutic target: selective CB2 agonists are able to modulate inflammation without triggering psychotropic effects. In this work, we report a new series of selective CB2 agonists based on a benzo[d]thiazol-2(3H)-one scaffold. This drug design project led to the discovery of compound 9, as a very potent CB2 agonist (Ki = 13.5 nM) with a good selectivity versus CB1. This compound showed no cytotoxicity, acceptable ADME-Tox parameters and demonstrates the ability to counteract colon inflammatory process in vivo.

A phenotypic approach to the discovery of compounds that promote non-amyloidogenic processing of the amyloid precursor protein: Toward a new profile of indirect ß-secretase inhibitors.

Dysregulation of the Amyloid Precursor Protein (APP) processing leading to toxic species of amyloid ß peptides (Aß) is central to Alzheimer's disease (AD) etiology. Aß peptides are produced by sequential cleavage of APP by ß-secretase (BACE-1) and γ-secretase. Lysosomotropic agent, chloroquine (CQ), has been reported to inhibit Aß peptide production. However, this effect is accompanied by an inhibition of lysosome-mediated degradation pathways. Following on from the promising activity of two series of APP metabolism modulators derived from CQ, we sought to develop new series of compounds that would retain the inhibitory effects on Aß production without altering lysosome functions. Herein, we applied a ligand-based pharmacophore modeling approach coupled with de novo design that led to the discovery of a series of biaryl compounds. Structure-activity relationship studies revealed that minor modifications like replacing a piperidine moiety of compound 30 by a cyclohexyl (compound 31) allowed for the identification of compounds with the desired profile. Further studies have demonstrated that compounds 30 and 31 act through an indirect mechanism to inhibit ß-secretase activity. This work shows that it is possible to dissociate the inhibitory effect on Aß peptide secretion of CQ-derived compounds from the lysosome-mediated degradation effect, providing a new profile of indirect ß-secretase inhibitors.

Toward the Discovery of a Novel Class of YAP⁻TEAD Interaction Inhibitors by Virtual Screening Approach Targeting YAP⁻TEAD Protein⁻Protein Interface.

Intrinsically disordered protein YAP (yes-associated protein) interacts with TEADs transcriptional factors family (transcriptional enhancer associated domain) creating three interfaces. Interface 3, between the Ω-loop of YAP and a shallow pocket of TEAD was identified as the most important TEAD zone for YAP-TEAD interaction. Using the first X-ray structure of the hYAP50⁻71-hTEAD1209⁻426 complex (PDB 3KYS) published in 2010, a protein-protein interaction inhibitors-enriched library (175,000 chemical compounds) was screened against this hydrophobic pocket of TEAD. Four different chemical families have been identified and evaluated using biophysical techniques (thermal shift assay, microscale thermophoresis) and in cellulo assays (luciferase activity in transfected HEK293 cells, RTqPCR in MDA-MB231 cells). A first promising hit with micromolar inhibition in the luciferase gene reporter assay was discovered. This hit also decreased mRNA levels of TEAD target genes.

Design and synthesis of 2,6-disubstituted-8-amino imidazo[1,2a]pyridines, a promising privileged structure.

Imidazo[1,2a]pyridines have gained much interest in the field of medicinal chemistry research. In the aim of accessing new privileged structure, we decided to design and synthesize 8-aminated-imidazo[1,2a]pyridines substituted on positions 2 and 6. This scaffold, rarely found in the literature, was obtained via palladium-catalyzed coupling reactions (Suzuki reaction or N-hydroxysuccinimidyl activated ester method) and tested on adenosine receptor A2A. We demonstrated how incorporation of an exocyclic amine enhanced affinity towards this receptor while maintaining low cytotoxicity.

New phenylaniline derivatives as modulators of amyloid protein precursor metabolism.

The chloroquinoline scaffold is characteristic of anti-malarial drugs such as chloroquine (CQ) or amodiaquine (AQ). These drugs are also described for their potential effectiveness against prion disease, HCV, EBV, Ebola virus, cancer, Parkinson or Alzheimer diseases. Amyloid precursor protein (APP) metabolism is deregulated in Alzheimer's disease. Indeed, CQ modifies amyloid precursor protein (APP) metabolism by precluding the release of amyloid-beta peptides (Aß), which accumulate in the brain of Alzheimer patients to form the so-called amyloid plaques. We showed that AQ and analogs have similar effects although having a higher cytotoxicity. Herein, two new series of compounds were synthesized by replacing 7-chloroquinolin-4-amine moiety of AQ by 2-aminomethylaniline and 2-aminomethylphenyle moieties. Their structure activity relationship was based on their ability to modulate APP metabolism, Aß release, and their cytotoxicity similarly to CQ. Two compounds 15a, 16a showed interesting and potent effect on the redirection of APP metabolism toward a decrease of Aß peptide release (in the same range compared to AQ), and a 3-10-fold increased stability of APP carboxy terminal fragments (CTFα and AICD) without obvious cellular toxicity at 100 µM.

Importance of the second extracellular loop for melatonin MT1 receptor function and absence of melatonin binding in GPR50.

BACKGROUND AND PURPOSE: Recent crystal structures of GPCRs have emphasized the previously unappreciated role of the second extracellular (E2) loop in ligand binding and gating and receptor activation. Here, we have assessed the role of the E2 loop in the activation of the melatonin MT1 receptor and in the inactivation of the closely related orphan receptor GPR50. EXPERIMENTAL APPROACH: Chimeric MT1 -GPR50 receptors were generated and functionally analysed in terms of 2-[125 I]iodomelatonin binding, Gi /cAMP signalling and ß-arrestin2 recruitment. We also used computational molecular dynamics (MD) simulations. KEY RESULTS: MD simulations of 300 ns revealed (i) the tight hairpin structure of the E2 loop of the MT1 receptor (ii) the most suitable features for melatonin binding in MT1 receptors and (iii) major predicted rearrangements upon MT1 receptor activation, stabilizing interaction networks between Phe179 or Gln181 in the E2 loop and transmembrane helixes 5 and 6. Functional assays confirmed these predictions, because reciprocal replacement of MT1 and GPR50 residues/domains led to the predicted loss- and gain-of-melatonin action of MT1 receptors and GPR50 respectively. CONCLUSIONS AND IMPLICATIONS: Our work demonstrated the crucial role of the E2 loop for MT1 receptor and GPR50 function by proposing a model in which the E2 loop is important in stabilizing active MT1 receptor conformations and by showing how evolutionary processes appear to have selected for modifications in the E2 loop in order to make GPR50 unresponsive to melatonin. LINKED ARTICLES: This article is part of a themed section on Recent Developments in Research of Melatonin and its Potential Therapeutic Applications. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.16/issuetoc.

Antagonists of the adenosine A2A receptor based on a 2-arylbenzoxazole scaffold: Investigation of the C5- and C7-positions to enhance affinity.

We have recently reported a series of 2-furoyl-benzoxazoles as potential A2A adenosine receptor (A2AR) antagonists. Two hits were identified with interesting pharmacokinetic properties but were find to bind the hA2AR receptor in the micromolar-range. Herein, in order to enhance affinity toward the hA2AR, we explored the C5- and C7-position of hits 1 and 2 based on docking studies. These modifications led to compounds with nanomolar-range affinity (e.g. 6a, Ki = 40 nM) and high antagonist activity (e.g. 6a, IC50 = 70.6 nM). Selected compounds also exhibited interesting in vitro DMPK (Drug Metabolism and Pharmacokinetics) properties including high solubility and low cytotoxicity. Therefore, the benzoxazole ring appears as a highly effective scaffold for the design of new A2A antagonists.

Design, synthesis and evaluation of 2-aryl benzoxazoles as promising hit for the A2A receptor.

The development of adenosine A2A receptor antagonists has received much interest in recent years for the treatment of neurodegenerative diseases. Based on docking studies, a new series of 2-arylbenzoxazoles has been identified as potential A2AR antagonists. Structure-affinity relationship was investigated in position 2, 5 and 6 of the benzoxazole heterocycle leading to compounds with a micromolar affinity towards the A2A receptor. Compound F1, with an affinity of 1 µm, presented good absorption, distribution, metabolism and excretion properties with an excellent aqueous solubility (184 µm) without being cytotoxic at 100 µm. This compound, along with low-molecular weight compound D1 (Ki = 10 µm), can be easily modulated and thus considered as relevant starting points for further hit-to-lead optimisation.

Synthesis and Biological Evaluation of N-[2-(4-Hydroxyphenylamino)-pyridin-3-yl]-4-methoxy-benzenesulfonamide (ABT-751) Tricyclic Analogues as Antimitotic and Antivascular Agents with Potent in Vivo Antitumor Activity.

Benzopyridothiadiazepine (2a) and benzopyridooxathiazepine (2b) were modified to produce tricyclic quinazolinone 15-18 or benzothiadiazine 26-27 derivatives. These compounds were evaluated in cytotoxicity and tubulin inhibition assays and led to potent inhibitors of tubulin polymerization. N-[2(4-Methoxyphenyl)ethyl]-1,2-dihydro-pyrimidino[2,1-b]quinazolin-6-one (16a) exhibited the best in vitro cytotoxic activity (GI50 10-66.9 nM) against the NCI 60 human tumor cell line and significant potency against tubulin assembly (IC50 0.812 µM). In mechanism studies, 16a was shown to block cell cycle in G2/M phase and to disrupt microtubule formation and displayed good antivascular properties as inhibition of cell migration, invasion, and endothelial tube formation. Compound 16a was evaluated in C57BL/6 mouse melanoma B16F10 xenograft model to validate its antitumor activity, in comparison with reference ABT-751 (1). Compound 16a displayed strong in vivo antitumor and antivascular activities at a dose of 5 mg/kg without obvious toxicity, whereas 1 needed a 10-fold higher concentration to reach similar effects.

Update on melatonin receptors: IUPHAR Review 20.

Melatonin receptors are seven transmembrane-spanning proteins belonging to the GPCR superfamily. In mammals, two melatonin receptor subtypes exist - MT1 and MT2 - encoded by the MTNR1A and MTNR1B genes respectively. The current review provides an update on melatonin receptors by the corresponding subcommittee of the International Union of Basic and Clinical Pharmacology. We will highlight recent developments of melatonin receptor ligands, including radioligands, and give an update on the latest phenotyping results of melatonin receptor knockout mice. The current status and perspectives of the structure of melatonin receptor will be summarized. The physiological importance of melatonin receptor dimers and biologically important and type 2 diabetes-associated genetic variants of melatonin receptors will be discussed. The role of melatonin receptors in physiology and disease will be further exemplified by their functions in the immune system and the CNS. Finally, antioxidant and free radical scavenger properties of melatonin and its relation to melatonin receptors will be critically addressed.