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.

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.

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.

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.

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.

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.

Design, synthesis and biological evaluation of potent FAAH inhibitors.

A new series of 3-carboxamido-5-aryl-isoxazoles was designed, synthesized and evaluated for their biological activity. Different pharmacomodulations have been explored and the lipophilicity of these compounds was assessed. Investigation of the in vitro biological activity led to the identification of 5 compounds as potent FAAH inhibitors, their good FAAH inhibition capacity is probably correlated with their suitable lipophilicity. Specifically, compound 25 showed similar inhibition potency against FAAH in comparison with URB597, one of the most potent FAAH inhibitor known to date.

Relationships between Th1 or Th2 iNKT cell activity and structures of CD1d-antigen complexes: meta-analysis of CD1d-glycolipids dynamics simulations.

A number of potentially bioactive molecules can be found in nature. In particular, marine organisms are a valuable source of bioactive compounds. The activity of an α-galactosylceramide was first discovered in 1993 via screening of a Japanese marine sponge (Agelas mauritanius). Very rapidly, a synthetic glycololipid analogue of this natural molecule was discovered, called KRN7000. Associated with the CD1d protein, this α-galactosylceramide 1 (KRN7000) interacts with the T-cell antigen receptor to form a ternary complex that yields T helper (Th) 1 and Th2 responses with opposing effects. In our work, we carried out molecular dynamics simulations (11.5 µs in total) involving eight different ligands (conducted in triplicate) in an effort to find out correlation at the molecular level, if any, between chemical modulation of 1 and the orientation of the known biological response, Th1 or Th2. Comparative investigations of human versus mouse and Th1 versus Th2 data have been carried out. A large set of analysis tools was employed including free energy landscapes. One major result is the identification of a specific conformational state of the sugar polar head, which could be correlated, in the present study, to the biological Th2 biased response. These theoretical tools provide a structural basis for predicting the very different dynamical behaviors of α-glycosphingolipids in CD1d and might aid in the future design of new analogues of 1.

3-Carboxamido-5-aryl-isoxazoles as new CB2 agonists for the treatment of colitis.

Recent investigations showed that anandamide, the main endogenous ligand of CB1 and CB2 cannabinoid receptors, possesses analgesic, antidepressant and anti-inflammatory effects. In the perspective to treat inflammatory bowel disease (IBD), our approach was to develop new selective CB2 receptor agonists without psychotropic side effects associated to CB1 receptors. In this purpose, a new series of 3-carboxamido-5-aryl-isoxazoles, never described previously as CB2 receptor agonists, was designed, synthesized and evaluated for their biological activity. The pharmacological results have identified great selective CB2 agonists with in vivo anti-inflammatory activity in a DSS-induced acute colitis mouse model.

Mixed metallic Ba(Co,Fe)X(0.2)O(3-δ) (X = F, Cl) hexagonal perovskites: drastic effect of Fe-incorporation on structural and electronic features.

Starting from the parent 10H-Ba(5)Co(5)X(1-x)O(13-δ) (trimeric strings of face-sharing CoO(6) octahedra with terminal CoO(4) tetrahedra, stacking sequence (chhch')(2)) and 6H-Ba(6)Co(6)X(1-x)O(16-δ) (similar with tetrameric strings, stacking sequence chhhch') hexagonal perovskites forms (X = F, Cl; c, h = [BaO(3)] layers ; h' = [BaOX(1-y)] layers), we show here that the Fe incorporation leads to large domains of solid solutions for both X = F and Cl but exclusively stabilizes the 10H-form independently of the synthesis method. In this form, the lowest concentration of h-layers is stabilized by a sensitive metal reduction with increasing the Fe ratio. In a more general context of competition between several hexagonal perovskite polymorphs available for most of the transition metals, this redox change is most probably the key factor driving 1D (face-sharing chains) to 3D (corner-sharing) connectivities. Strikingly, ND data evidence the location of oxygen deficiencies in the tetrahedral (Co/Fe) coordination. This effect is exaggerated at high temperature, while (Co/Fe)O(4-δ) coordinations are completed by the displacement of X(-) anions toward the (Co/Fe) sphere of coordination following a "push-and-pull" mechanism within h'-[BaOX(1-y)] layers. The Fe-incorporation is also accompanied by increasing conduction gaps with predominant 1D variable range hopping. The full series show antiferromagnetic behavior with increasing T(N) as [Fe] increases. For Fe-rich compounds T(N) is estimated about 600 K, as rarely observed for hexagonal perovskite compounds. Finally, magnetic structures of all iron-doped compounds show a site-to-site AFM ordering, different of the magnetic structure of Co-only parent compounds. Here, DFT calculations predict low-spin octahedral Co configurations, but high-spin Fe species in the same sites.

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.

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.

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.

New FAAH inhibitors based on 3-carboxamido-5-aryl-isoxazole scaffold that protect against experimental colitis.

Growing evidence suggests a role for the endocannabinoid (EC) system, in intestinal inflammation and compounds inhibiting anandamide degradation offer a promising therapeutic option for the treatment of inflammatory bowel diseases. In this paper, we report the first series of carboxamides derivatives possessing FAAH inhibitory activities. Among them, compound 39 displayed significant inhibitory FAAH activity (IC(50)=0.088 µM) and reduced colitis induced by intrarectal administration of TNBS.

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.

Mutations in components of complement influence the outcome of Factor I-associated atypical hemolytic uremic syndrome.

Genetic studies have shown that mutations of complement inhibitors such as membrane cofactor protein, Factors H, I, or B and C3 predispose patients to atypical hemolytic uremic syndrome (aHUS). Factor I is a circulating serine protease that inhibits complement by degrading C3b and up to now only a few mutations in the CFI gene have been characterized. In a large cohort of 202 patients with aHUS, we identified 23 patients carrying exonic mutations in CFI. Their overall clinical outcome was unfavorable, as half died or developed end-stage renal disease after their first syndrome episode. Eight patients with CFI mutations carried at least one additional known genetic risk factor for aHUS, such as a mutation in MCP, CFH, C3 or CFB; a compound heterozygous second mutation in CFI; or mutations in both the MCP and CFH genes. Five patients exhibited homozygous deletion of the Factor H-related protein 1 (CFHR-1) gene. Ten patients with aHUS had one mutation in their CFI gene (Factor I-aHUS), resulting in a quantitative or functional Factor I deficiency. Patients with a complete deletion of the CFHR-1 gene had a significantly higher risk of a bad prognosis compared with those with one Factor I mutation as their unique vulnerability feature. Our results emphasize the necessity of genetic screening for all susceptibility factors in patients with aHUS.

Genetic, molecular and functional analyses of complement factor I deficiency.

Complete deficiency of complement inhibitor factor I (FI) results in secondary complement deficiency due to uncontrolled spontaneous alternative pathway activation leading to susceptibility to infections. Current genetic examination of two patients with near complete FI deficiency and three patients with no detectable serum FI and also close family members revealed homozygous or compound heterozygous mutations in several domains of FI. These mutations were introduced into recombinant FI and the resulting proteins were purified for functional studies, while transient transfection was used to analyze expression and secretion. The G170V mutation resulted in a protein that was not expressed, whereas the mutations Q232K, C237Y, S250L, I339M and H400L affected secretion. Furthermore, the C237Y and the S250L mutants did not degrade C4b and C3b as efficiently as the WT. The truncated Q336x mutant could be expressed, in vitro, but was not functional because it lacks the serine protease domain. Furthermore, this truncated FI was not detected in serum of the patient. Structural investigations using molecular modeling were performed to predict the potential impact the mutations have on FI structure. This is the first study that investigates, at the functional level, the consequences of molecular defects identified in patients with full FI deficiency.

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.