Characterization of anti-GASP motif antibodies that inhibit the interaction between GPRASP1 and G protein-coupled receptors.

G protein-coupled receptor associated sorting protein 1 (GPRASP1) belongs to a family of 10 proteins that display sequence homologies in their C-terminal region. Several members including GPRASP1 also display a short repeated sequence called the GASP motif that is critically involved in protein-protein interactions with G protein-coupled receptors (GPCRs). Here, we characterized anti-GASP motif antibodies and investigated their potential inhibitory functions. We first showed that our in-house anti-GPRASP1 rabbit polyclonal serum contains anti-GASP motif antibodies and purified them by affinity chromatography. We further showed that these antibodies can detect GPRASP1 and GPRASP2 in Western blot, immunoprecipitation and immunofluorescence experiments while a mutant of GPRASP2, in which the most conserved hydrophobic core of the GASP motifs is mutated, was no more detected. Further characterization of anti-GASP motif antibodies by ELISA and Surface Plasmon Resonance assays suggests that GASP motifs function as multivalent epitopes. Finally, we set-up an Amplified Luminescent Proximity Homogeneous AlphaScreen® assay to detect the interaction between purified ADRB2 receptor and the central domain of GPRASP1 and showed that anti-GASP motif antibodies efficiently inhibit this interaction. Altogether, our results suggest that anti-GASP motif antibodies could represent a valuable tool to neutralize the interaction of GPRASP1 and GPRASP2 with different GPCRs.

Rapid and Highly Selective Fluorescent Labeling of Peptides via a Thia-Diels-Alder Cycloaddition: Application to Apelin.

Herein, we describe a catalyst-free thia-Diels-Alder cycloaddition for the chemoselective labeling of fully deprotected phosphonodithioester-peptides in solution with fluorophores functionalized with an exocyclic diene. The reaction was optimized on the model tripeptide 1 containing a lysine residue, which enabled its rapid and straightforward labeling with three different fluorophores (fluorescein, lissamine rhodamine B, and squaraine) in very mild conditions (H2O/iPrOH, 37 °C, 1 h). The reaction was then successfully applied to the chemoselective labeling of fully deprotected apelin-13 with squaraine dye. The resulting fluorescent ligand 18 exhibited a high affinity (0.17 ± 0.03 nM) for apelinR. It enabled the development of time-resolved FRET-based competition assays for high-throughput screening and drug discovery. Thanks to its fluorogenic properties, ligand 18 was also successfully involved in the live-cell optical imaging of apelinR in no-wash conditions.

Target-Focused Library Design by Pocket-Applied Computer Vision and Fragment Deep Generative Linking.

We here describe a computational approach (POEM: Pocket Oriented Elaboration of Molecules) to drive the generation of target-focused libraries while taking advantage of all publicly available structural information on protein-ligand complexes. A collection of 31 384 PDB-derived images with key shapes and pharmacophoric properties, describing fragment-bound microenvironments, is first aligned to the query target cavity by a computer vision method. The fragments of the most similar PDB subpockets are then directly positioned in the query cavity using the corresponding image transformation matrices. Lastly, suitable connectable atoms of oriented fragment pairs are linked by a deep generative model to yield fully connected molecules. POEM was applied to generate a library of 1.5 million potential cyclin-dependent kinase 8 inhibitors. By synthesizing and testing as few as 43 compounds, a few nanomolar inhibitors were quickly obtained with limited resources in just two iterative cycles.

Engineering Novel 3D Models to Recreate High-Grade Osteosarcoma and its Immune and Extracellular Matrix Microenvironment.

Osteosarcoma (OS) is the most common primary bone cancer, where the overall 5-year surviving rate is below 20% in resistant forms. Accelerating cures for those poor outcome patients remains a challenge. Nevertheless, several studies of agents targeting abnormal cancerous pathways have yielded disappointing results when translated into clinic because of the lack of accurate OS preclinical modeling. So, any effort to design preclinical drug testing may consider all inter-, intra-, and extra-tumoral heterogeneities throughout models mimicking extracellular and immune microenvironment. Therefore, the bioengineering of patient-derived models reproducing the OS heterogeneity, the interaction with tumor-associated macrophages (TAMs), and the modulation of oxygen concentrations additionally to recreation of bone scaffold is proposed here. Eight 2D preclinical models mimicking several OS clinical situations and their TAMs in hypoxic conditions are developed first and, subsequently, the paired 3D models faithfully preserving histological and biological characteristics are generated. It is possible to shape reproducibly M2-like macrophages cultured with all OS patient-derived cell lines in both dimensions. The final 3D models pooling all heterogeneity features are providing accurate proliferation and migration data to understand the mechanisms involved in OS and immune cells/biomatrix interactions and sustained such that engineered 3D preclinical systems will improve personalized medicine.

High-Throughput Screening for Extracellular Inhibitors of the FLT3 Receptor Tyrosine Kinase Reveals Chemically Diverse and Druggable Negative Allosteric Modulators.

Inhibiting receptor tyrosine kinases is commonly achieved by two main strategies targeting either the intracellular kinase domain by low molecular weight compounds or the extracellular ligand-binding domain by monoclonal antibodies. Identifying small molecules able to inhibit RTKs at the extracellular level would be highly desirable to gain exquisite selectivity but is believed to be challenging owing to the size of RTK endogenous ligands (cytokines, growth factors) and the topology of RTK extracellular domains. We here report the high-throughput screening of the French Chemical Library (48K compounds) for extracellular inhibitors of the Fms-like tyrosine kinase 3 (FLT3) receptor tyrosine kinase, by a homogeneous time-resolved fluorescence competition assay. A total of 679 small molecular weight ligands (1.4%) were confirmed to strongly inhibit (>75%) the binding of the fluorescent labeled FLT3 ligand (FL cytokine) to FLT3 overexpressed in HEK-293 cells, at two different concentrations (5 and 20 µM). Concentration-response curves, obtained for 111 lead-like molecules, confirmed the unexpected tolerance of the FLT3 extracellular domain for low molecular weight druggable inhibitors exhibiting submicromolar potencies, chemical diversity, and promising pharmacokinetic properties. Further investigation of one hit confirmed inhibitory properties in dorsal root ganglia neurons and in a mouse model of neuropathic pain.

Treating leishmaniasis in Amazonia, part 2: Multi-target evaluation of widely used plants to understand medicinal practices.

ETHNOPHARMACOLOGICAL RELEVANCE: Leishmaniasis are widely distributed among tropical and subtropical countries, and remains a crucial health issue in Amazonia. Indigenous groups across Amazonia have developed abundant knowledge about medicinal plants related to this pathology. AIM OF THE STUDY: We intent to explore the weight of different pharmacological activities driving taxa selection for medicinal use in Amazonian communities. Our hypothesis is that specific activity against Leishmania parasites is only one factor along other (anti-inflammatory, wound healing, immunomodulating, antimicrobial) activities. MATERIALS AND METHODS: The twelve most widespread plant species used against leishmaniasis in Amazonia, according to their cultural and biogeographical importance determined through a wide bibliographical survey (475 use reports), were selected for this study. Plant extracts were prepared to mimic their traditional preparations. Antiparasitic activity was evaluated against promastigotes of reference and clinical New-World strains of Leishmania (L. guyanensis, L. braziliensis and L. amazonensis) and L. amazonensis intracellular amastigotes. We concurrently assessed the extracts immunomodulatory properties on PHA-stimulated human PBMCs and RAW264.7 cells, and on L. guyanensis antigens-stimulated PBMCs obtained from Leishmania-infected patients, as well as antifungal activity and wound healing properties (human keratinocyte migration assay) of the selected extracts. The cytotoxicity of the extracts against various cell lines (HFF1, THP-1, HepG2, PBMCs, RAW264.7 and HaCaT cells) was also considered. The biological activity pattern of the extracts was represented through PCA analysis, and a correlation matrix was calculated. RESULTS: Spondias mombin L. bark and Anacardium occidentale L. stem and leaves extracts displayed high anti-promatigotes activity, with IC50 ≤ 32 µg/mL against L. guyanensis promastigotes for S. mombin and IC50 of 67 and 47 µg/mL against L. braziliensis and L. guyanensis promastigotes, respectively, for A. occidentale. In addition to the antiparasitic effect, antifungal activity measured against C. albicans and T. rubrum (MIC in the 16-64 µg/mL range) was observed. However, in the case of Leishmania amastigotes, the most active species were Bixa orellana L. (seeds), Chelonantus alatus (Aubl.) Pulle (leaves), Jacaranda copaia (Aubl.) D. Don. (leaves) and Plantago major L. (leaves) with IC50 < 20 µg/mL and infection rates of 14-25% compared to the control. Concerning immunomodulatory activity, P. major and B. orellana were highlighted as the most potent species for the wider range of cytokines in all tested conditions despite overall contrasting results depending on the model. Most of the species led to moderate to low cytotoxic extracts except for C. alatus, which exhibited strong cytotoxic activity in almost all models. None of the tested extracts displayed wound healing properties. CONCLUSIONS: We highlighted pharmacologically active extracts either on the parasite or on associated pathophysiological aspects, thus supporting the hypothesis that antiparasitic activities are not the only biological factor useful for antileishmanial evaluation. This result should however be supplemented by in vivo studies, and attracts once again the attention on the importance of the choice of biological models for an ethnophamacologically consistent study. Moreover, plant cultural importance, ecological status and availability were discussed in relation with biological results, thus contributing to link ethnobotany, medical anthropology and biology.

Antiallodynic action of phosphodiesterase inhibitors in a mouse model of peripheral nerve injury.

Neuropathic pain arises as a consequence of a lesion or disease affecting the somatosensory nervous system. It is accompanied by neuronal and non-neuronal alterations, including alterations in intracellular second messenger pathways. Cellular levels of 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP) are regulated by phosphodiesterase (PDE) enzymes. Here, we studied the impact of PDE inhibitors (PDEi) in a mouse model of peripheral nerve injury induced by placing a cuff around the main branch of the sciatic nerve. Mechanical hypersensitivity, evaluated using von Frey filaments, was relieved by sustained treatment with the non-selective PDEi theophylline and ibudilast (AV-411), with PDE4i rolipram, etazolate and YM-976, and with PDE5i sildenafil, zaprinast and MY-5445, but not by treatments with PDE1i vinpocetine, PDE2i EHNA or PDE3i milrinone. Using pharmacological and knock-out approaches, we show a preferential implication of delta opioid receptors in the action of the PDE4i rolipram and of both mu and delta opioid receptors in the action of the PDE5i sildenafil. Calcium imaging highlighted a preferential action of rolipram on dorsal root ganglia non-neuronal cells, through PDE4B and PDE4D inhibition. Rolipram had anti-neuroimmune action, as shown by its impact on levels of the pro-inflammatory cytokine tumor necrosis factor-α (TNFα) in the dorsal root ganglia of mice with peripheral nerve injury, as well as in human peripheral blood mononuclear cells (PBMCs) stimulated with lipopolysaccharides. This study suggests that PDEs, especially PDE4 and 5, may be targets of interest in the treatment of neuropathic pain.

Liposomal encapsulation of trans-crocetin enhances oxygenation in patients with COVID-19-related ARDS receiving mechanical ventilation.

Current therapeutic treatments improving the impaired transportation of oxygen in acute respiratory distress syndrome (ARDS) have been found to be relevant and beneficial for the therapeutic treatment of COVID-19 patients suffering from severe respiratory complications. Hence, we report the preclinical and the preliminary results of the Phase I/II clinical trial of LEAF-4L6715, a liposomal nanocarrier encapsulating the kosmotropic agent trans-crocetin (TC), which, once injected, enhance the oxygenation of vascular tissue and therefore has the potential to improve the clinical outcomes of ARDS and COVID-19 in severely impacted patients. We demonstrated that the liposomal formulation enabled to increase from 30 min to 48 h the reoxygenation properties of free TCs in vitro in endothelial cells, but also to improve the half-life of TC by 6-fold in healthy mice. Furthermore, we identified 25 mg/kg as the maximum tolerated dose in mice. This determined concentration led to the validation of the therapeutic efficacy of LEAF-4 L6715 in a sepsis mouse model. Finally, we report the preliminary outcomes of an open-label multicenter Phase I/II clinical trial (EudraCT 2020-001393-30; NCT04378920), which was aimed to define the appropriate schedule and dosage of LEAF-4L6715 and to confirm its tolerability profile and preliminary clinical activity in COVID-19 patients treated in intensive care unit.

Nile Red-Based GPCR Ligands as Ultrasensitive Probes of the Local Lipid Microenvironment of the Receptor.

The local lipid microenvironment of transmembrane receptors is an essential factor in G protein coupled receptor (GPCR) signaling. However, tools are currently missing for studying endogenously expressed GPCRs in primary cells and tissues. Here, we introduce fluorescent environment-sensitive GPCR ligands for probing the microenvironment of the receptor in living cells using fluorescence microscopy under no-wash conditions. We designed and synthesized antagonist ligands of the oxytocin receptor (OTR) by conjugating a high-affinity nonpeptidic OTR ligand PF-3274167 to the environment-sensitive fluorescent dye Nile Red. The length of the polar PEG spacer between the pharmacophore and the fluorophore was adjusted to lower the nonspecific interactions of the probe while preserving a strong fluorogenic response. We demonstrated that the new probes embed into the lipid bilayer in the vicinity of the receptor and convey information about the local polarity and the lipid order via the wavelength-shifting emission of the Nile Red fluorophore.

Overexpression of the MSK1 Kinase in Patients With Chronic Lung Allograft Dysfunction and Its Confirmed Role in a Murine Model.

BACKGROUND: Chronic lung allograft dysfunction (CLAD) and its obstructive form, the obliterative bronchiolitis (OB), are the main long-term complications related to high mortality rate postlung transplantation. CLAD treatment lacks a significant success in survival. Here, we investigated a new strategy through inhibition of the proinflammatory mitogen- and stress-activated kinase 1 (MSK1) kinase. METHODS: MSK1 expression was assessed in a mouse OB model after heterotopic tracheal allotransplantation. Pharmacological inhibition of MSK1 (H89, fasudil, PHA767491) was evaluated in the murine model and in a translational model using human lung primary fibroblasts in proinflammatory conditions. MSK1 expression was graded over time in biopsies from a cohort of CLAD patients. RESULTS: MSK1 mRNA progressively increased during OB (6.4-fold at D21 posttransplantation). Inhibition of MSK1 allowed to counteract the damage to the epithelium (56% restoration for H89), and abolished the recruitment of MHCII+ (94%) and T cells (100%) at the early inflammatory phase of OB. In addition, it markedly decreased the late fibroproliferative obstruction in allografts (48%). MSK1 inhibitors decreased production of IL-6 (whose transcription is under the control of MSK1) released from human lung fibroblasts (96%). Finally, we confirmed occurrence of a 2.9-fold increased MSK1 mRNA expression in lung biopsies in patients at 6 months before CLAD diagnosis as compared to recipients with stable lung function. CONCLUSIONS: These findings suggest the overall interest of the MSK1 kinase either as a marker or as a potential therapeutic target in lung dysfunction posttransplantation.

Design, Synthesis and Biological Evaluation of Arylpyridin-2-yl Guanidine Derivatives and Cyclic Mimetics as Novel MSK1 Inhibitors. An Application in an Asthma Model.

Mitogen- and Stress-Activated Kinase 1 (MSK1) is a nuclear kinase, taking part in the activation pathway of the pro-inflammatory transcription factor NF-kB and is demonstrating a therapeutic target potential in inflammatory diseases such as asthma, psoriasis and atherosclerosis. To date, few MSK1 inhibitors were reported. In order to identify new MSK1 inhibitors, a screening of a library of low molecular weight compounds was performed, and the results highlighted the 6-phenylpyridin-2-yl guanidine (compound 1a, IC50~18 µM) as a starting hit for structure-activity relationship study. Derivatives, homologues and rigid mimetics of 1a were designed, and all synthesized compounds were evaluated for their inhibitory activity towards MSK1. Among them, the non-cytotoxic 2-aminobenzimidazole 49d was the most potent at inhibiting significantly: (i) MSK1 activity, (ii) the release of IL-6 in inflammatory conditions in vitro (IC50~2 µM) and (iii) the inflammatory cell recruitment to the airways in a mouse model of asthma.

Efficacy Assessment of an Uncharged Reactivator of NOP-Inhibited Acetylcholinesterase Based on Tetrahydroacridine Pyridine-Aldoxime Hybrid in Mouse Compared to Pralidoxime.

(1) Background: Human exposure to organophosphorus compounds employed as pesticides or as chemical warfare agents induces deleterious effects due to cholinesterase inhibition. One therapeutic approach is the reactivation of inhibited acetylcholinesterase by oximes. While currently available oximes are unable to reach the central nervous system to reactivate cholinesterases or to display a wide spectrum of action against the variety of organophosphorus compounds, we aim to identify new reactivators without such drawbacks. (2) Methods: This study gathers an exhaustive work to assess in vitro and in vivo efficacy, and toxicity of a hybrid tetrahydroacridine pyridinaldoxime reactivator, KM297, compared to pralidoxime. (3) Results: Blood-brain barrier crossing assay carried out on a human in vitro model established that KM297 has an endothelial permeability coefficient twice that of pralidoxime. It also presents higher cytotoxicity, particularly on bone marrow-derived cells. Its strong cholinesterase inhibition potency seems to be correlated to its low protective efficacy in mice exposed to paraoxon. Ventilatory monitoring of KM297-treated mice by double-chamber plethysmography shows toxic effects at the selected therapeutic dose. This breathing assessment could help define the No Observed Adverse Effect Level (NOAEL) dose of new oximes which would have a maximum therapeutic effect without any toxic side effects.

Anti-Inflammatory, Antioxidant, and Wound-Healing Properties of Cyanobacteria from Thermal Mud of Balaruc-Les-Bains, France: A Multi-Approach Study.

Background: The Balaruc-les-Bains' thermal mud was found to be colonized predominantly by microorganisms, with cyanobacteria constituting the primary organism in the microbial biofilm observed on the mud surface. The success of cyanobacteria in colonizing this specific ecological niche can be explained in part by their taxa-specific adaptation capacities, and also the diversity of bioactive natural products that they synthesize. This array of components has physiological and ecological properties that may be exploited for various applications. Methods: Nine cyanobacterial strains were isolated from Balaruc thermal mud and maintained in the Paris Museum Collection (PMC). Full genome sequencing was performed coupled with targeted and untargeted metabolomic analyses (HPLC-DAD and LC-MS/MS). Bioassays were performed to determine antioxidant, anti-inflammatory, and wound-healing properties. Results: Biosynthetic pathways for phycobiliproteins, scytonemin, and carotenoid pigments and 124 metabolite biosynthetic gene clusters (BGCs) were characterized. Several compounds with known antioxidant or anti-inflammatory properties, such as carotenoids, phycobilins, mycosporine-like amino acids, and aeruginosins, and other bioactive metabolites like microginins, microviridins, and anabaenolysins were identified. Secretion of the proinflammatory cytokines TNF-α, IL-1ß, IL-6, and IL-8 appeared to be inhibited by crude extracts of Planktothricoides raciborskii PMC 877.14, Nostoc sp. PMC 881.14, and Pseudo-chroococcus couteii PMC 885.14. The extract of the Aliinostoc sp. PMC 882.14 strain was able to slightly enhance migration of HaCat cells that may be helpful in wound healing. Several antioxidant compounds were detected, but no significant effects on nitric oxide secretion were observed. There was no cytotoxicity on the three cell types tested, indicating that cyanobacterial extracts may have anti-inflammatory therapeutic potential without harming body cells. These data open up promising uses for these extracts and their respective molecules in drugs or thermal therapies.

Hypoxic Environment and Paired Hierarchical 3D and 2D Models of Pediatric H3.3-Mutated Gliomas Recreate the Patient Tumor Complexity.

BACKGROUND: Pediatric high-grade gliomas (pHGGs) are facing a very dismal prognosis and representative pre-clinical models are needed for new treatment strategies. Here, we examined the relevance of collecting functional, genomic, and metabolomics data to validate patient-derived models in a hypoxic microenvironment. METHODS: From our biobank of pediatric brain tumor-derived models, we selected 11 pHGGs driven by the histone H3.3K28M mutation. We compared the features of four patient tumors to their paired cell lines and mouse xenografts using NGS (next generation sequencing), aCGH (array comparative genomic hybridization), RNA sequencing, WES (whole exome sequencing), immunocytochemistry, and HRMAS (high resolution magic angle spinning) spectroscopy. We developed a multicellular in vitro model of cell migration to mimic the brain hypoxic microenvironment. The live cell technology Incucyte© was used to assess drug responsiveness in variable oxygen conditions. RESULTS: The concurrent 2D and 3D cultures generated from the same tumor sample exhibited divergent but complementary features, recreating the patient intra-tumor complexity. Genomic and metabolomic data described the metabolic changes during pHGG progression and supported hypoxia as an important key to preserve the tumor metabolism in vitro and cell dissemination present in patients. The neurosphere features preserved tumor development and sensitivity to treatment. CONCLUSION: We proposed a novel multistep work for the development and validation of patient-derived models, considering the immature and differentiated content and the tumor microenvironment of pHGGs.

SynAggreg: A Multifunctional High-Throughput Technology for Precision Study of Amyloid Aggregation and Systematic Discovery of Synergistic Inhibitor Compounds.

Numerous proteins can coalesce into amyloid self-assemblies, which are responsible for a class of diseases called amyloidoses, but which can also fulfill important biological functions and are of great interest for biotechnology. Amyloid aggregation is a complex multi-step process, poorly prone to detailed structural studies. Therefore, small molecules interacting with amyloids are often used as tools to probe the amyloid aggregation pathway and in some cases to treat amyloidoses as they prevent pathogenic protein aggregation. Here, we report on SynAggreg, an in vitro high-throughput (HT) platform dedicated to the precision study of amyloid aggregation and the effect of modulator compounds. SynAggreg relies on an accurate bi-fluorescent amyloid-tracer readout that overcomes some limitations of existing HT methods. It allows addressing diverse aspects of aggregation modulation that are critical for pathomechanistic studies, such as the specificity of compounds toward various amyloids and their effects on aggregation kinetics, as well as the co-assembly propensity of distinct amyloids and the influence of prion-like seeding on self-assembly. Furthermore, SynAggreg is the first HT technology that integrates tailored methodology to systematically identify synergistic compound combinations-an emerging strategy to improve fatal amyloidoses by targeting multiple steps of the aggregation pathway. To this end, we apply analytical combinatorial scores to rank the inhibition efficiency of couples of compounds and to readily detect synergism. Finally, the SynAggreg platform should be suited for the characterization of a broad class of amyloids, whether of interest for drug development purposes, for fundamental research on amyloid functions, or for biotechnological applications.

LIT-001, the First Nonpeptide Oxytocin Receptor Agonist that Improves Social Interaction in a Mouse Model of Autism.

Oxytocin (OT) and its receptor (OT-R) are implicated in the etiology of autism spectrum disorders (ASD), and OT-R is a potential target for therapeutic intervention. Very few nonpeptide oxytocin agonists have currently been reported. Their molecular and in vivo pharmacology remain to be clarified, and none of them has been shown to be efficient in improving social interaction in animal models relevant to ASD. In an attempt to rationalize the design of centrally active nonpeptide full agonists, we studied in a systematic way the structural determinants of the affinity and efficacy of representative ligands of the V1a and V2 vasopressin receptor subtypes (V1a-R and V2-R) and of the oxytocin receptor. Our results confirm the subtlety of the structure-affinity and structure-efficacy relationships around vasopressin/oxytocin receptor ligands and lead however to the first nonpeptide OT receptor agonist active in a mouse model of ASD after peripheral ip administration.

Discovery of a Locally and Orally Active CXCL12 Neutraligand (LIT-927) with Anti-inflammatory Effect in a Murine Model of Allergic Airway Hypereosinophilia.

We previously reported Chalcone-4 (1) that binds the chemokine CXCL12, not its cognate receptors CXCR4 or CXCR7, and neutralizes its biological activity. However, this neutraligand suffers from limitations such as poor chemical stability, solubility, and oral activity. Herein, we report on the discovery of pyrimidinone 57 (LIT-927), a novel neutraligand of CXCL12 which displays a higher solubility than 1 and is no longer a Michael acceptor. While both 1 and 57 reduce eosinophil recruitment in a murine model of allergic airway hypereosinophilia, 57 is the only one to display inhibitory activity following oral administration. Thereby, we here describe 57 as the first orally active CXCL12 neutraligand with anti-inflammatory properties. Combined with a high binding selectivity for CXCL12 over other chemokines, 57 represents a powerful pharmacological tool to investigate CXCL12 physiology in vivo and to explore the activity of chemokine neutralization in inflammatory and related diseases.

WNK1 kinase and its partners Akt, SGK1 and NBC-family Na+/HCO3- cotransporters are potential therapeutic targets for glioblastoma stem-like cells linked to Bisacodyl signaling.

Glioblastoma is a highly heterogeneous brain tumor. The presence of cancer cells with stem-like and tumor initiation/propagation properties contributes to poor prognosis. Glioblastoma cancer stem-like cells (GSC) reside in hypoxic and acidic niches favoring cell quiescence and drug resistance. A high throughput screening recently identified the laxative Bisacodyl as a cytotoxic compound targeting quiescent GSC placed in acidic microenvironments. Bisacodyl activity requires its hydrolysis into DDPM, its pharmacologically active derivative. Bisacodyl was further shown to induce tumor shrinking and increase survival in in vivo glioblastoma models. Here we explored the cellular mechanism underlying Bisacodyl cytotoxic effects using quiescent GSC in an acidic microenvironment and GSC-derived 3D macro-spheres. These spheres mimic many aspects of glioblastoma tumors in vivo, including hypoxic/acidic areas containing quiescent cells. Phosphokinase protein arrays combined with pharmacological and genetic modulation of signaling pathways point to the WNK1 serine/threonine protein kinase as a mediator of Bisacodyl cytotoxic effect in both cell models. WNK1 partners including the Akt and SGK1 protein kinases and NBC-family Na+/HCO3- cotransporters were shown to participate in the compound's effect on GSC. Overall, our findings uncover novel potential therapeutic targets for combatting glioblastoma which is presently an incurable disease.

Phenylpyridine-2-ylguanidines and rigid mimetics as novel inhibitors of TNFα overproduction: Beneficial action in models of neuropathic pain and of acute lung inflammation.

4-phenylpyridin-2-yl-guanidine (5b): a new inhibitor of the overproduction of pro-inflammatory cytokines (TNFα and Il1ß) was identified from a high-throughput screening of a chemical library on human peripheral blood mononuclear cells (PBMCs) after LPS stimulation. Derivatives, homologues and rigid mimetics of 5b were designed and synthesized, and their cytotoxicity and ability to inhibit TNFα overproduction were evaluated. Among them, compound 5b and its mimetic 12 (2-aminodihydroquinazoline) showed similar inhibitory activities, and were evaluated in vivo in models of lung inflammation and neuropathic pain in mice. In particular, compound 12 proved to be active (5 mg/kg, ip) in both models.

A Time-Resolved FRET Cell-Based Binding Assay for the Apelin Receptor.

Analogues of apelin-13 carrying diverse spacers and an ad hoc DY647-derived fluorophore were designed and synthesized by chemoselective acylation of α-hydrazinopeptides. The resulting probes retain very high affinity and efficacy for both the wild-type and SNAP-tagged apelin receptor (ApelinR). They give a time-resolved FRET (TR-FRET) signal with rare-earth lanthanides used as donor fluorophores grafted onto the SNAP-tagged receptor. This specific signal allowed the validation of a binding assay with a high signal-to-noise ratio. In such an assay, the most potent sub-nanomolar fluorescent probe was found to be competitively displaced by the endogenous apelin peptides with binding constants similar to those obtained in a classical radioligand assay. We have thus validated the first TR-FRET cell-based binding assay for ApelinR with potential high-throughput screening applications.