Ethaverine and Papaverine Target Cyclin-Dependent Kinase 5 and Inhibit Lung Cancer Cell Proliferation and Migration.

CDK5 kinase plays a central role in the regulation of neuronal functions, and its hyperactivation has been associated with neurodegenerative pathologies and more recently with several human cancers, in particular lung cancer. However, ATP-competitive inhibitors targeting CDK5 are poorly selective and suffer limitations, calling for new classes of inhibitors. In a screen for allosteric modulators of CDK5, we identified ethaverine and closely related derivative papaverine and showed that they inhibit cell proliferation and migration of non small cell lung cancer cell lines. Moreover the efficacy of these compounds is significantly enhanced when combined with the ATP-competitive inhibitor roscovitine, suggesting an additive dual mechanism of inhibition targeting CDK5. These compounds do not affect CDK5 stability, but thermodenaturation studies performed with A549 cell extracts infer that they interact with CDK5 in cellulo. Furthermore, the inhibitory potentials of ethaverine and papaverine are reduced in A549 cells treated with siRNA directed against CDK5. Taken together, our results provide unexpected and novel evidence that ethaverine and papaverine constitute promising leads that can be repurposed for targeting CDK5 in lung cancer.

Multitarget µ-Opioid Receptor Agonists─Neuropeptide FF Receptor Antagonists Induce Potent Antinociception with Reduced Adverse Side Effects.

The design of bifunctional compounds is a promising approach toward the development of strong analgesics with reduced side effects. We here report the optimization of the previously published lead peptide KGFF09, which contains opioid receptor agonist and neuropeptide FF receptor antagonist pharmacophores and is shown to induce potent antinociception and reduced side effects. We evaluated the novel hybrid peptides for their in vitro activity at MOP, NPFFR1, and NPFFR2 and selected four of them (DP08/14/32/50) for assessment of their acute antinociceptive activity in mice. We further selected DP32 and DP50 and observed that their antinociceptive activity is mostly peripherally mediated; they produced no respiratory depression, no hyperalgesia, significantly less tolerance, and strongly attenuated withdrawal syndrome, as compared to morphine and the recently FDA-approved TRV130. Overall, these data suggest that MOP agonist/NPFF receptor antagonist hybrids might represent an interesting strategy to develop novel analgesics with reduced side effects.

Identification of 8-Hydroxyquinoline Derivatives That Decrease Cystathionine Beta Synthase (CBS) Activity.

CBS encodes a pyridoxal 5'-phosphate-dependent enzyme that catalyses the condensation of homocysteine and serine to form cystathionine. Due to its implication in some cancers and in the cognitive pathophysiology of Down syndrome, the identification of pharmacological inhibitors of this enzyme is urgently required. However, thus far, attempts to identify such molecules have only led to the identification of compounds with low potency and limited selectivity. We consequently developed an original, yeast-based screening method that identified three FDA-approved drugs of the 8-hydroxyquinoline family: clioquinol, chloroxine and nitroxoline. These molecules reduce CBS enzymatic activity in different cellular models, proving that the molecular mechanisms involved in yeast phenotypic rescue are conserved in mammalian cells. A combination of genetic and chemical biology approaches also revealed the importance of copper and zinc intracellular levels in the regulation of CBS enzymatic activity-copper promoting CBS activity and zinc inhibiting its activity. Taken together, these results indicate that our effective screening approach identified three new potent CBS inhibitors and provides new findings for the regulation of CBS activity, which is crucial to develop new therapies for CBS-related human disorders.

New insights into the regulation of Cystathionine beta synthase (CBS), an enzyme involved in intellectual deficiency in Down syndrome.

Down syndrome (DS), the most frequent chromosomic aberration, results from the presence of an extra copy of chromosome 21. The identification of genes which overexpression contributes to intellectual disability (ID) in DS is important to understand the pathophysiological mechanisms involved and develop new pharmacological therapies. In particular, gene dosage of Dual specificity tyrosine phosphorylation Regulated Kinase 1A (DYRK1A) and of Cystathionine beta synthase (CBS) are crucial for cognitive function. As these two enzymes have lately been the main targets for therapeutic research on ID, we sought to decipher the genetic relationship between them. We also used a combination of genetic and drug screenings using a cellular model overexpressing CYS4, the homolog of CBS in Saccharomyces cerevisiae, to get further insights into the molecular mechanisms involved in the regulation of CBS activity. We showed that overexpression of YAK1, the homolog of DYRK1A in yeast, increased CYS4 activity whereas GSK3ß was identified as a genetic suppressor of CBS. In addition, analysis of the signaling pathways targeted by the drugs identified through the yeast-based pharmacological screening, and confirmed using human HepG2 cells, emphasized the importance of Akt/GSK3ß and NF-κB pathways into the regulation of CBS activity and expression. Taken together, these data provide further understanding into the regulation of CBS and in particular into the genetic relationship between DYRK1A and CBS through the Akt/GSK3ß and NF-κB pathways, which should help develop more effective therapies to reduce cognitive deficits in people with DS.

Comprehensive overview of biased pharmacology at the opioid receptors: biased ligands and bias factors.

One of the main challenges in contemporary medicinal chemistry is the development of safer analgesics, used in the treatment of pain. Currently, moderate to severe pain is still treated with the "gold standard" opioids whose long-term often leads to severe side effects. With the discovery of biased agonism, the importance of this area of pharmacology has grown exponentially over the past decade. Of these side effects, tolerance, opioid misuse, physical dependence and substance use disorder (SUD) stand out, since these have led to many deaths over the past decades in both USA and Europe. New therapeutic molecules that induce a biased response at the opioid receptors (MOR, DOR, KOR and NOP receptor) are able to circumvent these side effects and, consequently, serve as more advantageous therapies with great promise. The concept of biased signaling extends far beyond the already sizeable field of GPCR pharmacology and covering everything would be vastly outside the scope of this review which consequently covers the biased ligands acting at the opioid family of receptors. The limitation of quantifying bias, however, makes this a controversial subject, where it is dependent on the reference ligand, the equation or the assay used for the quantification. Hence, the major issue in the field of biased ligands remains the translation of the in vitro profiles of biased signaling, with corresponding bias factors to in vivo profiles showing the presence or the lack of specific side effects. This review comprises a comprehensive overview of biased ligands in addition to their bias factors at individual members of the opioid family of receptors, as well as bifunctional ligands.

Identification of an N-acylated-DArg-Leu-NH2 Dipeptide as a Highly Selective Neuropeptide FF1 Receptor Antagonist That Potently Prevents Opioid-Induced Hyperalgesia.

RFamide-related peptide-3 (RFRP-3) and neuropeptide FF (NPFF) target two different receptor subtypes called neuropeptide FF1 (NPFF1R) and neuropeptide FF2 (NPFF2R) that modulate several functions. However, the study of their respective role is severely limited by the absence of selective blockers. We describe here the design of a highly selective NPFF1R antagonist called RF3286, which potently blocks RFRP-3-induced hyperalgesia in mice and luteinizing hormone release in hamsters. We then showed that the pharmacological blockade of NPFF1R in mice prevents the development of fentanyl-induced hyperalgesia while preserving its analgesic effect. Altogether, our data indicate that RF3286 represents a useful pharmacological tool to study the involvement of the NPFF1R/RFRP-3 system in different functions and different species. Thanks to this compound, we showed that this system is critically involved in the development of opioid-induced hyperalgesia, suggesting that NPFF1R antagonists might represent promising therapeutic tools to improve the use of opioids in the treatment of chronic pain.

Anti-prion Drugs Targeting the Protein Folding Activity of the Ribosome Reduce PABPN1 Aggregation.

Prion diseases are caused by the propagation of PrPSc, the pathological conformation of the PrPC prion protein. The molecular mechanisms underlying PrPSc propagation are still unsolved and no therapeutic solution is currently available. We thus sought to identify new anti-prion molecules and found that flunarizine inhibited PrPSc propagation in cell culture and significantly prolonged survival of prion-infected mice. Using an in silico therapeutic repositioning approach based on similarities with flunarizine chemical structure, we tested azelastine, duloxetine, ebastine, loperamide and metixene and showed that they all have an anti-prion activity. Like flunarizine, these marketed drugs reduced PrPSc propagation in cell culture and in mouse cerebellum organotypic slice culture, and inhibited the protein folding activity of the ribosome (PFAR). Strikingly, some of these drugs were also able to alleviate phenotypes due to PABPN1 nuclear aggregation in cell and Drosophila models of oculopharyngeal muscular dystrophy (OPMD). These data emphasize the therapeutic potential of anti-PFAR drugs for neurodegenerative and neuromuscular proteinopathies.

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.

Diastereoselective Synthesis of Nonplanar 3-Amino-1,2,4-oxadiazine Scaffold: Structure Revision of Alchornedine.

Herein, we report the diastereoselective synthesis of a 3-amino-1,2,4-oxadiazine (AOXD) scaffold. The presence of a N-O bond in the ring prevents the planar geometry of the aromatic system and induces a strong decrease in the basicity of the guanidine moiety. While DIBAL-H appeared to be the most efficient reducing agent because it exhibited high diastereoselectivity, we observed various behaviors of the Mitsunobu reaction on the resulting ß-aminoalcohol, leading to either inversion or retention of the configuration depending on the steric hindrance in the vicinity of the hydroxy group. The physicochemical properties (pKa and log D) and hepatic stability of several AOXD derivatives were experimentally determined and found that the AOXD scaffold possesses promising properties for drug development. Moreover, we synthesized alchornedine, the only natural product with the AOXD scaffold. Based on a comparison of the analytical data, we found that the reported structure of alchornedine was incorrect and hypothesized a new one.

From Electronic Waste to Suzuki-Miyaura Cross-Coupling Reaction in Water: Direct Valuation of Recycled Palladium in Catalysis.

Invited for this month's cover is the group of Damien Bourgeois at the Marcoule Institute for Separation Chemistry (ICSM). The image shows how a short process chain can efficiently transform our waste into an active catalyst. The Full Paper itself is available at 10.1002/cssc.202001155.

From Electronic Waste to Suzuki-Miyaura Cross-Coupling Reaction in Water: Direct Valuation of Recycled Palladium in Catalysis.

From electronic waste to Pd-catalyzed reaction! The straightforward valuation of palladium recovered from electronic waste is reported here. Following a classical leaching stage, palladium is selectively extracted from a complex aqueous mixture of metallic cations into an organic phase. Afterwards, the judicious choice of a surfactant enables stabilization of palladium during back extraction cycles, and the direct preparation of an aqueous micellar solution, which can be employed in a model Suzuki-Miyaura cross-coupling reaction. Clean phase separation is observed, and distribution of all components between organic and aqueous phases is mastered. The proposed process avoids several waste generating steps dedicated to palladium isolation and ultimate purification, as well as the preparation of palladium pre-catalyst. This novel approach enables a better use of both natural resources and industrial wastes, through new cycles in circular economy.

Mitochondria modulatory effects of new TSPO ligands in a cellular model of tauopathies.

Translocator protein 18 kDa (TSPO) is a mitochondrial protein located in the outer membrane and involved in cholesterol translocation, a prerequisite for steroid biosynthesis. TSPO modulation also appears to play a role in other mitochondrial functions, including mitochondrial respiration and cell survival. In the central nervous system, its expression is up-regulated in neuropathology such as Alzheimer's disease (AD). Previously, we demonstrated that two new TSPO ligands, named 2a and 2b, stimulated pregnenolone synthesis and ATP production in a cellular model of AD overproducing ß-amyloid peptide. The present study aimed to evaluate the impact of the new TSPO ligands on mitochondrial dysfunction in a cellular model of AD-related tauopathy (human neuroblastoma cells SH-SY5Y stably overexpressing the P301L-mutant Tau) presenting mitochondrial impairments, including a decreased ATP synthesis and mitochondrial membrane potential, as well as a decrease in pregnenolone synthesis compared to control cells. The effects of our new ligands were compared with those of TSPO ligands described in the literature (XBD173, SSR-180,575 and Ro5-4864). The TSPO ligands 2a and 2b exerted beneficial mitochondrial modulatory effects by increasing ATP levels and mitochondrial membrane potential, paralleled by an increase of pregnenolone levels in mutant Tau cells, as well as in control cells. The compounds 2a and 2b showed effects on mitochondrial activity similar to those obtained with the TSPO ligands of reference. These findings indicate that the new TSPO ligands modulate the mitochondrial bioenergetic phenotype as well as the de novo synthesis of neurosteroids in a cellular model of AD-related tauopathy, suggesting that these compounds could be potential new therapeutic tools for the treatment of AD.

TSPO Ligands Boost Mitochondrial Function and Pregnenolone Synthesis.

Translocator protein 18 kDa (TSPO) is located in the mitochondrial outer membrane and plays an important role in steroidogenesis and cell survival. In the central nervous system (CNS), its expression is upregulated in neuropathologies such as Alzheimer's disease (AD). Previously, we demonstrated that two new TSPO ligands based on an imidazoquinazolinone termed 2a and 2b, stimulated pregnenolone synthesis and ATP production in vitro. In the present study, we compared their effects to those of TSPO ligands described in the literature (XBD173, SSR-180,575, and Ro5-4864) by profiling the mitochondrial bioenergetic phenotype before and after treatment and investigating the protective effects of these ligands after oxidative injury in a cellular model of AD overexpressing amyloid-ß (Aß). Of note, ATP levels increased with rising pregnenolone levels suggesting that the energetic performance of mitochondria is linked to an increased production of this neurosteroid via TSPO modulation. Our results further demonstrate that the TSPO ligands 2a and 2b exerted neuroprotective effects by improving mitochondrial respiration, reducing reactive oxygen species and thereby decreasing oxidative stress-induced cell death as well as lowering Aß levels. The compounds 2a and 2b show similar or even better functional effects than those obtained with the reference TSPO ligands XBD173 and SSR-180.575. These findings indicate that the new TSPO ligands modulate mitochondrial bioenergetic phenotype and protect against oxidative injury probably through the de novo synthesis of neurosteroids, suggesting that these compounds could be potential new therapeutic tools for the treatment of neurodegenerative disease.

Design and synthesis of 3-aminophthalazine derivatives and structural analogues as PDE5 inhibitors: anti-allodynic effect against neuropathic pain in a mouse model.

Neuropathic pain is a chronic pain caused by a lesion or disease affecting the somatosensory nervous system. To date, no specific treatment has been developed to cure this pain. Antidepressants and anticonvulsant drugs are used, but they do not demonstrate universal efficacy, and they often cause detrimental adverse effects. Some studies highlighted the efficacy of sildenafil, a well-known inhibitor of phosphodiesterase 5 (PDE5, (IC50 = 3.3 nM)), in models of pain. Based on these results, we focused our attention on MY 5445, another known PDE5 inhibitor. Homologues, isosteres and structural analogues of MY 5445 were designed and all synthesized compounds were evaluated for their inhibitory activity toward PDE5. Selectivity profiles towards other PDE1-4 isoenzymes, water solubility and stability in acidic medium of the most potent PDE5 inhibitors were determined and the aminophthalazine 16h and its mimetic 41n (3-aminoindazole) were evaluated in comparison to MY 5445 (4b) in vivo in a model of neuropathic pain induced by sciatic nerve cuffing in mice (3 and 0.5 mg/kg, ip twice a day). Both compounds showed the same efficacy on neuropathic allodynia as MY 5445, and thus produced a significant relief of mechanical hypersensitivity after 12 days of treatment.

Evolution of coumaroyl conjugate 3-hydroxylases in land plants: lignin biosynthesis and defense.

Multiple adaptations were necessary when plants conquered the land. Among them were soluble phenylpropanoids related to plant protection and lignin necessary for upright growth and long-distance water transport. Cytochrome P450 monooxygenase 98 (CYP98) catalyzes a rate-limiting step in phenylpropanoid biosynthesis. Phylogenetic reconstructions suggest that a single copy of CYP98 founded each major land plant lineage (bryophytes, lycophytes, monilophytes, gymnosperms and angiosperms), and was maintained as a single copy in all lineages but the angiosperms. In angiosperms, a series of independent gene duplications and losses occurred. Biochemical assays in four angiosperm species tested showed that 4-coumaroyl-shikimate, a known intermediate in lignin biosynthesis, was the preferred substrate of one member in each species, while independent duplicates in Populus trichocarpa and Amborella trichopoda each showed broad substrate ranges, accepting numerous 4-coumaroyl-esters and -amines, and were thus capable of producing a wide range of hydroxycinnamoyl conjugates. The gymnosperm CYP98 from Pinus taeda showed a broad substrate range, but preferred 4-coumaroyl-shikimate as its best substrate. In contrast, CYP98s from the lycophyte Selaginella moellendorffii and the fern Pteris vittata converted 4-coumaroyl-shikimate poorly in vitro, but were able to use alternative substrates, in particular 4-coumaroyl-anthranilate. Thus, caffeoyl-shikimate appears unlikely to be an intermediate in monolignol biosynthesis in non-seed vascular plants, including ferns. The best substrate for CYP98A34 from the moss Physcomitrella patens was also 4-coumaroyl-anthranilate, while 4-coumaroyl-shikimate was converted to lower extents. Despite having in vitro activity with 4-coumaroyl-shikimate, CYP98A34 was unable to complement the Arabidopsis thaliana cyp98a3 loss-of-function phenotype, suggesting distinct properties also in vivo.

Fukuyama Cross-Coupling Approach to Isoprekinamycin: Discovery of the Highly Active and Bench-Stable Palladium Precatalyst POxAP.

An efficient and user-friendly palladium(II) precatalyst, POxAP (post-oxidative-addition precatalyst), was identified for use in Fukuyama cross-coupling reactions. Suitable for storage under air, the POxAP precatalyst allowed reaction between thioesters and organozinc reagents with turnover numbers of ∼90000. A series of 23 ketones were obtained with yields ranging from 53 to 99%. As proof of efficacy, an alternative approach was developed for the synthesis of a key precursor of the natural product isoprekinamycin.

Induction of ATP Release, PPIX Transport, and Cholesterol Uptake by Human Red Blood Cells Using a New Family of TSPO Ligands.

Two main isoforms of the Translocator Protein (TSPO) have been identified. TSPO1 is ubiquitous and is mainly present at the outer mitochondrial membrane of most eukaryotic cells, whereas, TSPO2 is specific to the erythroid lineage, located at the plasma membrane, the nucleus, and the endoplasmic reticulum. The design of specific tools is necessary to determine the molecular associations and functions of TSPO, which remain controversial nowadays. We recently demonstrated that TSPO2 is involved in a supramolecular complex of the erythrocyte membrane, where micromolar doses of the classical TSPO ligands induce ATP release and zinc protoporphyrin (ZnPPIX) transport. In this work, three newly-designed ligands (NCS1016, NCS1018, and NCS1026) were assessed for their ability to modulate the functions of various erythrocyte's and compare them to the TSPO classical ligands. The three new ligands were effective in reducing intraerythrocytic Plasmodium growth, without compromising erythrocyte survival. While NCS1016 and NCS1018 were the most effective ligands in delaying sorbitol-induced hemolysis, NCS1016 induced the highest uptake of ZnPPIX and NCS1026 was the only ligand inhibiting the cholesterol uptake. Differential effects of ligands are probably due, not only, to ligand features, but also to the dynamic interaction of TSPO with various partners at the cell membrane. Further studies are necessary to fully understand the mechanisms of the TSPO's complex activation.

Human erythrocytes release ATP by a novel pathway involving VDAC oligomerization independent of pannexin-1.

We previously demonstrated that the translocase protein TSPO2 together with the voltage-dependent anion channel (VDAC) and adenine nucleotide transporter (ANT) were involved in a membrane transport complex in human red blood cells (RBCs). Because VDAC was proposed as a channel mediating ATP release in RBCs, we used TSPO ligands together with VDAC and ANT inhibitors to test this hypothesis. ATP release was activated by TSPO ligands, and blocked by inhibitors of VDAC and ANT, while it was insensitive to pannexin-1 blockers. TSPO ligand increased extracellular ATP (ATPe) concentration by 24-59% over the basal values, displaying an acute increase in [ATPe] to a maximal value, which remained constant thereafter. ATPe kinetics were compatible with VDAC mediating a fast but transient ATP efflux. ATP release was strongly inhibited by PKC and PKA inhibitors as well as by depleting intracellular cAMP or extracellular Ca2+, suggesting a mechanism involving protein kinases. TSPO ligands favoured VDAC polymerization yielding significantly higher densities of oligomeric bands than in unstimulated cells. Polymerization was partially inhibited by decreasing Ca2+ and cAMP contents. The present results show that TSPO ligands induce polymerization of VDAC, coupled to activation of ATP release by a supramolecular complex involving VDAC, TSPO2 and ANT.

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.

Aminoguanidine Hydrazone Derivatives as Nonpeptide NPFF1 Receptor Antagonists Reverse Opioid Induced Hyperalgesia.

Neuropeptide FF receptors (NPFF1R and NPFF2R) and their endogenous ligand neuropeptide FF have been shown previously to display antiopioid properties and to play a critical role in the adverse effects associated with chronic administrations of opiates including the development of opioid-induced hyperalgesia and analgesic tolerance. In this work, we sought to identify novel NPFF receptors ligands by focusing our interest in a series of heterocycles as rigidified nonpeptide NPFF receptor ligands, starting from already described aminoguanidine hydrazones (AGHs). Binding experiments and functional assays highlighted AGH 1n and its rigidified analogue 2-amino-dihydropyrimidine 22e for in vivo experiments. As shown earlier with the prototypical dipeptide antagonist RF9, both 1n and 22e reduced significantly the long lasting fentanyl-induced hyperalgesia in rodents. Altogether these data indicate that AGH rigidification maintains nanomolar affinities for both NPFF receptors, while improving antagonist character toward NPFF1R.