Perspectives on obesity imaging: [18F]2FNQ1P a specific 5-HT6 brain PET radiotracer.

BACKGROUND: Estimates suggest that approximatively 25% of the world population will be overweight in 2025. Better understanding of the pathophysiology of obesity will help to develop future therapeutics. Serotonin subtype 6 receptors (5-HT6) have been shown to be critically involved in appetite reduction and weight loss. However, it is not known if the pathological cascade triggered by obesity modifies the density of 5-HT6 receptors in the brain. METHODS: Influence of diet-induced obesity (DIO) in Wistar rats was explored using MRI (whole-body fat) and PET ([18F]2FNQ1P as a specific 5-HT6 radiotracer). The primary goal was to monitor the 5-HT6 receptor density before and after a 10-week diet (DIO group). The secondary goal was to compare 5-HT6 receptor densities between DIO group, Wistar control diet group, Zucker rats (with genetic obesity) and Zucker lean strain rats. RESULTS: Wistar rats fed with high-fat diet showed higher body fat gain than Wistar control diet rats on MRI. [18F]2FNQ1P PET analysis highlighted significant clusters of voxels (located in hippocampus, striatum, cingulate, temporal cortex and brainstem) with increased binding after high-fat diet (p < 0.05, FWE corrected). CONCLUSION: This study sheds a new light on the influence of high-fat diet on 5-HT6 receptors. This study also positions [18F]2FNQ1P PET as an innovative tool to explore neuronal consequences of obesity or eating disorder pathophysiology.

[18F]RS-127445 radiosynthesis and evaluation as a 5-HT2B receptor PET radiotracer in rat brain.

Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter involved in many physiological and pathological mechanisms through its numerous receptors. Among these, the 5-HT2B receptor is known to play a key role in multiple brain disorders but remains poorly understood. Positron emission tomography (PET) can contribute to a better understanding of pathophysiological mechanisms regulated by the 5-HT2B receptor. To develop the first PET radiotracer for the 5-HT2B receptor, RS-127445, a well-known 5-HT2B receptor antagonist, was labeled with fluorine-18. [18F]RS-127445 was synthesized in a high radiochemical purity and with a good molar activity and radiochemical yield. Preliminary PET scans in rats showed good brain penetration of [18F]RS-127445. However, competition experiments and in vitro autoradiography showed high non-specific binding, especially to brain white matter.

Synthesis and biological evaluation of Haspin inhibitors: Kinase inhibitory potency and cellular activity.

Haspin (haploid germ cell-specific nuclear protein kinase) offers a potential target for the development of new anticancer drugs. Thus, the identification of new inhibitors targeting this protein kinase is of high interest. However, Haspin inhibitors developed to date show a poor selectivity profile over other protein kinases of the human kinome. Here, we identified a new pyridoquinazoline based inhibitor (4), with excellent inhibitory activity and selectivity for Haspin (IC50 of 50 nM). We describe the structure-activity relationship study including the evaluation of this inhibitor on a large panel of 486 kinases as well as on immortalized or cancer cell lines. In addition, we determined the binding mode of analog 2a in complex with Haspin using X-ray crystallography.

Targeting different binding sites in the CFTR structures allows to synergistically potentiate channel activity.

Recent evidence shows that combination of correctors and potentiators, such as the drug ivacaftor (VX-770), can significantly restore the functional expression of mutated Cystic Fibrosis Transmembrane conductance Regulator (CFTR), an anion channel which is mutated in cystic fibrosis (CF). The success of these combinatorial therapies highlights the necessity of identifying a broad panel of specific binding mode modulators, occupying several distinct binding sites at structural level. Here, we identified two small molecules, SBC040 and SBC219, which are two efficient cAMP-independent potentiators, acting at low concentration of forskolin with EC50 close to 1 µM and in a synergic way with the drug VX-770 on several CFTR mutants of classes II and III. Molecular dynamics simulations suggested potential SBC binding sites at the vicinity of ATP-binding sites, distinct from those currently proposed for VX-770, outlining SBC molecules as members of a new family of potentiators.

Kinase inhibitions in pyrido[4,3-h] and [3,4-g]quinazolines: Synthesis, SAR and molecular modeling studies.

New pyrido[3,4-g]quinazoline derivatives were prepared and evaluated for their inhibitory potency toward 5 protein kinases (CLK1, DYRK1A, GSK3, CDK5, CK1). A related pyrido[4,3-h]quinazoline scaffold with an angular structure was also synthesized and its potency against the same protein kinase panel was compared to the analogous pyrido[3,4-g]quinazoline. Best results were obtained for 10-nitropyrido[3,4-g]quinazoline 4 toward CLK1 with nanomolar activities.

New pyrido[3,4-g]quinazoline derivatives as CLK1 and DYRK1A inhibitors: synthesis, biological evaluation and binding mode analysis.

Cdc2-like kinase 1 (CLK1) and dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) are involved in the regulation of alternative pre-mRNA splicing. Dysregulation of this process has been linked to cancer progression and neurodegenerative diseases, making CLK1 and DYRK1A important therapeutic targets. Here we describe the synthesis of new pyrido[3,4-g]quinazoline derivatives and the evaluation of the inhibitory potencies of these compounds toward CDK5, CK1, GSK3, CLK1 and DYRK1A. Introduction of aminoalkylamino groups at the 2-position resulted in several compounds with low nanomolar affinity and selective inhibition of CLK1 and/or DYRK1A. Their evaluation on several immortalized or cancerous cell lines showed varying degree of cell viability reduction. Co-crystal structures of CLK1 with two of the most potent compounds revealed two alternative binding modes of the pyrido[3,4-g]quinazoline scaffold that can be exploited for future inhibitor design.

QSAR Model of Indeno[1,2-b]indole Derivatives and Identification of N-isopentyl-2-methyl-4,9-dioxo-4,9-Dihydronaphtho[2,3-b]furan-3-carboxamide as a Potent CK2 Inhibitor.

Casein kinase II (CK2) is an intensively studied enzyme, involved in different diseases, cancer in particular. Different scaffolds were used to develop inhibitors of this enzyme. Here, we report on the synthesis and biological evaluation of twenty phenolic, ketonic, and para-quinonic indeno[1,2-b]indole derivatives as CK2 inhibitors. The most active compounds were 5-isopropyl-1-methyl-5,6,7,8-tetrahydroindeno[1,2-b]indole-9,10-dione 4h and 1,3-dibromo-5-isopropyl-5,6,7,8-tetrahydroindeno[1,2-b]indole-9,10-dione 4w with identical IC50 values of 0.11 µM. Furthermore, the development of a QSAR model based on the structure of indeno[1,2-b]indoles was performed. This model was used to predict the activity of 25 compounds with naphtho[2,3-b]furan-4,9-dione derivatives, which were previously predicted as CK2 inhibitors via a molecular modeling approach. The activities of four naphtho[2,3-b]furan-4,9-dione derivatives were determined in vitro and one of them (N-isopentyl-2-methyl-4,9-dioxo-4,9-dihydronaphtho[2,3-b]furan-3-carboxamide) turned out to inhibit CK2 with an IC50 value of 2.33 µM. All four candidates were able to reduce the cell viability by more than 60% after 24 h of incubation using 10 µM.

Involvement of the Prion Protein in the Protection of the Human Bronchial Epithelial Barrier Against Oxidative Stress.

Aim: Bronchial epithelium acts as a defensive barrier against inhaled pollutants and microorganisms. This barrier is often compromised in inflammatory airway diseases that are characterized by excessive oxidative stress responses, leading to bronchial epithelial shedding, barrier failure, and increased bronchial epithelium permeability. Among proteins expressed in the junctional barrier and participating to the regulation of the response to oxidative and to environmental stresses is the cellular prion protein (PrPC). However, the role of PrPC is still unknown in the bronchial epithelium. Herein, we investigated the cellular mechanisms by which PrPC protein participates into the junctional complexes formation, regulation, and oxidative protection in human bronchial epithelium. Results: Both PrPC messenger RNA and mature protein were expressed in human epithelial bronchial cells. PrPC was localized in the apical domain and became lateral, at high degree of cell polarization, where it colocalized and interacted with adherens (E-cadherin/γ-catenin) and desmosomal (desmoglein/desmoplakin) junctional proteins. No interaction was detected with tight junction proteins. Disruption of such interactions induced the loss of the epithelial barrier. Moreover, we demonstrated that PrPC protection against copper-associated oxidative stress was involved in multiple processes, including the stability of adherens and desmosomal junctional proteins. Innovation: PrPC is a pivotal protein in the protection against oxidative stress that is associated with the degradation of adherens and desmosomal junctional proteins. Conclusion: Altogether, these results demonstrate that the loss of the integrity of the epithelial barrier by oxidative stress is attenuated by the activation of PrPC expression, where deregulation might be associated with respiratory diseases.

6-Chloropurine Ribonucleosides from Chloropyrimidines: One-Pot Synthesis.

A one-pot glycosylation and cyclization procedure is described for the synthesis of 6-chloropurine ribonucleosides from chloropyrimidines. From such a procedure and modification of the obtained chloropurine ribonucleosides, many drug candidates or molecular tools for biological study designed from their similarity to naturally occurring nucleosides could be obtained. The synthesis begins by preparation of several amidinoaminochloropyrimidines as precursors for the one-pot procedure. Then, by adding trimethylsilyl trifluoromethanesulfonate (TMSOTf) to a mixture of a pyrimidine and 1-O-acetyl-2,3,5-tri-O-benzoyl-ß-D-ribose, different 6-chloropurine ribonucleosides are obtained. This methodology allows the straightforward introduction of an alkyl substituent at position 8 of purine ribonucleosides, which then can be functionalized at positions 2 and 6. © 2018 by John Wiley & Sons, Inc.

In Silico, in Vitro, and in Vivo Evaluation of New Candidates for α-Synuclein PET Imaging.

Accumulation of α-synuclein (α-syn) is a neuropathological hallmark of synucleinopathies. To date, no selective α-syn positron emission tomography (PET) radiotracer has been identified. Our objective was to develop the first original, selective, and specific α-syn PET radiotracer. Chemical design inspired from three structural families that demonstrated interesting α-syn binding characteristics was used as a starting point. Bioinformatics modeling of α-syn fibrils was then employed to select the best molecular candidates before their syntheses. An in vitro binding assay was performed to evaluate the affinity of the compounds. Radiotracer specificity and selectivity were assessed by in vitro autoradiography and in vivo PET studies in animal (rodents) models. Finally, gold standard in vitro autoradiography with patients' postmortem tissues was performed to confirm/infirm the α-syn binding characteristics. Two compounds exhibited a good brain availability and bound to α-syn and Aß fibrils in a rat model. In contrast, no signal was observed in a mouse model of synucleinopathy. Experiments in human tissues confirmed these negative results.

Amyloid-Beta Radiotracer [18F]BF-227 Does Not Bind to Cytoplasmic Glial Inclusions of Postmortem Multiple System Atrophy Brain Tissue.

The accumulation of aggregated alpha-synuclein (α-syn) in multiple brain regions is a neuropathological hallmark of synucleinopathies. Multiple system atrophy (MSA) is a synucleinopathy characterized by the predominant cerebral accumulation of aggregated α-syn as cytoplasmic glial inclusions (CGI). A premortem diagnosis tool would improve early diagnosis and help monitoring disease progression and therapeutic efficacy. One Positron Emission Tomography (PET) study suggested [11C]BF-227 as a promising radiotracer for monitoring intracellular α-syn deposition in MSA patients. We sought to confirm the binding of this radiotracer to α-syn using state-of-the-art autoradiography. Medulla sections were obtained from 9 MSA patients and 9 controls (London Neurodegenerative Diseases Brain Bank). [18F]BF-227, chemically identical to [11C]BF-227, was used at nanomolar concentrations to perform in vitro autoradiography assays. Autoradiograms were superimposed on fluorescent staining from the conformational anti-α-syn antibody 5G4 and quantified after immunofluorescence-driven definition of regions of interest. Autoradiography showed no specific signals in MSA patients in comparison to controls despite widespread pathology detected by immunofluorescence. Autoradiography does not support a significant binding of [18F]BF-227 to CGI at concentrations typically achieved in PET experiments.

A One-Pot Synthesis of Highly Functionalized Purines.

Highly substituted purines were synthesized in good to high yields through a one-pot straightforward metal-free scalable method, using the Traube synthesis adapted to Vilsmeier-type reagents. From 5-amino-4-chloropyrimidines, new 9-aryl-substituted chloropurines and intermediates for peptide nucleic acid synthesis were prepared. Variant procedures allowing a rapid synthesis of ribonucleosides and 7-benzylpurine from 5-amidino-6-aminopyrimidines are also reported to illustrate the high potential of this versatile toolbox. This route appears to be particularly interesting in the field of nucleic acids for a direct and rapid access to various new 8-alkylpurine nucleosides.

Synthesis and preliminary in vitro kinase inhibition evaluation of new diversely substituted pyrido[3,4-g]quinazoline derivatives.

The synthesis of new diversely substituted pyrido[3,4-g]quinazolines is described. The inhibitory potencies of prepared compounds toward a panel of five CMGC protein kinases (CDK5, CLK1, DYRK1A, CK1, GSK3), that are known to play a potential role in Alzheimer's disease, were evaluated. The best overall kinase inhibition profile was found for nitro compound 4 bearing an ethyl group at the 5-position.

Discovery of pyrido[3,4-g]quinazoline derivatives as CMGC family protein kinase inhibitors: Design, synthesis, inhibitory potency and X-ray co-crystal structure.

The design and synthesis of new pyrido[3,4-g]quinazoline derivatives is described as well as their protein kinase inhibitory potencies toward five CMGC family members (CDK5, CK1, GSK3, CLK1 and DYRK1A). The interest for this original tricyclic heteroaromatic scaffold as modulators of CLK1/DYRK1A activity was validated by nanomolar potencies (compounds 12 and 13). CLK1 co-crystal structures with two inhibitors revealed the binding mode of these compounds within the ATP-binding pocket.

Converting potent indeno[1,2-b]indole inhibitors of protein kinase CK2 into selective inhibitors of the breast cancer resistance protein ABCG2.

A series of indeno[1,2-b]indole-9,10-dione derivatives were synthesized as human casein kinase II (CK2) inhibitors. The most potent inhibitors contained a N(5)-isopropyl substituent on the C-ring. The same series of compounds was found to also inhibit the breast cancer resistance protein ABCG2 but with totally different structure-activity relationships: a N(5)-phenethyl substituent was critical, and additional hydrophobic substituents at position 7 or 8 of the D-ring or a methoxy at phenethyl position ortho or meta also contributed to inhibition. The best ABCG2 inhibitors, such as 4c, 4h, 4i, 4j, and 4k, behaved as very weak inhibitors of CK2, whereas the most potent CK2 inhibitors, such as 4a, 4p, and 4e, displayed limited interaction with ABCG2. It was therefore possible to convert, through suitable substitutions of the indeno[1,2-b]indole-9,10-dione scaffold, potent CK2 inhibitors into selective ABCG2 inhibitors and vice versa. In addition, some of the best ABCG2 inhibitors, which displayed a very low cytotoxicity, thus giving a high therapeutic ratio, and appeared not to be transported, constitute promising candidates for further investigations.

Inverse relationship between hSHBG affinity for testosterone and hSHBG concentration revealed by surface plasmon resonance.

A wide range of human sex hormone-binding globulin (hSHBG) affinity constants for testosterone (KA_hSHBG) has been reported in literature. To bring new insight on the KA_hSHBG value, we implemented a study of the molecular interactions occurring between testosterone and its plasma transport proteins by using surface plasmon resonance. The immobilization on the sensorchip of a testosterone derivative was performed by an oligoethylene glycol linker. For different plasmas with hSHBG concentrations, an assessment of the KA_hSHBG was obtained from a set of sensorgrams and curve-fitting these data. We observed that KA_hSHBG decreased, from at least two decades, when the plasma hSHBG concentration increased from 4.4 to 680 nmol/L. Our study shows a wide biological variability of KA_hSHBG that is related to the hSHBG concentration. These unexpected results may have a physiological significance and question the validity of current methods that are recommended for calculating free testosterone concentrations to evaluate androgen disorders in humans.

Preparation and characterization of CK2 inhibitor-loaded cyclodextrin nanoparticles for drug delivery.

Casein Kinase 2 (CK2) is a ubiquitous kinase protein currently targeted for the treatment of some cancers. Recently, the series of indeno[1,2-b]indoles has revealed great interest as potent and selective CK(2) ATP-competitive inhibitors. Among them, 1-amino-5-isopropyl-5,6,7,8-tetrahydroindeno[1,2-b]indole-9,10-dione (CM1) was selected for an encapsulation study in order to improve its biodisponibility. Its complexation was evaluated at the molecular scale, with a series of fluorinated or hydrocarbonated amphiphilic cyclodextrins (CDs). Then the encapsulation of CM1 within CD nanoparticles at the supramolecular level was achieved. Nanoparticles formed between CM1 and hexakis[6-deoxy-6-(3-perfluorohexylpropanethio)-2,3-di-O-methyl]-α-cyclodextrin, a fluorinated amphiphilic α-cyclodextrin, gave the best results in terms of encapsulation rate, stability and drug release. These nanospheres showed an encapsulation efficiency of 65% and a sustained release of the entrapped drug over 3h. Based on these results, encapsulation within fluorinated amphiphilic CD nanoparticles could be considered as a potential drug delivery system for indenoindole-type CK2 inhibitors, allowing better biodisponibility and offering perspectives for tumor targeting development.

Progesterone-adenine hybrids as bivalent inhibitors of P-glycoprotein-mediated multidrug efflux: design, synthesis, characterization and biological evaluation.

Bivalent ligands were designed on the basis of the described close proximity of the ATP-site and the putative steroid-binding site of P-glycoprotein (ABCB1). The syntheses of 19 progesterone-adenine hybrids are described. Their abilities to inhibit P-glycoprotein-mediated daunorubicin efflux in K562/R7 human leukemic cells overexpressing P-glycoprotein were evaluated versus progesterone. The hybrid with a hexamethylene linker chain showed the best inhibitory potency. The efficiency of these progesterone-adenine hybrids depends on two main factors: (i) the nature of the linker and (ii) its attachment point on the steroid skeleton.

Design, synthesis and evaluation of progesterone-adenine hybrids as bivalent inhibitors of P-glycoprotein-mediated multidrug efflux.

Steroidal bivalent ligands were designed on the basis of the described closer proximity of the ATP-site and the putative steroid-binding site of P-glycoprotein (ABCB1). The syntheses of seven progesterone-adenine hybrids were described. Their abilities to inhibit P-glycoprotein-mediated daunorubicin efflux in K562/R7 human leukemic cells overexpressing P-glycoprotein were evaluated versus progesterone.

Synthesis and modulation properties of imidazo[4,5-b]pyridin-7-one and indazole-4,7-dione derivatives towards the Cryptosporidium parvum CpABC3 transporter.

The syntheses of new N-polysubstituted imidazo[4,5-b]pyridine-7-one (IP, 5 and 8a-8f) and indazole-4,7-dione (ID, 9 and 10) derivatives are described. The binding affinity of IP and ID towards the recombinant Nucleotide Binding Domain NBD1 of Cryptosporidium parvum CpABC3 was evaluated by intrinsic fluorescence quenching. IP induced a moderate quenching of the intrinsic fluorescence of H6-NBD1 whereas IDs 9 and 10 showed a binding affinity comparable to the ATP analogue TNP-ATP. In addition, 8d, 8e and 10 were shown to be competitive inhibitors of the ATPase activity, but with low affinity. These compounds could thus act like some flavonoid derivatives, which can partly overlap both the nucleotide-binding site and the adjacent hydrophobic steroid-binding region of mammalian P-glycoproteins.