Discovery of Bis-Imidazoline Derivatives as New CXCR4 Ligands.

The chemokine receptor CXCR4 and its ligand CXCL12 regulate leukocyte trafficking, homeostasis and functions and are potential therapeutic targets in many diseases such as HIV-1 infection and cancers. Here, we identified new CXCR4 ligands in the CERMN chemical library using a FRET-based high-throughput screening assay. These are bis-imidazoline compounds comprising two imidazole rings linked by an alkyl chain. The molecules displace CXCL12 binding with submicromolar potencies, similarly to AMD3100, the only marketed CXCR4 ligand. They also inhibit anti-CXCR4 mAb 12G5 binding, CXCL12-mediated chemotaxis and HIV-1 infection. Further studies with newly synthesized derivatives pointed out to a role of alkyl chain length on the bis-imidazoline properties, with molecules with an even number of carbons equal to 8, 10 or 12 being the most potent. Interestingly, these differ in the functions of CXCR4 that they influence. Site-directed mutagenesis and molecular docking predict that the alkyl chain folds in such a way that the two imidazole groups become lodged in the transmembrane binding cavity of CXCR4. Results also suggest that the alkyl chain length influences how the imidazole rings positions in the cavity. These results may provide a basis for the design of new CXCR4 antagonists targeting specific functions of the receptor.

Toward Understanding Mcl-1 Promiscuous and Specific Binding Mode.

Mcl-1, which is an anti-apoptotic member of the Bcl-2 protein family, is overexpressed in various cancers and promotes the aberrant survival of tumor cells. To inhibit Mcl-1, and initiate apoptosis, an interaction between BH3-only proteins and Mcl-1 anti-apoptotic protein is necessary. These protein-protein interactions exhibit some selectivity: Mcl-1 binds specifically to Noxa, whereas Bim and Puma bind strongly to all anti-apoptotic proteins. Even if the three-dimensional (3D) structures of several Mcl-1/BH3-only complexes have been solved, the BH3-only binding specificity to Mcl-1 is still not completely understood. In this study, molecular dynamics simulations were used to elucidate the molecular basis of the interactions with Mcl-1. Our results corroborate the importance of four conserved hydrophobic residues and a conserved aspartic acid on BH3-only as a common binding pattern. Furthermore, our results highlight the contribution of the fifth hydrophobic residue in the C-terminal part and a negatively charged patch in the N-terminal of BH3-only peptides as important for their fixation to Mcl-1. We hypothesize that this negatively charged patch will be an Mcl-1 specific binding pattern.

The Toxicity of a Novel Antifungal Compound Is Modulated by Endoplasmic Reticulum-Associated Protein Degradation Components.

In a search for new antifungal compounds, we screened a library of 4,454 chemicals for toxicity against the human fungal pathogen Aspergillus fumigatus. We identified sr7575, a molecule that inhibits growth of the evolutionary distant fungi A. fumigatus, Cryptococcus neoformans, Candida albicans, and Saccharomyces cerevisiae but lacks acute toxicity for mammalian cells. To gain insight into the mode of inhibition, sr7575 was screened against 4,885 S. cerevisiae mutants from the systematic collection of haploid deletion strains and 977 barcoded haploid DAmP (decreased abundance by mRNA perturbation) strains in which the function of essential genes was perturbed by the introduction of a drug resistance cassette downstream of the coding sequence region. Comparisons with previously published chemogenomic screens revealed that the set of mutants conferring sensitivity to sr7575 was strikingly narrow, affecting components of the endoplasmic reticulum-associated protein degradation (ERAD) stress response and the ER membrane protein complex (EMC). ERAD-deficient mutants were hypersensitive to sr7575 in both S. cerevisiae and A. fumigatus, indicating a conserved mechanism of growth inhibition between yeast and filamentous fungi. Although the unfolded protein response (UPR) is linked to ERAD regulation, sr7575 did not trigger the UPR in A. fumigatus and UPR mutants showed no enhanced sensitivity to the compound. The data from this chemogenomic analysis demonstrate that sr7575 exerts its antifungal activity by disrupting ER protein quality control in a manner that requires ERAD intervention but bypasses the need for the canonical UPR. ER protein quality control is thus a specific vulnerability of fungal organisms that might be exploited for antifungal drug development.

Tampering with cell division by using small-molecule inhibitors of CDK-CKS protein interactions.

Cyclin-dependent kinases (CDKs) control many cellular processes and are considered important therapeutic targets. Large collections of inhibitors targeting CDK active sites have been discovered, but their use in chemical biology or drug development has been often hampered by their general lack of specificity. An alternative approach to develop more specific inhibitors is targeting protein interactions involving CDKs. CKS proteins interact with some CDKs and play important roles in cell division. We discovered two small-molecule inhibitors of CDK-CKS interactions. They bind to CDK2, do not inhibit its enzymatic activity, inhibit the proliferation of tumor cell lines, induce an increase in G1 and/or S-phase cell populations, and cause a decrease in CDK2, cyclin A, and p27(Kip1) levels. These molecules should help decipher the complex contributions of CDK-CKS complexes in the regulation of cell division, and they might present an interesting therapeutic potential.

Antileishmanial pharmacomodulation in 8-nitroquinolin-2(1H)-one series.

An antileishmanial pharmacomodulation at position 4 of 8-nitroquinolin-2(1H)-one was conducted by using the Sonogashira and Suzuki-Miyaura coupling reactions. A series of 25 derivatives was tested in vitro on the promastigote stage of Leishmania donovani along with an in vitro cytotoxicity evaluation on the human HepG2 cell line. Only the derivatives bearing a phenyl moiety at position 4 of the quinoline ring displayed interesting biologic profile, when the phenyl moiety was substituted at the para position by a Br or Cl atom, or by a CF3 group. Among them, molecules 17 and 19 were the most selective and were then tested in vitro on the intracellular amastigote stage of both L. donovani and Leishmania infantum, in parallel with complementary in vitro cytotoxicity assays on the macrophage cell lines THP-1 and J774A.1. Molecule 19 showed no activity on the amastigote stages of the parasites and some cytotoxicity on the J774A.1 cell line while molecule 17, less cytotoxic than 19, showed anti-amastigote activity in L. infantum, being 3 times less active than miltefosine but more active and selective than pentamidine. Nevertheless, hit-molecule 17 did not appear as selective as the parent compound.

Small-animal single-photon emission computed tomographic imaging of the brain serotoninergic systems in wild-type and mdr1a knockout rats.

The pharmacokinetic properties of radiotracers are crucial for successful in vivo single-photon emission computed tomographic (SPECT) imaging. Our goal was to determine if MDR1A-deficient animals could allow better SPECT imaging outcomes than wild-type (WT) animals for a selection of serotoninergic radioligands. Thus, we compared the performances of 123I-p-MPPI, 123I-R91150, 123I-SB207710, and 123I-ADAM radioligands, for imaging of their respective targets (5-hydroxytryptamine [5-HT]1A, 5-HT2A, 5-HT4, and serotonin transporter [SERT]), in WT and Mdr1a knockout (KO) rats. With 123I-SB207710, virtually no SPECT signal was recorded in the brain of WT or KO animals. For 123I-p-MPPI, low nondisplaceable binding potentials (BPND, mean ± SD) were observed in WT (0.49 ± 0.25) and KO (0.89 ± 0.52) animals. For 123I-ADAM, modest imaging contrast was observed in WT (1.27 ± 0.02) and KO (1.31 ± 0.09) animals. For 123I-R91150, the BPND were significantly higher in Mdr1a KO (3.98 ± 0.65) animals compared to WT animals (1.22 ± 0.26). The pharmacokinetics of 123I-SB207710 and 123I-p-MPPI do not make them ideal tracers for preclinical SPECT neuroimaging. 123I-ADAM showed adequate brain uptake regardless of Mdr1a expression and appeared suitable for preclinical SPECT neuroimaging in both animal strains. The use of Mdr1a KO animals significantly improved the brain penetration of 123I-R91150, making this animal strain an interesting option when considering SPECT neuroimaging of 5-HT2A receptors in rat.

Synthesis and evaluation of cytotoxic activities of new guanidines derived from carbazoles.

Several new alkylguanidines derived from carbazole have been synthesized in a simple one-pot reaction starting from 3-aminocarbazole derivatives. The aminocarbazoles were reacted with ethoxycarbonylisothiocyanate, to give thiourea intermediates, followed by the addition of an alkylamine and HgCl2 to give ethoxycarbonylguanidine intermediates. The reaction mixture was then heated at 160 °C to give the N-(1,4-dimethyl-9H-carbazol-3-yl)-N'-alkylguanidines. The cytotoxic activity of all the synthesized guanidines was evaluated against different cell lines.

Dual histamine H3R/serotonin 5-HT4R ligands with antiamnesic properties: pharmacophore-based virtual screening and polypharmacology.

In recent years, preclinical and clinical studies have generated considerable interest in the development of histamine H3 receptor (H3R) antagonists as novel treatment for degenerative disorders associated with impaired cholinergic function. To identify novel scaffolds for H3R antagonism, a common feature-based pharmacophore model was developed and used to screen the 17,194 compounds of the CERMN (Centre d'Etudes et de Recherche sur le Médicament de Normandie) chemical library. Out of 268 virtual hits which have been gathered in 34 clusters, we were particularly interested in tricyclic derivatives also exhibiting a potent 5HT4R affinity. Benzo[h][1,6]naphthyridine derivatives showed the highest H3R affinity, and compound 17 (H3R Ki = 41.6 nM; 5-HT4R Ki = 208 nM) completely reversed the amnesiant effect of scopolamine at 3 mg/kg in a spatial working memory experiment. For the first time we demonstrated the feasibility to combine H3R and 5-HT4R activities in a single molecule, raising the exciting possibility that dual H3R antagonist/5HT4R agonist have potential for the treatment of neurodegenerative diseases such as Alzheimer's disease.

Conformation control of abiotic α-helical foldamers.

With the aim to find new protein-protein inhibitors, a three part methodology was applied to oligophenylpyridines. Theoretical ring twist angle predictions have been validated by X-ray diffraction and molecular dynamics simulations with NMR constraints. Careful choice of substituent and nitrogen positions in oligophenylpyridyl foldamer units opens the way to conformational control of the side chain distribution of this α-helix mimic.

Use of allostery to identify inhibitors of calmodulin-induced activation of Bacillus anthracis edema factor.

Allostery plays a key role in the regulation of the activity and function of many biomolecules. And although many ligands act through allostery, no systematic use is made of it in drug design strategies. Here we describe a procedure for identifying the regions of a protein that can be used to control its activity through allostery. This procedure is based on the construction of a plausible conformational path, which describes protein transition between known active and inactive conformations. The path is calculated by using a framework approach that steers and markedly improves the conjugate peak refinement method. The evolution of conformations along this path was used to identify a putative allosteric site that could regulate activation of Bacillus anthracis adenylyl cyclase toxin (EF) by calmodulin. Conformations of the allosteric site at different steps along the path from the inactive (free) to the active (bound to calmodulin) forms of EF were used to perform virtual screenings and propose candidate EF inhibitors. Several candidates then proved to inhibit calmodulin-induced activation in an in vitro assay. The most potent compound fully inhibited EF at a concentration of 10 microM. The compounds also inhibited the related adenylyl cyclase toxin from Bordetella pertussis (CyaA). The specific homology between the putative allosteric sites in both toxins supports that these pockets are the actual binding sites of the selected inhibitors.

Structural characterizations of oligopyridyl foldamers, α-helix mimetics.

Protein-protein interactions are central to many biological processes, from intracellular communication to cytoskeleton assembly, and therefore represent an important class of targets for new therapeutics. The most common secondary structure in natural proteins is an α-helix. Small molecules seem to be attractive candidates for stabilizing or disrupting protein-protein interactions based on α-helices. In our study, we assessed the ability of oligopyridyl scaffolds to mimic the α-helical twist. The theoretical as well as experimental studies (X-ray diffraction and NMR) on conformations of bipyridines in the function of substituent and pyridine nitrogen positions were carried out. Furthermore, the experimental techniques showed that the conformations observed in bipyridines are maintained within a longer oligopyridyl scaffold (quaterpyridines). The alignment of the synthesized quaterpyridine with two methyl substituents showed that it is an α-helix foldamer; their methyl groups overlap very well with side chain positions, i and i + 3, of an ideal α-helix.

New hypotheses for the binding mode of 4- and 7-substituted indazoles in the active site of neuronal nitric oxide synthase.

Taking into account the potency of 4- and 7-nitro and haloindazoles as nNOS inhibitors previously reported in the literature by our team, a multidisciplinary study, described in this article, has recently been carried out to elucidate their binding mode in the enzyme active site. Firstly, nitrogenous fastening points on the indazole building block have been investigated referring to molecular modeling hypotheses and thanks to the in vitro biological evaluation of N(1)- and N(2)-methyl and ethyl-4-substituted indazoles on nNOS. Secondly, we attempted to confirm the importance of the substitution in position 4 or 7 by a hydrogen bond acceptor group thanks to the synthesis and the in vitro biological evaluation of a new analogous 4-substituted derivative, the 4-cyanoindazole. Finally, by opposition to previous hypotheses describing NH function in position 1 of the indazole as a key fastening point, the present work speaks in favour of a crucial role of nitrogen in position 2.

Antiproliferative activity of some 1,4-dimethylcarbazoles on cells that express estrogen receptors: part I.

Several 9H-carbazole derivatives are used for various pharmacological applications. Many of these compounds demonstrated cytotoxic and anticancer activities. In this work, we have investigated the cytotoxic activity of some substituted carbazoles against cancer cell lines (MCF-7, and ISK). The derivative 2a showed the highest inhibitory activity against both cell lines.

Interpretation of honeybees contact toxicity associated to acetylcholinesterase inhibitors.

The widespread use of different pesticides generates adverse effects on non target organisms like honeybees. Organophosphorous and carbamates kill honeybees through the inactivation of acetylcholinesterase (AChE), thereby interfering with nerve signaling and function. For this class of pesticides, it is fundamental to understand the relationship between their structures and the contact toxicity for honeybees. A Quantitative Structure-Activity Relationship (QSAR) study was carried out on 45 derivatives by a genetic algorithm approach starting from more than 2500 descriptors. In parallel, a new 3D model of AChE associated to honeybees was defined. Physicochemical properties of the receptor and docking studies of the derivatives allow understanding the meaningful of three descriptors and the implication of several amino acids in the overall toxicity of the pesticides.

A new synthetic route to original sulfonamide derivatives in 2-trichloromethylquinazoline series: a structure-activity relationship study of antiplasmodial activity.

We report herein a simple and efficient two-step synthetic approach to new 2-trichloromethylquinazolines possessing a variously substituted sulfonamide group at position 4 used to prepare new quinazolines with antiparasitic properties. Thus, an original series of 20 derivatives was synthesized, which proved to be less-toxic than previously synthesized hits on the human HepG2 cell line, but did not display significant antiplasmodial activity. A brief Structure-Activity Relationship (SAR) evaluation shows that a more restricted conformational freedom is probably necessary for providing antiplasmodial activity.

Selective ortho-bromination of substituted benzaldoximes using Pd-catalyzed C-H activation: application to the synthesis of substituted 2-bromobenzaldehydes.

Substituted 2-bromobenzaldehydes were synthesized from benzaldehydes using a three-step sequence involving a selective palladium-catalyzed ortho-bromination as the key step. O-Methyloxime serves as a directing group in this reaction. A rapid deprotection of substituted 2-bromobenzaldoximes afforded substituted 2-bromobenzaldehydes with good overall yields.

4-Thiophenoxy-2-trichloromethyquinazolines display in vitro selective antiplasmodial activity against the human malaria parasite Plasmodium falciparum.

A series of original quinazolines bearing a 4-thiophenoxy and a 2-trichloromethyl group was synthesized in a convenient and efficient way and was evaluated toward its in vitro antiplasmodial potential. The series revealed global good activity against the K1-multi-resistant Plasmodium falciparum strain, especially with hit compound 5 (IC(50)=0.9 µM), in comparison with chloroquine and doxycycline chosen as reference-drugs. Both the in vitro cytotoxicity study which was conducted on the human HepG2 cell line and the in vitro antitoxoplasmic screening against Toxoplasma gondii indicate that this series presents an interesting selective antiplasmodial profile. Structure-activity- and toxicity relationships highlight that the trichloromethyl group plays a key role in the antiplasmodial activity and also show that the modulation of the thiophenol moiety influences the toxicity/activity ratio.

A New Thienopyrimidinone Chemotype Shows Multistage Activity against Plasmodium falciparum, Including Artemisinin-Resistant Parasites.

Human malaria infection begins with a one-time asymptomatic liver stage followed by a cyclic symptomatic blood stage. For decades, the research for novel antimalarials focused on the high-throughput screening of molecules that only targeted the asexual blood stages. In a search for new effective compounds presenting a triple action against erythrocytic and liver stages in addition to the ability to block the transmission of the disease via the mosquito vector, 2-amino-thienopyrimidinone derivatives were synthesized and tested for their antimalarial activity. One molecule, named gamhepathiopine (denoted as "M1" herein), was active at submicromolar concentrations against both erythrocytic (50% effective concentration [EC50] = 0.045 µM) and liver (EC50 = 0.45 µM) forms of Plasmodium falciparum. Furthermore, gamhepathiopine efficiently blocked the development of the sporogonic cycle in the mosquito vector by inhibiting the exflagellation step. Moreover, M1 was active against artemisinin-resistant forms (EC50 = 0.227 µM), especially at the quiescent stage. Nevertheless, in mice, M1 showed modest activity due to its rapid metabolization by P450 cytochromes into inactive derivatives, calling for the development of new parent compounds with improved metabolic stability and longer half-lives. These results highlight the thienopyrimidinone scaffold as a novel antiplasmodial chemotype of great interest to search for new drug candidates displaying multistage activity and an original mechanism of action with the potential to be used in combination therapies for malaria elimination in the context of artemisinin resistance. IMPORTANCE This work reports a new chemical structure that (i) displays activity against the human malaria parasite Plasmodium falciparum at 3 stages of the parasitic cycle (blood stage, hepatic stage, and sexual stages), (ii) remains active against parasites that are resistant to the first-line treatment recommended by the World Health Organization (WHO) for the treatment of severe malaria (artemisinins), and (iii) reduces transmission of the parasite to the mosquito vector in a mouse model. This new molecule family could open the way to the conception of novel antimalarial drugs with an original multistage mechanism of action to fight against Plasmodium drug resistance and block interhuman transmission of malaria.

Two-step synthesis of substituted 3-aminoindazoles from 2-bromobenzonitriles.

A general two-step synthesis of substituted 3-aminoindazoles from 2-bromobenzonitriles involving a palladium-catalyzed arylation of benzophenone hydrazone followed by an acidic deprotection/cyclization sequence is described. This procedure offers a general and efficient alternative to the typical S(N)Ar reaction of hydrazine with o-fluorobenzonitriles.

Receptor- and ligand-based study on novel 2,2'-bithienyl derivatives as non-peptidic AANAT inhibitors.

Arylalkylamine N-acetyl transferase (serotonin N-acetyl transferase, AANAT) is a critical enzyme in the light-mediated regulation of melatonin production and circadian rythm. With the objective of discovering new chemical entities with inhibitory potencies against AANAT, a medium-throughput screening campaign was performed on a chemolibrary. We found a class of molecules based on a 2,2'-bithienyl scaffold, and compound 1 emerged as a first hit. Herein, we describe our progress from hit discovery and to optimization of this new class of compounds. To complete the study, computational approaches were carried out: a docking study which provided insights into the plausible binding modes of these new AANAT inhibitors and a three-dimensional quantitative structure-activity relationship study that applied comparative molecular field analysis (CoMFA) methodology. Several CoMFA models were developed (variable alignments and options), and the best predictive one yields good statistical results (q(2) = 0.744, r(2) = 0.891, and s = 0.273). The resulting CoMFA contour maps were used to illustrate the pharmacomodulations relevant to the biological activities in this series of analogs and to design new active inhibitors. This novel series of 2,2'-bithienyl derivatives gives new insights into the design of AANAT inhibitors.