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.

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.

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.

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.

Virtual screening discovery of new acetylcholinesterase inhibitors issued from CERMN chemical library.

In our quest to find new inhibitors able to inhibit acetylcholinesterase (AChE) and, at the same time, to protect neurons from beta amyloid toxicity, i.e., inhibitors interacting with the catalytic anionic subsite as well as with the peripherical anionic site of AChE, a virtual screening of the Centre d'Etudes et de Recherche sur le Medicament de Normandie (CERMN) chemical library was carried out. Two complementary approaches were applied, i.e., a ligand- and a structure-based screening. Each screening led to the selection of different compounds, but only two were present in both screening results. In vitro tests on AChE showed that one of those compounds presented a very good inhibition activity, of the same order as Donepezil. This result shows the real complementary of both methods for the discovery of new ligands.

3D Pharmacophore, hierarchical methods, and 5-HT4 receptor binding data.

5-Hydroxytryptamine subtype-4 (5-HT(4)) receptors have stimulated considerable interest amongst scientists and clinicians owing to their importance in neurophysiology and potential as therapeutic targets. A comparative analysis of hierarchical methods applied to data from one thousand 5-HT(4) receptor-ligand binding interactions was carried out. The chemical structures were described as chemical and pharmacophore fingerprints. The definitions of indices, related to the quality of the hierarchies in being able to distinguish between active and inactive compounds, revealed two interesting hierarchies with the Unity (1 active cluster) and pharmacophore fingerprints (4 active clusters). The results of this study also showed the importance of correct choice of metrics as well as the effectiveness of a new alternative of the Ward clustering algorithm named Energy (Minimum E-Distance method). In parallel, the relationship between these classifications and a previously defined 3D 5-HT(4) antagonist pharmacophore was established.

3-(Dipropylamino)-5-hydroxybenzofuro[2,3-f]quinazolin-1(2H)-one (DPA-HBFQ-1) plays an inhibitory role on breast cancer cell growth and progression.

A series of unknown 3-(alkyl(dialkyl)amino)benzofuro[2,3-f]quinazolin-1(2H)-ones 4-17 has been synthesized as new ellipticine analogs, in which the carbazole moiety and the pyridine ring were replaced by a dibenzofuran residue and a pyrimidine ring, respectively. The synthesis of these benzofuroquinazolinones 4-17 was performed in a simple one-pot reaction using 3-aminodibenzofuran or its 2-methoxy derivative, as starting materials. From 3-(dipropylamino)-5-methoxybenzofuro[2,3-f] quinazolin-1(2H)-one (13), we prepared 3-(dipropylamino)-5-hydroxybenzofuro[2,3-f]quinazolin-1(2H)-one (18), referred to as DPA-HBFQ-1. The cytotoxic activities of all the synthesized compounds, tested in different human breast cancer cell lines, revealed that DPA-HBFQ-1 was the most active compound. In particular, the latter was able to inhibit anchorage-dependent and -independent cell growth and to induce apoptosis in estrogen receptor alpha (ERα)-positive and -negative breast cancer cells. It did not affect proliferation and apoptotic responses in MCF-10A normal breast epithelial cells. The observed effects have been ascribed to an enhanced p21(Cip1/WAF1) expression in a p53-dependent manner of tumor suppressor and to a selective inhibition of human topoisomerase II. In addition, DPA-HBFQ-1 exerted growth inhibitory effects also in other cancer cell lines, even though with a lower cytotoxic activity. Our results indicate DPA-HBFQ-1 as a good candidate to be useful as cancer therapeutic agent, particularly for breast cancer.

Design of a serotonin 4 receptor radiotracer with decreased lipophilicity for single photon emission computed tomography.

With the aim to develop a suitable radiotracer for the brain imaging of the serotonin 4 receptor subtype (5-HT4R) using single photon emission computed tomography (SPECT), we synthesized and evaluated a library of di- and triazaphenanthridines with lipophilicity values which were in the range expected to favour brain penetration, and which demonstrated specific binding to the target of interest. Adding additional nitrogen atoms to previously described phenanthridine ligands exhibiting a high unspecific binding, we were able to design a radioiodinated compound [(125)I]14. This compound exhibited a binding affinity value of 0.094 nM toward human 5-HT4R and a high selectivity over other serotonin receptor subtypes (5-HTR). In vivo SPECT imaging studies and competition experiments demonstrated that the decreased lipophilicity (in comparison with our previously reported compounds 4 and 5) allowed a more specific labelling of the 5-HT4R brain-containing regions.

Boronic species as promising inhibitors of the Staphylococcus aureus NorA efflux pump: study of 6-substituted pyridine-3-boronic acid derivatives.

In response to the extensive use of antibiotics, bacteria have evolved numerous mechanisms of defense against antimicrobial agents. Among them, extrusion of the antimicrobial agents outside the bacterial cell through efflux pumps is a major cause of concern. At first limited to one or few structurally-related antibiotics, bacterial resistance have then progressed towards cross-resistance between different classes of antibiotics, leading to multidrug-resistant microorganisms. Emergence of these pathogens requires development of novel therapeutic strategies and inhibition of efflux pumps appears to be a promising strategy that could restore the potency of existing antibiotics. NorA is the most studied chromosomal efflux pump of Staphylococcus aureus; it is known to be implied in resistance of Methicillin-resistant S. aureus (MRSA) strains against a wide range of unrelated substrates, including hydrophilic fluoroquinolones. Starting from 6-benzyloxypyridine-3-boronic acid I that we previously identified as a potential inhibitor of the NorA efflux pump against the NorA-overexpressing S. aureus 1199B strain (SA1199B), we describe here the synthesis and biological evaluation of a series of 6-(aryl)alkoxypyridine-3-boronic acids. 6-(3-Phenylpropoxy)pyridine-3-boronic acid 3i and 6-(4-phenylbutoxy)pyridine-3-boronic acid 3j were found to potentiate ciprofloxacin activity by a 4-fold increase compared to the parent compound I. In addition, it has been shown that both compounds promote Ethidium Bromide (EtBr) accumulation in SA1199B, thus corroborating their potential mode of action as NorA inhibitors.

Discovery of new thienopyrimidinone derivatives displaying antimalarial properties toward both erythrocytic and hepatic stages of Plasmodium.

A preliminary in vitro screening of compounds belonging to various chemical families from our library revealed the thieno[3,2-d]pyrimidin-4(3H)-one scaffold displayed a promising profile against Plasmodium falciparum. Then, 120 new derivatives were synthesized and evaluated in vitro; compared to drug references, 40 showed good activity toward chloroquine sensitive (IC50 35-344 nM) and resistant (IC50 45-800 nM) P. falciparum strains. They were neither cytotoxic (CC50 15-50 µM) toward HepG2 and CHO cells, nor mutagenic. Structure-activity relationships were defined. The lead-compound also appeared active against the Plasmodium liver stages (Plasmodium yoelii IC50 = 35 nM) and a preliminary in vivo evaluation indicated the in vitro activity was preserved (45% reduction in parasitemia compared to untreated infected mice). A mechanistic study demonstrated these molecules do not involve any of the pathways described for commercial drugs and exert a specific activity on the ring and trophozoite stages.

First identification of boronic species as novel potential inhibitors of the Staphylococcus aureus NorA efflux pump.

Overexpression of efflux pumps is an important mechanism of bacterial resistance that results in the extrusion of antimicrobial agents outside the bacterial cell. Inhibition of such pumps appears to be a promising strategy that could restore the potency of existing antibiotics. The NorA efflux pump of Staphylococcus aureus confers resistance to a wide range of unrelated substrates, such as hydrophilic fluoroquinolones, leading to a multidrug-resistance phenotype. In this work, approximately 150 heterocyclic boronic species were evaluated for their activity against susceptible and resistant strains of S. aureus. Twenty-four hit compounds, although inactive when tested alone, were found to potentiate ciprofloxacin activity by a 4-fold increase at concentrations ranging from 0.5 to 8 µg/mL against S. aureus 1199B, which overexpresses NorA. Boron-free analogues showed no biological activity, thus revealing that the boron atom is crucial for biological activity. This work describes the first reported efflux pump inhibitory activity of boronic acid derivatives.

Synthesis and in vitro evaluation of 4-trichloromethylpyrrolo[1,2-a]quinoxalines as new antiplasmodial agents.

Thanks to a preliminary in vitro screening of several CCl3-substituted-nitrogen containing heterocycles belonging to our chemical library, the 2-trichloromethylquinoxaline scaffold appeared to be of potential interest for developing new antiplasmodial agents. Then, combining these experimental results to the antimalarial properties reported for various pyrrolo[1,2-a]quinoxaline derivatives, an original series of fifteen 7-substituted-4-trichoromethylpyrrolo[1,2-a]quinoxalines was synthesized in a 4 to 5 reaction steps pathway. All molecules were evaluated in vitro toward both their antiplasmodial activity on the K1 multi-resistant Plasmodium falciparum strain and their cytotoxicity on the HepG2 human cell line. Thus, 3 hit molecules were identified, displaying IC50 values in the micromolar range and low cytotoxicity values, reaching good selectivity indexes, in comparison with the reference drugs chloroquine and doxycycline. Structure-activity relationship studies showed that the pyrrolo[1,2-a]quinoxaline scaffold can support selective antiplasmodial activity when substituted at position 4 by a CCl3 group. However, substitution at position 7 of the same scaffold is neither beneficial for cytotoxicity nor favourable for the solubility in the biological media.

MR22388, a novel anti-cancer agent with a strong FLT-3 ITD kinase affinity.

This work describes the study of the mechanism of action and spectrum of activity of MR22388, a novel anti-cancer agent belonging to the tripentone series. MR22388 is highly cytotoxic (within the nanomolar range) against numerous cancer cell lines and studies of its cytotoxicity mechanisms show that it is a weak inhibitor of the polymerization of tubulin and that it induces apoptosis via the MAP kinase pathways. Further MR22388 is a very strong inhibitor of several kinases including the tyrosine kinase FLT3-ITD. FLT3-ITD is a mutated form of the tyrosine kinase receptor (RTK) FLT3, resulting in the constitutive activation of the kinase, occurring in about 25% of normal karyotypes' Acute Myeloid Leukemia (AML) and is linked to a bad prognosis. Consecutively, MR22388 appears as a novel promising anticancer lead agent especially for AML therapy.

Targeting the proangiogenic VEGF-VEGFR protein-protein interface with drug-like compounds by in silico and in vitro screening.

Protein-protein interactions play a central role in medicine, and their modulation with small organic compounds remains an enormous challenge. Because it has been noted that the macromolecular complexes modulated to date have a relatively pronounced binding cavity at the interface, we decided to perform screening experiments over the vascular endothelial growth factor receptor (VEGFR), a validated target for antiangiogenic treatments with a very flat interface. We focused the study on the VEGFR-1 D2 domain, and 20 active compounds were identified. These small compounds contained a (3-carboxy-2-ureido)thiophen unit and had IC(50) values in the low micromolar range. The most potent compound inhibited the VEGF-induced VEGFR-1 transduction pathways. Our findings suggest that our best hit may be a promising scaffold to probe this macromolecular complex and for the development of treatments of VEGFR-1-dependent diseases.

Novel aminoethylbiphenyls as 5-HT7 receptor ligands.

The synthesis of a series of aminoethylbiphenyls as novel 5-HT(7) receptor ligands is described. The novel derivatives exhibit high affinity for the 5-HT(7) receptor with selectivity toward 5-HT(1A) receptor.

Definition of new pharmacophores for nonpeptide antagonists of human urotensin-II. Comparison with the 3D-structure of human urotensin-II and URP.

Starting from nonpeptide agonists and antagonists of human urotensin-II (hU-II), several pharmacophores were designed and compared to the structure of hU-II. NMR and dynamic studies were realized on hU-II and urotensin-II-related peptide to check the conformation flexibilities of these peptides and the relationships between their potential 3D structures and the pharmacophores. In parallel, a virtual screening was carried out, leading to the discovery of six new derivatives with micromolar affinities. This last result shows the interest of these pharmacophores for the discovery of new ligands.

Molecular modeling studies focused on 5-HT7 versus 5-HT1A selectivity. Discovery of novel phenylpyrrole derivatives with high affinity for 5-HT7 receptors.

The present study discusses the well-known 5-HT7/5-HT1A selectivity issue through a new series of phenylpyrrole derivatives. The first hits emerged from a virtual screening performed on a chemolibrary. Further study led to an optimization of a preliminary 5-HT7 pharmacophore model. The importance of each pharmacophoric feature is confirmed, but these characteristics have to be coupled to geometric constraints in order to achieve a 5-HT7 selectivity. Indeed, 5-HT1A affinity probably arises from extended conformations, whereas a bent one appears to be best suited for 5-HT7 selectivity.