In silico chemical library screening and experimental validation of novel compounds with potential varroacide activities.

The mite Varroa destructor is an ectoparasite and has been identified as a major cause of worldwide honey bee colony losses. The use of yearly treatments for the control of varroosis is the most common answer to prevent collapses of honey bee colonies due to the mite. However, the number of effective acaricides is small and the mite tends to become resistant to these few active molecules. In this study, we have been looking for a new original varroacide treatment inhibiting selectively Varroa destructor AChE (vdAChE) with respect to Apis mellifera AChE (amAChE). To do this an original drug design methodology was used applying virtual screening of the CERMN chemolibrary, starting from a vdAChE homology sequence model. By combining the in silico screening with in vitro experiments, two promising compounds were found. In vitro tests of AChE inhibition for both species have confirmed good selectivity toward the mite vdAChE. Moreover, an in vivo protocol was performed and highlighted a varroacide activity without acute consequences on honey bee survival. The two compounds discovered have the potential to become new drug leads for the development of new treatments against the mite varroa. The method described here clearly shows the potential of a drug-design approach to develop new solutions to safeguard honey bee health.

Protective effects of caffeic acid against hypothalamic neuropeptides alterations induced by malathion in rat.

Exposure to pesticides is suspected to cause human health problems. Our study aimed to evaluate preventive effects of caffeic acid (3,4-dihydroxycinnamic acid) in the hypothalamus against malathion-induced neuropeptides gene expression alterations. Malathion at 100 mg/kg was administered intragastrically to rats alone or in combination with caffeic acid at 100 mg/kg during 4 weeks. A molecular expression of hypothalamic neuropeptides and plasmatic cholinesterase activity was investigated. Furthermore, we used in silico analysis, known as computational docking, to highlight the nature of acetylcholinesterase-malathion/caffeic acid interactions. Our findings showed differences in the responses and indicate that caffeic acid reversed malathion-induced decrease in corticotropin-releasing hormone mRNA but not brain-derived neurotrophic factor which presented an increased tendency. We suggest that caffeic acid can interact with acetylcholinesterase as the primary target of organophosphorus compounds. Results predict that caffeic acid can block partly the acetylcholinesterase gorge entrance via π-π stacking interaction with Tyr 124 and Trp 286 residues of the peripheral site leading to its stricture. Under this condition, we suggested that acetylcholine trafficking toward the catalytic site is ameliorated compared to malaoxon according to their sizes.

Protecting honey bees: identification of a new varroacide by in silico, in vitro, and in vivo studies.

Varroa destructor is the main concern related to the gradual decline of honeybees. Nowadays, among the various acaricides used in the control of V. destructor, most presents increasing resistance. An interesting alternative could be the identification of existent molecules as new acaricides with no effect on honeybee health. We have previously constructed the first 3D model of AChE for honeybee. By analyzing data concerning amino acid mutations implicated in the resistance associated to pesticides, it appears that pirimicarb should be a good candidate for varroacide. To check this hypothesis, we characterized the AChE gene of V. destructor. In the same way, we proposed a 3D model for the AChE of V. destructor. Starting from the definition of these two 3D models of AChE in honeybee and varroa, a comparison between the gorges of the active site highlighted some major differences and particularly different shapes. Following this result, docking studies have shown that pirimicarb adopts two distinct positions with the strongest intermolecular interactions with VdAChE. This result was confirmed with in vitro and in vivo data for which a clear inhibition of VdAChE by pirimicarb at 10 µM (contrary to HbAChE) and a 100% mortality of varroa (dose corresponding to the LD50 (contact) for honeybee divided by a factor 100) were observed. These results demonstrate that primicarb could be a new varroacide candidate and reinforce the high relationships between in silico, in vitro, and in vivo data for the design of new selective pesticides.

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.

Synthesis and structure-affinity relationships of selective high-affinity 5-HT(4) receptor antagonists: application to the design of new potential single photon emission computed tomography tracers.

The work described herein aims at finding new potential ligands for the brain imaging of 5-HT(4) receptors (5-HT(4)Rs) using single-photon emission computed tomography (SPECT). Starting from the nonsubstituted phenanthridine compound 4a, exhibiting a K(i) value of 51 nM on the 5-HT(4)R, we explored the structure-affinity in this series. We found that substitution in position 4 of the tricycle with a fluorine atom gave the best result. Introduction of an additional nitrogen atom inside the tricyclic framework led to an increase of both the affinity and selectivity for 5-HT(4)R, suggesting the design of the antagonist 4v, exhibiting a high affinity of 0.04 nM. Several iodinated analogues were then synthesized as potential SPECT tracers. The iodinated compound 11d was able to displace the reference radioiodinated 5-HT(4)R antagonist (1-butylpiperidin-4-yl)methyl-8-amino-7-iodo[(123)I]-2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate {[(123)I]1, [(123)I]SB 207710} both in vitro and in vivo in brain. Compound 11d was radiolabeled with [(125)I]iodine, providing a potential SPECT candidate for brain imaging of 5-HT(4)R.

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.

Structure-function perturbation and dissociation of tetrameric urate oxidase by high hydrostatic pressure.

Structure-function relationships in the tetrameric enzyme urate oxidase were investigated using pressure perturbation. As the active sites are located at the interfaces between monomers, enzyme activity is directly related to the integrity of the tetramer. The effect of hydrostatic pressure on the enzyme was investigated by x-ray crystallography, small-angle x-ray scattering, and fluorescence spectroscopy. Enzymatic activity was also measured under pressure and after decompression. A global model, consistent with all measurements, discloses structural and functional details of the pressure-induced dissociation of the tetramer. Before dissociating, the pressurized protein adopts a conformational substate characterized by an expansion of its substrate binding pocket at the expense of a large neighboring hydrophobic cavity. This substate should be adopted by the enzyme during its catalytic mechanism, where the active site has to accommodate larger intermediates and product. The approach, combining several high-pressure techniques, offers a new (to our knowledge) means of exploring structural and functional properties of transient states relevant to protein mechanisms.

P3 peptide, a truncated form of A beta devoid of synaptotoxic effect, does not assemble into soluble oligomers.

In previously proposed models of A beta soluble oligomers, the N-terminal domain A beta(1-16), which is missing in p3 peptides, protects the hydrophobic core of the oligomers from the solvent. Without this N-terminal part, oligomers of p3 peptides would likely expose hydrophobic residues to water and would consequently be less stable. We thus suggest, based on theoretical and experimental results, that p3 peptides would have a low propensity to assemble into stable oligomers, evolving then directly to fibrillar aggregates. These properties may explain why p3 would be devoid of any impact on synaptic function and moreover, strengthen the hypothesis that A beta oligomers are the principal synaptotoxic forms of A beta peptides in Alzheimer disease.

Sequence-based modeling of Abeta42 soluble oligomers.

Abeta fibrils, which are central to the pathology of Alzheimer's disease, form a cross-beta-structure that contains likely parallel beta-sheets with a salt bridge between residues Asp23 and Lys28. Recent studies suggest that soluble oligomers of amyloid peptides have neurotoxic effects in cell cultures, raising the interest in studying the structures of these intermediate forms. Here, we present three models of possible soluble Abeta forms based on the sequences similarities, assumed to support local structural similarities, of the Abeta peptide with fragments of three proteins (adhesin, Semliki Forest virus capsid protein, and transthyretin). These three models share a similar structure in the C-terminal region composed of two beta-strands connected by a loop, which contain the Asp23-Lys28 salt bridge. This segment is also structurally well conserved in Abeta fibril forms. Differences between the three monomeric models occur in the N-terminal region and in the C-terminal tail. These three models might sample some of the most stable conformers of the soluble Abeta peptide within oligomeric assemblies, which were modeled here in the form of dimers, trimers, tetramers, and hexamers. The consistency of these models is discussed with respect to available experimental and theoretical data.

Arginine 260 of the amino-terminal domain of NR1 subunit is critical for tissue-type plasminogen activator-mediated enhancement of N-methyl-D-aspartate receptor signaling.

Tissue-type plasminogen activator (tPA) has been involved in both physiological and pathological glutamatergic-dependent processes, such as synaptic plasticity, seizure, trauma, and stroke. In a previous study, we have shown that the proteolytic activity of tPA enhances the N-methyl-D-aspartate (NMDA) receptor-mediated signaling in neurons (Nicole, O., Docagne, F., Ali, C., Margaill, I., Carmeliet, P., MacKenzie, E. T., Vivien, D., and Buisson, A. (2001) Nat. Med. 7, 59-64). Here, we show that tPA forms a direct complex with the amino-terminal domain (ATD) of the NR1 subunit of the NMDA receptor and cleaves this subunit at the arginine 260. Furthermore, point mutation analyses show that arginine 260 is necessary for both tPA-induced cleavage of the ATD of NR1 and tPA-induced potentiation of NMDA receptor signaling. Thus, tPA is the first binding protein described so far to interact with the ATD of NR1 and to modulate the NMDA receptor function.