Computational Tool to Design Small Synthetic Inhibitors Selective for XIAP-BIR3 Domain.

X-linked inhibitor of apoptosis protein (XIAP) exercises its biological function by locking up and inhibiting essential caspase-3, -7 and -9 toward apoptosis execution. It is overexpressed in multiple human cancers, and it plays an important role in cancer cells' death skipping. Inhibition of XIAP-BIR3 domain and caspase-9 interaction was raised as a promising strategy to restore apoptosis in malignancy treatment. However, XIAP-BIR3 antagonists also inhibit cIAP1-2 BIR3 domains, leading to serious side effects. In this study, we worked on a theoretical model that allowed us to design and optimize selective synthetic XIAP-BIR3 antagonists. Firstly, we assessed various MM-PBSA strategies to predict the XIAP-BIR3 binding affinities of synthetic ligands. Molecular dynamics simulations using hydrogen mass repartition as an additional parametrization with and without entropic term computed by the interaction entropy approach produced the best correlations. These simulations were then exploited to generate 3D pharmacophores. Following an optimization with a training dataset, five features were enough to model XIAP-BIR3 synthetic ligands binding to two hydrogen bond donors, one hydrogen bond acceptor and two hydrophobic groups. The correlation between pharmacophoric features and computed MM-PBSA free energy revealed nine residues as crucial for synthetic ligand binding: Thr308, Glu314, Trp323, Leu307, Asp309, Trp310, Gly306, Gln319 and Lys297. Ultimately, and three of them seemed interesting to use to improve XIAP-BR3 versus cIAP-BIR3 selectivity: Lys297, Thr308 and Asp309.

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.

Straightforward Access to a New Class of Dual DYRK1A/CLK1 Inhibitors Possessing a Simple Dihydroquinoline Core.

The DYRK (Dual-specificity tyrosine phosphorylation-regulated kinase) family of protein kinases is involved in the pathogenesis of several neurodegenerative diseases. Among them, the DYRK1A protein kinase is thought to be implicated in Alzheimer's disease (AD) and Down syndrome, and as such, has emerged as an appealing therapeutic target. DYRKs are a subset of the CMGC (CDK, MAPKK, GSK3 and CLK) group of kinases. Within this group of kinases, the CDC2-like kinases (CLKs), such as CLK1, are closely related to DYRKs and have also sparked great interest as potential therapeutic targets for AD. Based on inhibitors previously described in the literature (namely TG003 and INDY), we report in this work a new class of dihydroquinolines exhibiting inhibitory activities in the nanomolar range on hDYRK1A and hCLK1. Moreover, there is overwhelming evidence that oxidative stress plays an important role in AD. Pleasingly, the most potent dual kinase inhibitor 1p exhibited antioxidant and radical scavenging properties. Finally, drug-likeness and molecular docking studies of this new class of DYRK1A/CLK1 inhibitors are also discussed in this article.

Synthesis and Physicochemical Properties of Cryptophazane-A Soluble and Functionalizable C1 -Symmetrical Cryptophane.

The first example of a cryptophazane, a cryptophane functionalized with a nitrogen atom replacing one of the methylene bridges, is obtained with a 28 % overall yield over 8 steps, through the preparation of a C1 -symmetrical aza-cyclotriveratrylene (aza-CTV). Herein, we demonstrate that the introduction of a nitrogen atom on this part of the cryptophane core enhances the solubility in organic media of both the cryptophane and the synthetic intermediates, while presenting the same conformation as known cryptophanes. Cryptophazane was prepared on a multigram scale and easily functionalized. We also investigated its ability to encapsulate xenon atoms using hyperpolarized 129 Xe (HP 129 Xe) NMR spectroscopy. We found that both its affinity and exchange kinetics were in the appropriate range for applications in 129 Xe magnetic resonance imaging (MRI). Combined with the wide range of possible functionalization, this makes cryptophazane an excellent candidate for targeted HP 129 Xe MRI.

Development of Novel Potential Pleiotropic Compounds of Interest in Alzheimer's Disease Treatment through Rigidification Strategy.

The development of Multi-Target Directed Ligand is of clear interest for the treatment of multifactorial pathology such as Alzheimer's disease (AD). In this context, acetylcholinesterase (AChE) inhibitors have been modulated in order to generate novel pleiotropic compounds targeting a second protein of therapeutic interest in AD. Among them, donecopride was the first example of a dual acetylcholinesterase inhibitor and 5-HT4 receptor agonist. In order to explore the structural diversity around this preclinical candidate we have explored the preparation of novel constrained analogs through late-stage rigidification strategy. A series of phenylpyrazoles was prepared in a late-stage functionalization process and all compounds were evaluated in vitro towards AChE and 5-HTRs. A docking study was performed in order to better explain the observed SAR towards AChE, 5-HT4R and 5-HT6R and this study led to the description of novel ligand targeting both AChE and 5-HT6R.

Insights into Mcl-1 Conformational States and Allosteric Inhibition Mechanism from Molecular Dynamics Simulations, Enhanced Sampling, and Pocket Crosstalk Analysis.

In this study, we explored the structural dynamics of Mcl-1, an anti-apoptotic protein. On the basis of structural ensembles, the essential dynamics was extracted and showed two major axes of variability: a breathing motion at the binding interface and a correlated motion through the internal loops. A free energy surface characterizing the breathing motion at the binding interface was generated and suggested an equilibrium between a closed conformation and a "ready to bind" conformation as the predominant states of Mcl-1 in solution. Moreover, the analysis of the dynamics along the internal loops revealed a hidden communication network of transient and cryptic pockets controlling the allosteric inhibition of Mcl-1. A detailed model joining the pocket crosstalk and salt bridge networks along the internal loops was proposed and allowed us to shed light on the key interactions governing Mcl-1's allosteric inhibition.

First Synthesis of Racemic Trans Propargylamino-Donepezil, a Pleiotrope Agent Able to Both Inhibit AChE and MAO-B, with Potential Interest against Alzheimer's Disease.

Alzheimer's disease (AD) is a multifactorial neurodegenerative disease towards which pleiotropic approach using Multi-Target Directed Ligands is nowadays recognized as probably convenient. Among the numerous targets which are today validated against AD, acetylcholinesterase (ACh) and Monoamine Oxidase-B (MAO-B) appear as particularly convincing, especially if displayed by a sole agent such as ladostigil, currently in clinical trial in AD. Considering these results, we wanted to take benefit of the structural analogy lying in donepezil (DPZ) and rasagiline, two indane derivatives marketed as AChE and MAO-B inhibitors, respectively, and to propose the synthesis and the preliminary in vitro biological characterization of a structural compromise between these two compounds, we called propargylaminodonepezil (PADPZ). The synthesis of racemic trans PADPZ was achieved and its biological evaluation established its inhibitory activities towards both (h)AChE (IC50 = 0.4 µM) and (h)MAO-B (IC50 = 6.4 µM).

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.

Inhibiting Acetylcholinesterase to Activate Pleiotropic Prodrugs with Therapeutic Interest in Alzheimer's Disease.

Alzheimer's disease (AD) is a multifactorial neurodegenerative disease which is still poorly understood. The drugs currently used against AD, mainly acetylcholinesterase inhibitors (AChEI), are considered clinically insufficient and are responsible for deleterious side effects. AChE is, however, currently receiving renewed interest through the discovery of a chaperone role played in the pathogenesis of AD. But AChE could also serve as an activating protein for pleiotropic prodrugs. Indeed, inhibiting central AChE with brain-penetrating designed carbamates which are able to covalently bind to the enzyme and to concomitantly liberate active metabolites in the brain could constitute a clinically more efficient approach which, additionally, is less likely to cause peripheral side effects. We aim in this article to pave the road of this new avenue with an in vitro and in vivo study of pleiotropic prodrugs targeting both the 5-HT4 receptor and AChE, in order to display a neuroprotective activity associated with a sustained restoration of the cholinergic neurotransmission and without the usual peripheral side effects associated with classic AChEI. This plural activity could bring to AD patients effective, relatively safe, symptomatic and disease-modifying therapeutic benefits.

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.

Conformation and Dynamics of Human Urotensin II and Urotensin Related Peptide in Aqueous Solution.

Conformation and dynamics of the vasoconstrictive peptides human urotensin II (UII) and urotensin related peptide (URP) have been investigated by both unrestrained and enhanced-sampling molecular-dynamics (MD) simulations and NMR spectroscopy. These peptides are natural ligands of the G-protein coupled urotensin II receptor (UTR) and have been linked to mammalian pathophysiology. UII and URP cannot be characterized by a single structure but exist as an equilibrium of two main classes of ring conformations, open and folded, with rapidly interchanging subtypes. The open states are characterized by turns of various types centered at K8Y9 or F6W7 predominantly with no or only sparsely populated transannular hydrogen bonds. The folded conformations show multiple turns stabilized by highly populated transannular hydrogen bonds comprising centers F6W7K8 or W7K8Y9. Some of these conformations have not been characterized previously. The equilibrium populations that are experimentally difficult to access were estimated by replica-exchange MD simulations and validated by comparison of experimental NMR data with chemical shifts calculated with density-functional theory. UII exhibits approximately 72% open:28% folded conformations in aqueous solution. URP shows very similar ring conformations as UII but differs in an open:folded equilibrium shifted further toward open conformations (86:14) possibly arising from the absence of folded N-terminal tail-ring interaction. The results suggest that the different biological effects of UII and URP are not caused by differences in ring conformations but rather by different interactions with UTR.

A Three-Site Mechanism for Agonist/Antagonist Selective Binding to Vasopressin Receptors.

Molecular-dynamics simulations with metadynamics enhanced sampling reveal three distinct binding sites for arginine vasopressin (AVP) within its V2 -receptor (V2 R). Two of these, the vestibule and intermediate sites, block (antagonize) the receptor, and the third is the orthosteric activation (agonist) site. The contacts found for the orthosteric site satisfy all the requirements deduced from mutagenesis experiments. Metadynamics simulations for V2 R and its V1a R-analog give an excellent correlation with experimental binding free energies by assuming that the most stable binding site in the simulations corresponds to the experimental binding free energy in each case. The resulting three-site mechanism separates agonists from antagonists and explains subtype selectivity.

Room-temperature ortho-alkoxylation and -halogenation of N-tosylbenzamides by using palladium(II)-catalyzed C-H activation.

The N-tosylcarboxamide group can direct the room-temperature palladium-catalyzed C-H alkoxylation and halogenation of substituted arenes in a simple and mild procedure. The room-temperature stoichiometric cyclopalladation of N-tosylbenzamide was first studied, and the ability of the palladacycle to react with oxidants to form C-X and C-O bonds under mild conditions was demonstrated. The reaction conditions were then adapted to promote room-temperature ortho-alkoxylations and ortho-halogenations of N-tosylbenzamides using palladium as catalyst. The scope and limitation of both alkoxylations and halogenations was studied and the subsequent functional transformation of the N-tosylcarboxamide group through nucleophilic additions was evaluated. This methodology offers a simple and mild route to diversely functionalized arenes.

Discovery of a pocket network on the domain 5 of the TrkB receptor - A potential new target in the quest for the new ligands.

The important role that the neurotrophin tyrosine kinase receptor - TrkB has in the pathogenesis of several neurodegenerative conditions such are Alzheimer's disease, Parkinson's disease, Huntington's disease, has been well described. This shouldn't be a surprise, since in the physiological conditions, once activated by brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5), the TrkB receptor promotes neuronal survival, differentiation and synaptic function. Considering that the natural ligands for TrkB receptor are large proteins, it is a challenge to discover small molecule capable to mimic their effects. Even though, the surface of receptor that is interacting with BDNF or NT-4/5 is known, there was always a question which pocket and interaction is responsible for activation of it. In order to answer this challenging question, we have used molecular dynamic (MD) simulations and Pocketron algorithm which enabled us to detect, for the first time, a pocket network existing in the interacting domain (d5) of the receptor; to describe them and to see how they are communicating with each other. This new discovery gave us potential new areas on receptor that can be targeted and used for structure-based drug design approach in the development of the new ligands.

Design, synthesis and preliminary biological evaluation of rivastigmine-INDY hybrids as multitarget ligands against Alzheimer's disease by targeting butyrylcholinesterase and DYRK1A/CLK1 kinases.

Based on a multitarget approach implementing rivastigmine-INDY hybrids 1, we identified a set of pseudo-irreversible carbamate-type inhibitors of eqBuChE that, after carbamate transfer at the active site serine residue, released the corresponding INDY analogues 2 endowed with hDYRK1A/hCLK1 kinases inhibitory properties. A SAR study and molecular docking investigation of both series of compounds 1 and 2 revealed that appropriate structural modifications at the carbamate moiety and at the N-appendage of the benzothiazole core led to potent and selective eqBuChE inhibitors with IC50 up to 27 nM and potent hDYRK1A and hCLK1 inhibitors with IC50 up to 106 nM and 17 nM respectively. Pleasingly, identification of the matched pair of compounds 1b/2b with a good balance between inhibition of eqBuChE and hDYRK1A/hCLK1 kinases (IC50 = 68 nM and IC50 = 529/54 nM, respectively) further validated our multitarget approach based on a sequential mechanism of action. In addition, target compound 1b exhibited a suitable ADMET profile, including good brain permeability and high stability in PBS, encouraging further biological investigation as a drug candidate.

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.

Gold(I)-Mediated Radioiododecarboxylation of Arenes.

A novel radioiodination method is developed using carboxylic acids as radiolabeling precursors. This method involves decarboxylation and organogold(I) intermediate formation, enabling efficient radioiodination of (hetero)arenes and cinnamic and phenylpropiolic acids. Additionally, we demonstrated the prolonged stability of crude gold(I) organometallic compounds, showcasing their enduring radiolabeling capabilities.

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.

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.

On the Tracks of the Aggregation Mechanism of the PHF6 Peptide from Tau Protein: Molecular Dynamics, Energy, and Interaction Network Investigations.

The formation of neurofibrillary tangles (NFTs), composed of tau protein aggregates, is a hallmark of some neurodegenerative diseases called tauopathies. NFTs are composed of paired helical filaments (PHFs) of tau protein with a dominant ß-sheet secondary structuration. The NFT formation mechanism is not known yet. This study focuses on PHF6, a crucial hexapeptide responsible for tau aggregation. A 2 µs molecular dynamics simulation was launched to determine the keys of the PHF6 aggregation mechanism. Hydrogen bonding, van der Waals, and other non-covalent interactions as π-stacking were investigated. Parallel aggregation was slightly preferred due to its adaptability, but antiparallel aggregation remained widely present during the PHF6 aggregation. The analysis highlighted the leading role of hydrogen bonds identified at the atomic level for each aggregation process. The aggregation study emphasized the importance of Tyr310 during the ß-sheets' complexation through π-stacking.