Assembly of a π-π Stack of ligands in the binding site of an acetylcholine-binding protein

Acetylcholine-binding protein is a water-soluble homologue of the extracellular ligand-binding domain of cys-loop receptors. It is used as a structurally accessible prototype for studying ligand binding to these pharmaceutically important pentameric ion channels, in particular to nicotinic acetylcholine receptors, due to conserved binding site residues present at the interface between two subunits. Here we report that an aromatic conjugated small molecule binds acetylcholine-binding protein in an ordered π-π stack of three identical molecules per binding site, two parallel and one antiparallel. Acetylcholine-binding protein stabilizes the assembly of the stack by aromatic contacts. Thanks to the plasticity of its ligand-binding site, acetylcholine-binding protein can accommodate the formation of aromatic stacks of different size by simple loop repositioning and minimal adjustment of the interactions. This type of supramolecular binding provides a novel paradigm in drug design. © 2013 Macmillan Publishers Limited. All rights reserved

Alkynamide phthalazinones as a new class of TbrPDEB1 inhibitors

Several 3′,5′-cyclic nucleotide phosphodiesterases (PDEs) have been validated as good drug targets for a large variety of diseases. Trypanosoma brucei PDEB1 (TbrPDEB1) has been designated as a promising drug target for the treatment of human African trypanosomiasis. Recently, the first class of selective nanomolar TbrPDEB1 inhibitors was obtained by targeting the parasite specific P-pocket. However, these biphenyl-substituted tetrahydrophthalazinone-based inhibitors did not show potent cellular activity against Trypanosoma brucei (T. brucei) parasites, leaving room for further optimization. Herein, we report the discovery of a new class of potent TbrPDEB1 inhibitors that display improved activities against T. brucei parasites. Exploring different linkers between the reported tetrahydrophthalazinone core scaffold and the amide tail group resulted in the discovery of alkynamide phthalazinones as new TbrPDEB1 inhibitors, which exhibit submicromolar activities versus T. brucei parasites and no cytotoxicity to human MRC-5 cells. Elucidation of the crystal structure of alkynamide 8b (NPD-048) bound to the catalytic domain of TbrPDEB1 shows a bidentate interaction with the key-residue Gln874 and good directionality towards the P-pocket. Incubation of trypanosomes with alkynamide 8b results in an increase of intracellular cAMP, validating a PDE-mediated effect in vitro and providing a new interesting compound series for further studies towards selective TbrPDEB1 inhibitors with potent phenotypic activity

Targeting a Subpocket in Trypanosoma brucei Phosphodiesterase B1 (TbrPDEB1) Enables the Structure-Based Discovery of Selective Inhibitors with Trypanocidal Activity

Several trypanosomatid cyclic nucleotide phosphodiesterases (PDEs) possess a unique, parasite-specific cavity near the ligand-binding region that is referred to as the P-pocket. One of these enzymes, Trypanosoma brucei PDE B1 (TbrPDEB1), is considered a drug target for the treatment of African sleeping sickness. Here, we elucidate the molecular determinants of inhibitor binding and reveal that the P-pocket is amenable to directed design. By iterative cycles of design, synthesis, and pharmacological evaluation and by elucidating the structures of inhibitor-bound TbrPDEB1, hPDE4B, and hPDE4D complexes, we have developed 4a,5,8,8a-tetrahydrophthalazinones as the first selective TbrPDEB1 inhibitor series. Two of these, 8 (NPD-008) and 9 (NPD-039), were potent (Ki = 100 nM) TbrPDEB1 inhibitors with antitrypanosomal effects (IC50 = 5.5 and 6.7 ?M, respectively). Treatment of parasites with 8 caused an increase in intracellular cyclic adenosine monophosphate (cAMP) levels and severe disruption of T. brucei cellular organization, chemically validating trypanosomal PDEs as therapeutic targets in trypanosomiasis

Escherichia coli O157 infection associated with a petting zoo.

A young child was admitted to hospital with haemolytic-uraemic syndrome caused by infection with a Shiga toxin 2-producing strain of Escherichia coli (STEC) O157. Five days before he became ill, the child had visited a small petting zoo. STEC O157 strains were isolated from faecal samples from goats and sheep housed on the farm. The human and the animal isolates were indistinguishable by molecular subtyping. The petting zoo voluntarily closed temporarily to prevent further cases of infection. Two out of 11 other, randomly selected petting zoos (including one deer park) visited subsequently, tested positive. Furthermore, during the study period there was one more notification of STEC O157 infection possibly linked with a farm visit. Although STEC O157 was indeed found in the petting zoo associated with this patient, transmission through animal contact could not be confirmed because the human isolate was not available for subtyping. The case study and the results of the other on-farm investigations highlight the risk of acquiring severe zoonotic infections during visits to petting zoos

Fragment growing induces conformational changes in acetylcholine-binding protein: A structural and thermodynamic analysis

Optimization of fragment hits toward high-affinity lead compounds is a crucial aspect of fragment-based drug discovery (FBDD). In the current study, we have successfully optimized a fragment by growing into a ligand-inducible subpocket of the binding site of acetylcholine-binding protein (AChBP). This protein is a soluble homologue of the ligand binding domain (LBD) of Cys-loop receptors. The fragment optimization was monitored with X-ray structures of ligand complexes and systematic thermodynamic analyses using surface plasmon resonance (SPR) biosensor analysis and isothermal titration calorimetry (ITC). Using site-directed mutagenesis and AChBP from different species, we find that specific changes in thermodynamic binding profiles, are indicative of interactions with the ligand-inducible subpocket of AChBP. This study illustrates that thermodynamic analysis provides valuable information on ligand binding modes and is complementary to affinity data when guiding rational structure- and fragment-based discovery approaches

To Target or Not to Target <i>Schistosoma mansoni</i> Cyclic Nucleotide Phosphodiesterase 4A?

Schistosomiasis is a neglected tropical disease with high morbidity. Recently, the Schistosoma mansoni phosphodiesterase SmPDE4A was suggested as a putative new drug target. To support SmPDE4A targeted drug discovery, we cloned, isolated, and biochemically characterized the full-length and catalytic domains of SmPDE4A. The enzymatically active catalytic domain was crystallized in the apo-form (PDB code: 6FG5) and in the cAMP- and AMP-bound states (PDB code: 6EZU). The SmPDE4A catalytic domain resembles human PDE4 more than parasite PDEs because it lacks the parasite PDE-specific P-pocket. Purified SmPDE4A proteins (full-length and catalytic domain) were used to profile an in-house library of PDE inhibitors (PDE4NPD toolbox). This screening identified tetrahydrophthalazinones and benzamides as potential hits. The PDE inhibitor NPD-0001 was the most active tetrahydrophthalazinone, whereas the approved human PDE4 inhibitors roflumilast and piclamilast were the most potent benzamides. As a follow-up, 83 benzamide analogs were prepared, but the inhibitory potency of the initial hits was not improved. Finally, NPD-0001 and roflumilast were evaluated in an in vitro anti-S. mansoni assay. Unfortunately, both SmPDE4A inhibitors were not effective in worm killing and only weakly affected the egg-laying at high micromolar concentrations. Consequently, the results with these SmPDE4A inhibitors strongly suggest that SmPDE4A is not a suitable target for anti-schistosomiasis therapy

Perceived Sodium Reduction Barriers Among Patients with Chronic Kidney Disease: Which Barriers Are Important and Which Patients Experience Barriers?

Purpose: The purposes of this study were to assess the importance of perceived sodium reduction barriers among patients with chronic kidney disease (CKD) and identify associated sociodemographic, clinical, and psychosocial factors. Method: A total of 156 patients with CKD completed a questionnaire assessing sodium reduction barriers (18 self-formulated items), depressive symptoms (Beck Depression Inventory), perceived autonomy support (Modified Health Care Climate Questionnaire), and self-efficacy (Partners in Health Questionnaire). Factor analysis was used to identify barrier domains. Correlation coefficients were computed to examine relationships between barrier domains and patient characteristics. Results: Nine barrier domains were identified. Barriers perceived as important were as follows: high sodium content in products, lack of sodium feedback, lack of goal setting and discussing strategies for sodium reduction, and not experiencing CKD-related symptoms (mean scores > 3.0 on 5-point scales, ranging from 1 ‘no barrier’ to 5 ‘very important barrier’). Other barriers (knowledge, attitude, coping skills when eating out, and professional support) were rated as moderately important (rated around midpoint), and the barrier ‘intrinsic motivation’ was rated as somewhat important (mean score = 1.9). Sodium reduction barrier domains were not associated with gender and kidney function, but were associated with age, level of education, number of comorbidities, perceived autonomy support, depressive symptoms, and self-efficacy (range r = 0.17–0.35). Patients with lower self-efficacy and perceived autonomy support scores experienced most sodium reduction barriers. Conclusion: Patients with CKD experience multiple important sodium reduction barriers and could benefit from support strategies that target various sodium reduction barriers and strengthen beliefs regarding self-efficacy and autonomy support. Additionally, environmental interventions should be implemented to reduce sodium levels in processed foods