Design of a General‐Purpose European Compound Screening Library for EU‐OPENSCREEN

This work describes a collaborative effort to define and apply a protocol for the rational selection of a general‐purpose screening library, to be used by the screening platforms affiliated with the EU‐OPENSCREEN initiative. It is designed as a standard source of compounds for primary screening against novel biological targets, at the request of research partners. Given the general nature of the potential applications of this compound collection, the focus of the selection strategy lies on ensuring chemical stability, absence of reactive compounds, screening‐compliant physicochemical properties, loose compliance to drug‐likeness criteria (as drug design is a major, but not exclusive application), and maximal diversity/coverage of chemical space, aimed at providing hits for a wide spectrum of drugable targets. Finally, practical availability/cost issues cannot be avoided. The main goal of this publication is to inform potential future users of this library about its conception, sources, and characteristics. The outline of the selection procedure, notably of the filtering rules designed by a large committee of European medicinal chemists and chemoinformaticians, may be of general methodological interest for the screening/medicinal chemistry community. The selection task of 200K molecules out of a pre‐filtered set of 1.4M candidates was shared by five independent European research groups, each picking a subset of 40K compounds according to their own in‐house methodology and expertise. An in‐depth analysis of chemical space coverage of the library serves not only to characterize the collection, but also to compare the various chemoinformatics‐driven selection procedures of maximal diversity sets. Compound selections contributed by various participating groups were mapped onto general‐purpose self‐organizing maps (SOMs) built on the basis of marketed drugs and bioactive reference molecules. In this way, the occupancy of chemical space by the EU‐OPENSCREEN library could be directly compared with distributions of known bioactives of various classes. This mapping highlights the relevance of the selection and shows how the consensus reached by merging the five different 40K selections contributes to achieve this relevance. The approach also allows one to readily identify subsets of target‐ or target‐class‐oriented compounds from the EU‐OPENSCREEN library to suit the needs of the diverse range of potential users. The final EU‐OPENSCREEN library, assembled by merging five independent selections of 40K compounds from various expert groups, represents an excellent example of a Europe‐wide collaborative effort toward the common objective of building best‐in‐class European open screening platforms.     

Nonlinear Absorption of Submicrometer Grain‐Size Cobalt‐Doped Magnesium Aluminate Transparent Ceramics

Transparent cobalt‐doped magnesium aluminate spinel (Co:MgAl₂O₄) ceramics with a submicrometer grain size were prepared by spark plasma sintering. For the first time, the nonlinear absorption of Co:MgAl₂O₄ transparent ceramics was experimentally demonstrated. Both ground state absorption (σ_GSA) and excited state absorption (σ_ESA) were estimated using the solid‐state slow saturable absorber model based on absorption saturation measurements performed at 1.535 μm. σ_GSA and σ_ESA for 0.03 at.% Co:MgAl₂O₄ were found to be 4.1 × 10^?19 cm² and 4.0 × 10^?20 cm², respectively. In the case of 0.06 at.% Co:MgAl₂O₄ ceramics, σ_GSA = 2.6 × 10^?19 cm² and σ_ESA= 5.3 × 10^?20 cm² were determined.     

Effects of a Protic Ionic Liquid on the Reaction Pathway during Non-Aqueous Sol–Gel Synthesis of Silica: A Raman Spectroscopic Investigation

The reaction pathway during the formation of silica via a two-component “non-aqueou” sol-gel synthesis is studied by in situ time-resolved Raman spectroscopy. This synthetic route is followed with and without the addition of the protic ionic liquid 1-ethylimidazolium bis(trifluoromethanesulfonyl)imide (C₂HImTFSI) in order to investigate its effect on the reaction pathway. We demonstrate that Raman spectroscopy is suitable to discriminate between different silica intermediates, which are produced and consumed at different rates with respect to the point of gelation. We find that half-way to gelation monomers and shorter chains are the most abundant silica species, while the formation of silica rings strongly correlates to the sol-to-gel transition. Thus, curling up of linear chains is here proposed as a plausible mechanism for the formation of small rings. These in turn act as nucleation sites for the condensation of larger rings and thus the formation of the open and polymeric silica network. We find that the protic ionic liquid does not change the reaction pathway per se, but accelerates the cyclization process, intermediated by the faster inclusion of monomeric species.     

Influence of Unmodified and β-Glycerophosphate Cross-Linked Chitosan on Anti-Candida Activity of Clotrimazole in Semi-Solid Delivery Systems

The combination of an antifungal agent and drug carrier with adjunctive antimicrobial properties represents novel strategy of complex therapy in pharmaceutical technology. The goal of this study was to investigate the unmodified and ion cross-linked chitosan’s influence on anti-Candida activity of clotrimazole used as a model drug in hydrogels. It was particularly crucial to explore whether the chitosans’ structure modification by β-glycerophosphate altered its antifungal properties. Antifungal studies (performed by plate diffusion method according to CLSI reference protocol) revealed that hydrogels obtained with chitosan/β-glycerophosphate displayed lower anti-Candida effect, probably as a result of weakened polycationic properties of chitosan in the presence of ion cross-linker. Designed chitosan hydrogels with clotrimazole were found to be more efficient against tested Candida strains and showed more favorable drug release profile compared to commercially available product. These observations indicate that novel chitosan formulations may be considered as promising semi-solid delivery system of clotrimazole.     

Compensatory fertilization of Scots pine stands polluted by heavy metals

The results from four compensatory fertilization experiments located at different distances (0.5, 2, 4 and 8 km) along a heavy metal deposition gradient extending from the Harjavalta Cu-Ni smelter in SW Finland are presented. The experiments were established in middle-age Scots pine stands growing on dryish sites of sorted glaciofluvial sediments. The soil type in all the experiments is ferric podsol. The treatments in the experiments consisted of liming, a powdered slow-release mineral mixture and stand-specific fertilization which comprised at least methylene urea and ammonium nitrate.Monitoring of deposition and soil solution and studies on soil chemical and microbiological properties, on the nutrient status of trees and needle litterfall, on fine root dynamics and on the growth of the tree stands were carried out during a 5-year period.There was a severe shortage of exchangeable Ca and Mg in the organic layer of the most polluted stands. Although the uppermost mineral soil layer had relatively high exchangeable Ca and Mg concentrations, the trees were not able to utilize these nutrient reserves presumably due to the toxic effects of Cu and Ni on the plant roots and mycorrhizas.The treatments that included limestone markedly decreased the Cu and Ni concentrations in the soil solution and soil organic layer, presumably due to immobilisation through precipitation or absorption. The Ca and Mg concentrations correspondingly increased, which certainly contributed to the partial recovery of fine root and stand growth. The powdered mineral mixture and the combination of methylene urea and ammonium nitrate had no short-term effect on the microbial biomass and activity. All the fertilizer treatments increased volume growth in the most polluted stand. The stand-specific fertilization increased needle mass in heavily polluted stands, but the response of the needle mass to fertilizer treatments was low in the less polluted stands. No clear evidence was found to support the role of nutrient status in tree resistance.     

Two‐Step Protein Labeling by Using Lipoic Acid Ligase with Norbornene Substrates and Subsequent Inverse‐Electron Demand Diels–Alder Reaction

Inverse‐electron‐demand Diels–Alder cycloaddition (DA_inv) between strained alkenes and tetrazines is a highly bio‐orthogonal reaction that has been applied in the specific labeling of biomolecules. In this work we present a two‐step labeling protocol for the site‐specific labeling of proteins based on attachment of a highly stable norbornene derivative to a specific peptide sequence by using a mutant of the enzyme lipoic acid ligase A (LplA^W37V), followed by the covalent attachment of tetrazine‐modified fluorophores to the norbornene moiety through the bio‐orthogonal DA_inv . We investigated 15 different norbornene derivatives for their selective enzymatic attachment to a 13‐residue lipoic acid acceptor peptide (LAP) by using a standardized HPLC protocol. Finally, we used this two‐step labeling strategy to label proteins in cell lysates in a site‐specific manner and performed cell‐surface labeling on living cells.     

Investigation of Serine‐Proteinase‐Catalyzed Peptide Splicing in Analogues of Sunflower Trypsin Inhibitor 1 (SFTI‐1)

Serine‐proteinase‐catalyzed peptide splicing was demonstrated in analogues of the trypsin inhibitor SFTI‐1: both single peptides and two‐peptide chains (C‐ and N‐terminal peptide chains linked by a disulfide bridge). In the second series, peptide splicing with catalytic amount of proteinase was observed only when formation of acyl–enzyme intermediate was preceded by hydrolysis of the substrate Lys–Ser peptide bond. Here we demonstrate that with an equimolar amount of the proteinase, splicing occurs in all the two‐peptide‐chain analogues. This conclusion was supported by high resolution crystal structures of selected analogues in complex with trypsin. We showed that the process followed a direct transpeptidation mechanism. Thus, the acyl–enzyme intermediate was formed and was immediately used for a new peptide bond formation; products associated with the hydrolysis of the acyl–enzyme were not observed. The peptide splicing was sequence‐ not structure‐specific.     

Synthesis and Antiplasmodial Activity of Novel Chloroquine Analogues with Bulky Basic Side Chains

Chloroquine is commonly used in the treatment and prevention of malaria, but Plasmodium falciparum, the main species responsible for malaria‐related deaths, has developed resistance against this drug. Twenty‐seven novel chloroquine (CQ) analogues characterized by a side chain terminated with a bulky basic head group, i.e., octahydro‐2H‐quinolizine and 1,2,3,4,5,6‐hexahydro‐1,5‐methano‐8H‐pyrido[1,2‐a][1,5]diazocin‐8‐one, were synthesized and tested for activity against D‐10 (CQ‐susceptible) and W‐2 (CQ‐resistant) strains of P. falciparum. Most compounds were found to be active against both strains with nanomolar or sub‐micromolar IC₅₀ values. Eleven compounds were found to be 2.7‐ to 13.4‐fold more potent than CQ against the W‐2 strain; among them, four cytisine derivatives appear to be of particular interest, as they combine high potency with low cytotoxicity against two human cell lines (HMEC‐1 and HepG2) along with easier synthetic accessibility. Replacement of the 4‐NH group with a sulfur bridge maintained antiplasmodial activity at a lower level, but produced an improvement in the resistance factor. These compounds warrant further investigation as potential drugs for use in the fight against malaria.     

Identification of Aminoimidazole and Aminothiazole Derivatives as Src Family Kinase Inhibitors

Src family kinases (SFKs) are a family of non‐receptor tyrosine kinases (TKs) implicated in the regulation of many cellular processes. The aberrant activity of these TKs has been associated with the growth and progression of cancer. In particular, c‐Src is overexpressed or hyperactivated in a variety of solid tumors and is most likely a strong promoting factor for the development of metastasis. Herein, the synthesis of new 4‐aminoimidazole and 2‐aminothiazole derivatives and their in vitro biological evaluation are described for their potential use as SFK inhibitors. Initially, 2‐aminothiazole analogues of dasatinib and 4‐aminoimidazole derivatives were synthesized and tested against the SFKs Src, Fyn, Lyn, and Yes. Five hits were identified as the most promising compounds, with K_i values in the range of 90–480 nm . A combination of molecular docking, homology modeling, and molecular dynamics were then used to investigate the possible binding mode of such compounds within the ATP binding site of the SFKs. Finally, the antiproliferative activities of the best candidates were evaluated against SH‐SY5Y and K562 cell lines. Compound 3 b [2‐(4‐{2‐methyl‐6‐[(5‐phenylthiazol‐2‐yl)amino]pyrimidin‐4‐yl}piperazin‐1‐yl)ethanol] was found to be the most active inhibitor.