Design and Synthesis of Galactosylated Bifurcated Ligands with Nanomolar Affinity for Lectin LecA from Pseudomonas aeruginosa

Lectin A (LecA) from Pseudomonas aeruginosa is an established virulence factor. Glycoclusters that target LecA and are able to compete with human glycoconjugates present on epithelial cells are promising candidates to treat P. aeruginosa infection. A family of 32 glycodendrimers of generation 0 and 1 based on a bifurcated bis‐galactoside motif have been designed to interact with LecA. The influences both of the central multivalent core and of the aglycon of these glycodendrimers on their affinity toward LecA have been evaluated by use of a microarray technique, both qualitatively for rapid screening of the binding properties and also quantitatively (K_d). This has led to high‐affinity LecA ligands with K_d values in the low nanomolar range (K_d=22 nm for the best one).     

Going Forward Laterally: Transmembrane Passage of Hydrophobic Molecules through Protein Channel Walls

Regular phospholipid bilayers do not pose efficient barriers for the transport of hydrophobic molecules. The outer membrane (OM) surrounding Gram‐negative bacteria is a nontypical, asymmetric bilayer with an outer layer of lipopolysaccharide (LPS). The sugar molecules of the LPS layer prevent spontaneous diffusion of hydrophobic molecules across the OM. As regular OM channels such as porins do not allow passage of hydrophobic molecules, specialized OM transport proteins are required for their uptake. Such proteins, exemplified by channels of the FadL family, transport their substrates according to a lateral diffusion mechanism. Here, substrates diffuse from the lumen of the β‐barrel laterally into the OM, through a stable opening in the wall of the barrel. In this way, the lipopolysaccharide barrier is bypassed and, by depositing the substrates into the OM, a driving force for uptake is provided. Lateral diffusion through protein channel walls also occurs in α‐helical inner membrane proteins, and could represent a widespread mechanism for proteins that transport and interact with hydrophobic substrates.     

Characterization of the Aminotransferase ThdN from Thienodolin Biosynthesis in Streptomyces albogriseolus

In Streptomyces albogriseolus the indolethiophen alkaloid thienodolin is derived from tryptophan. The first step in thienodolin biosynthesis is the regioselective chlorination of tryptophan in the 6‐position of the indole ring. The second step is catalyzed by the aminotransferase ThdN. ThdN shows sequence homology (up to 69 % similarity) with known pyridoxal 5′‐phosphate‐dependent aminotransferases of the aspartate aminotransferase family from Gram‐positive bacteria. thdN was heterologously expressed in Pseudomonas fluorescens, and the enzyme was purified by nickel‐affinity chromatography. ThdN is a homodimeric enzyme with a mass of 90 600 kDa and catalyzes the conversion of l ‐tryptophan and a number of chlorinated and brominated l ‐tryptophans. The lowest K_M values were found for 6‐bromo‐ and 6‐chlorotryptophan (40 and 66 μm , respectively). For l ‐tryptophan it was 454 μm, which explains why thienodolin is the major product and dechlorothienodolin is only a minor component. The turnover number (k_cat) for 7‐chlorotryptophan (128 min^?1) was higher than that for the natural substrate 6‐chlorotryptophan (88 min^?1).     

Synthesis and Structure–Activity Relationship Studies of 2‐(1,3,4‐Oxadiazole‐2(3H)‐thione)‐3‐amino‐5‐arylthieno[2,3‐b]pyridines as Inhibitors of DRAK2

In recent years, DAPK‐related apoptosis‐inducing protein kinase 2 (DRAK2) has emerged as a promising target for the treatment of a variety of autoimmune diseases and for the prevention of graft rejection after organ transplantation. However, medicinal chemistry optimization campaigns for the discovery of novel small‐molecule inhibitors of DRAK2 have not yet been published. Screening of a proprietary compound library led to the discovery of a benzothiophene analogue that displays an affinity constant (K_d) value of 0.25 μM . Variation of the core scaffold and of the substitution pattern afforded a series of 5‐arylthieno[2,3‐b]pyridines with strong binding affinity (K_d=0.008 μM for the most potent representative). These compounds also show promising activity in a functional biochemical DRAK2 enzyme assay, with an IC₅₀ value of 0.029 μM for the most potent congener. Selectivity profiling of the most potent compounds revealed that they lack selectivity within the DAPK family of kinases. However, one of the less potent analogues is a selective ligand for DRAK2 and can be used as starting point for the synthesis of selective and potent DRAK2 inhibitors.     

Estimation of interaction between polyanions and bovine serum albumin by means of affinity chromatography

The ligand binding of some polyanions to bovine serum albumin immobilized on Sepharose 4B has been studied by column affinity chromatography. Frontal chromatography using a polyanion of low concentration on an affinity adsorbent gave the dissociation constant K_d of the polyanion-immobilized ligand complex. K_d values determined under various concentrations enabled us to discuss in detail the interactions of bovine serum albumin and polyanions.     

Synthesis and antimicrobial activity of the Schiff base from polyoxyalkylene‐montmorillonite nanocomposites and aldehydes

Vanillin (4‐hydroxy‐3‐methoxy benzaldehyde) and 5‐formylamino salicylic acid microbicides were reacted with polyoxyalkylene‐montmorillonite (D_230–2000‐MMT) nanocomposites. The microstructure of these Schiff base nanocomposites was characterized by TEM and XRD. D_230–2000‐MMT nanocomposites were prepared by an ion exchange process of sodium montmorillonite (Na‐MMT) and NH₃ ⁺ groups in polyoxyalkylene amine hydrochloride with three different molecular masses of D₂₃₀, D₄₀₀, and D₂₀₀₀. Wide‐angle X‐ray diffraction confirms the intercalation of the polymer between the silicate layers. Electrostatic interaction between the positively charged NH₃ ⁺ groups and the negatively charged surface of MMT was observed. The nanocomposites were tested for antimicrobial activity against the Gram‐negative bacteria (Escherichia coli NCIM 2065), Gram‐positive bacteria (Bacillus subtillus ATCC), and fungi (Candida albicans SC5314 and Cryptococcus neoformans). The D₂₀₀₀‐MMT/vanillin Schiff base nanocomposite strongly inhibited the growth of all microorganisms that can be used in different applications. The amount of loaded polymer and the structure of the nanocomposite play an important role in inhibiting the bacterial and fungal strains. It is found that the Schiff base nanocomposite affect the morphology, oxygen consumption, and the release of cytoplasmic constituents such as potassium (K⁺), sodium (Na⁺), and calcium (Ca²⁺) ions leading to death of the cells. POLYM. COMPOS., 2012. © 2012 Society of Plastics Engineers     

The Discovery of Phenylbenzamide Derivatives as Grb7‐Based Antitumor Agents

Grb7 is a non‐catalytic protein, the overexpression of which has been associated with the proliferative and migratory potentials of cancer cells. Virtual screening strategies involving a shape‐based similarity search, molecular docking, and 2D‐similarity searches complemented by experimental binding studies (Thermofluor and isothermal titration calorimetry) resulted in the identification of nine novel phenylbenzamide‐based antagonists of the Grb7 SH2 domain. Moderate binding affinities were observed, ranging from K_d=32.3 μM for lead phenylbenzamide NSC 104999 ( 1 ) to K_d=1.1 μM for a structurally related compound, NSC 57148 ( 2 ). Deconvolution of the affinity data into its components revealed differences in lead binding, from being entropy based (lead 1 ) to enthalpically driven (NSC 100874 ( 3 ), NSC 55158 ( 4 ), and compound 2 ). Finally, the lead compound 1 was found to decrease the growth of MDA‐MB‐468 breast cancer cells, with an IC₅₀ value of 39.9 μM . It is expected that these structures will serve as novel leads in the development of Grb7‐based anticancer therapeutics.     

Thermophiles as Potential Source of Novel Endotoxin Antagonists: the Full Structure and Bioactivity of theLipo‐oligosaccharide from Thermomonas hydrothermalis

Thermomonas hydrothermalis is a Gram‐negative thermophilic bacterium that is able to live at 50 °C. This ability is attributed to chemical modifications, involving those to bacterial cell‐wall components, such as proteins and (glyco)lipids. As the main component of the outer membrane of Gram‐negative bacteria, lipopolysaccharides (LPSs) are exposed to the environment, thus they can undergo structural chemical changes to allow thermophilic bacteria to live at their optimal growth temperature. Furthermore, as one of the major target of the eukaryotic innate immune system, LPS elicits host immune response in a structure‐dependent mode; thus the uncommon chemical features of thermophilic bacterial LPSs might exert a different biological action on the innate immune system—an antagonistic effect, as shown in studies of LPS structure–activity relationship in the ongoing research into antagonist LPS candidates. Here, we report the complete structural and biological activity analysis of the lipo‐oligosaccharide isolated from Thermomonas hydrothermalis, achieved by a multidisciplinary approach (chemical analysis, NMR, MALDI MS and cellular immunology). We demonstrate a tricky and interesting structure combined with a very interesting effect on human innate immunity.     

Kinetic Analysis of the Interaction of Mos1 Transposase with its Inverted Terminal Repeats Reveals New Insight into the Protein–DNA Complex Assembly

Transposases are specific DNA‐binding proteins that promote the mobility of discrete DNA segments. We used a combination of physicochemical approaches to describe the association of MOS1 (an eukaryotic transposase) with its specific target DNA, an event corresponding to the first steps of the transposition cycle. Because the kinetic constants of the reaction are still unknown, we aimed to determine them by using quartz crystal microbalance on two sources of recombinant MOS1: one produced in insect cells and the other produced in bacteria. The prokaryotic‐expressed MOS1 showed no cooperativity and displayed a K_d of about 300 nM . In contrast, the eukaryotic‐expressed MOS1 generated a cooperative system, with a lower K_d (～2 nm) . The origins of these differences were investigated by IR spectroscopy and AFM imaging. Both support the conclusion that prokaryotic‐ and eukaryotic‐expressed MOS1 are not similarly folded, thereby resulting in differences in the early steps of transposition.     

C6‐Unsubstituted Pyrazolo[3,4‐d]pyrimidines Are Dual Src/Abl Inhibitors Effective against Imatinib Mesylate Resistant Chronic Myeloid Leukemia Cell Lines

Docking simulations were used to predict the most favorable interaction between the T315I mutated form of Abl (invariably associated with resistance to the tyrosine kinase inhibitor imatinib mesylate, IM) and C6‐unsubstituted and substituted pyrazolo[3,4‐d]pyrimidines previously found to be dual Src/Abl inhibitors. Two C6‐unsubstituted ( 1 and 2 ) and eight C6‐substituted compounds ( 3 – 10 ) were selected and assayed for their effects on the Ba/F3 cell line transducing the wild‐type p210Bcr–Abl construct, which is IM‐sensitive, or three of the most common mutations associated with IM resistance in vivo (T315I, Y253F, and E255K), and driven to drug resistance by saturating doses of IL‐3 or by the expression of the Bcr–Abl construct coding for the p185 protein of acute lymphoblastic leukemia. Compounds 1 and 2 were active against all cell lines assayed (LD₅₀ range: 0.7–4.3 μM ), whereas C6‐substituted compounds exhibited lower activity (LD₅₀～8 μM for compound 3 toward the T315I mutant). Notably, 1 and 2 were also effective toward the T315I mutation, which is insensitive to dual Src/Abl inhibitors. The cytotoxic effects of 1 and 2 on IM‐sensitive and IM‐resistant Ba/F3 cells were attributable, at least in part, to their pro‐apoptotic activity. Taken together, such findings suggest that C6‐unsubstituted pyrazolo[3,4‐d]pyrimidines may represent useful inhibitors to target IM‐resistant chronic myeloid leukemia.     

In vitro Characterization of Enzymes Involved in the Synthesis of Nonproteinogenic Residue (2S,3S)‐β‐Methylphenylalanine in Glycopeptide Antibiotic Mannopeptimycin

Mannopeptimycin, a potent drug lead, has superior activity against difficult‐to‐treat multidrug‐resistant Gram‐positive pathogens such as methicillin‐resistant Staphylococcus aureus (MRSA). (2S,3S)‐β‐Methylphenylalanine is a residue in the cyclic hexapeptide core of mannopeptimycin, but the synthesis of this residue is far from clear. We report here on the reaction order and the stereochemical course of reaction in the formation of (2S,3S)‐β‐methylphenylalanine. The reaction is executed by the enzymes MppJ and TyrB, an S‐adenosyl methionine (SAM)‐dependent methyltransferase and an (S)‐aromatic‐amino‐acid aminotransferase, respectively. Phenylpyruvic acid is methylated by MppJ at its benzylic position at the expense of one equivalent of SAM. The resulting β‐methyl phenylpyruvic acid is then converted to (2S,3S)‐β‐methylphenylalanine by TyrB. MppJ was further determined to be regioselective and stereoselective in its catalysis of the formation of (3S)‐β‐methylphenylpyruvic acid. The binding constant (K_D) of MppJ versus SAM is 26 μM . The kinetic constants with respect to k_{cat Ppy} and K_{M Ppy}, and k_{cat SAM} and K_{M SAM} are 0.8 s^?1 and 2.5 mM , and 8.15 s^?1 and 0.014 mM , respectively. These results suggest SAM has higher binding affinity for MppJ than Ppy, and the C? C bond formation in βmPpy might be the rate‐limiting step, as opposed to the C? S bond breakage in SAM.     

Research on the preparation and antibacterial properties of 2‐N‐thiosemicarbazide‐6‐O‐hydroxypropyl chitosan membranes with iodine

The 2‐N‐thiosemicarbazide‐6‐O‐hydroxypropyl chitosan (ATU‐HPCS) was prepared by chitosan grafted hydroxypropyl and thiosemicarbazide through the method of “amino protection‐graft‐deprotection,” while the ATU‐HPCS gel membranes were obtained from gelatin and polyvinyl pyrrolidone as additives, and the ATU‐HPCS membranes with iodine (ATU‐HPCS‐I₂‐M) were prepared by adding the ethanol solution of iodine in the ATU‐HPCS gel membranes. The ATU‐HPCS‐I₂‐M were characterized to evaluate their potential applications as antibacterial materials. The iodine releasing rule of ATU‐HPCS‐I₂‐M showed a sustained‐release effect of iodine, the maximum emission was approximately 0.80%. The inhibition zone diameters of ATU‐HPCS‐I₂‐M against Staphylococcus aureus (as Gram‐positive bacteria) and Escherichia coli (as Gram‐negative bacteria) were both greater than 15 mm, it demonstrated significant antibacterial activity compared with the ATU‐HPCS gel membranes. The double effects of the biocompatibility of chitosan and the sustained‐release of iodine provided an ideal healing environment for wound surface. These properties have made ATU‐HPCS‐I₂‐M highly potential as a novel natural macromolecule antimicrobial material preventing the bacteria from burns, surgery wounds, etc. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40535.     

Isolation of a Small‐Molecule Inhibitor of the Antiapoptotic Protein Bcl‐xL from a DNA‐Encoded Chemical Library

Bcl‐xL is an antiapoptotic member of the Bcl‐2 protein family and an attractive target for the development of anticancer agents. Here we describe the isolation of binders to Bcl‐xL from a DNA‐encoded chemical library using affinity‐capture selections and massively parallel high‐throughput sequencing of >30 000 sequence tags of library members. The most potent binder identified, compound 19 / 93 [(R)‐3‐(amido indomethacin)‐4‐(naphthalen‐1‐yl)butanoic acid], bound to Bcl‐xL with a dissociation constant (K_d) of 930 nM and was able to compete with a Bak‐derived BH3 peptide, an antagonist of Bcl‐xL function.     

A DNA‐Encoded Library of Chemical Compounds Based on Common Scaffolding Structures Reveals the Impact of Ligand Geometry on Protein Recognition

A DNA‐encoded chemical library (DECL) with 1.2 million compounds was synthesized by combinatorial reaction of seven central scaffolds with two sets of 343×492 building blocks. Library screening by affinity capture revealed that for some target proteins, the chemical nature of building blocks dominated the selection results, whereas for other proteins, the central scaffold also crucially contributed to ligand affinity. Molecules based on a 3,5‐bis(aminomethyl)benzoic acid core structure were found to bind human serum albumin with a K_d value of 6 nm , while compounds with the same substituents on an equidistant but flexible l ‐lysine scaffold showed 140‐fold lower affinity. A 18 nm tankyrase‐1 binder featured l ‐lysine as linking moiety, while molecules based on d ‐Lysine or (2S,4S)‐amino‐l ‐proline showed no detectable binding to the target. This work suggests that central scaffolds which predispose the orientation of chemical building blocks toward the protein target may enhance the screening productivity of encoded libraries.     

Using Chemical Probes to Assess the Feasibility of Targeting SecA for Developing Antimicrobial Agents against Gram‐Negative Bacteria

With the widespread emergence of drug resistance, there is an urgent need to search for new antimicrobials, especially those against Gram‐negative bacteria. Along this line, the identification of viable targets is a critical first step. The protein translocase SecA is commonly believed to be an excellent target for the development of broad‐spectrum antimicrobials. In recent years, we developed three structural classes of SecA inhibitors that have proven to be very effective against Gram‐positive bacteria. However, we have not achieved the same level of success against Gram‐negative bacteria, despite the potent inhibition of SecA in enzyme assays by the same inhibitors. In this study, we use representative inhibitors as chemical probes to gain an understanding as to why these inhibitors were not effective against Gram‐negative bacteria. The results validate our initial postulation that the major difference in effectiveness against Gram‐positive and Gram‐negative bacteria is in the additional permeability barrier posed by the outer membrane of Gram‐negative bacteria. We also found that the expression of efflux pumps, which are responsible for multidrug resistance (MDR), have no effect on the effectiveness of these SecA inhibitors. Identification of an inhibitor‐resistant mutant and complementation tests of the plasmids containing secA in a secAts mutant showed that a single secA‐azi‐9 mutation increased the resistance, providing genetic evidence that SecA is indeed the target of these inhibitors in bacteria. Such results strongly suggest SecA as an excellent target for developing effective antimicrobials against Gram‐negative bacteria with the intrinsic ability to overcome MDR. A key future research direction should be the optimization of membrane permeability.     

Physicochemical characterization of ATP binding to human 5-lipoxygenase

Human 5-lipoxygenase requires ATP as a stimulatory factor. At the two preferred concentrations of the free Ca²⁺, 0.02 μM with a resting cell and 20 μM with a stimulated cell, Scatchard analysis revealed that 5-lipoxygenase has one affinity ATP binding site with aK _d of 4.6 μM at the low Ca²⁺ concentration but has two affinity ATP binding sites with a higherK _d of 4.4 μM and a lowerK _d of 14.5 μM at the high Ca²⁺ concentration. In contrast, in a Tween 20 reaction system, 5-lipoxygenase had similar activation coefficients for ATP at both Ca²⁺ concentrations; these were 12.7 μM at the low Ca²⁺ concentration and 12.0 μM at the high Ca²⁺ concentration. These results showed that 5-lipoxygenase has an ATP binding site and suggest that self-association of 5-lipoxygenase in 20 μM Ca²⁺ may affect ATP binding affinity as measured by Scatchard analysis.     

Effects of the Surface Densities of Glycoclusters on the Determination of Their IC50 and Kd Value Determination by Using a Microarray

Pseudomonas aeruginosa (PA) is an opportunistic bacterium involved in 10–30 % of nosocomial diseases. It causes severe lung injury to cystic fibrosis patients, often leading to patient death. PA strains are multidrug resistant, thus making the design of new therapeutics a challenge for public health. One promising therapeutic option is to design glycoclusters that target the virulence factor of PA. LecA is a galactose‐specific lectin that might be involved in adhesion and biofilm formation by PA. The DNA‐directed immobilization (DDI) microarray is a powerful tool for screening and understanding of structure–activity relationships between glycoclusters and lectins. High‐throughput and multiplexed analysis of lectin–glycocluster interactions on a DDI microarray allows measurement of IC₅₀ and dissociation constant (K_d) values with minute amounts of material. In order to study the robustness of the DDI microarray in determination of IC₅₀ and K_d values, the impact of glycocluster surface density was investigated. The data obtained show that measured IC₅₀ values were influenced by glycocluster surface density: as the density of glycoclusters increases, the measured IC₅₀ values increase too. In contrast, the measured K_d values were not affected by glycocluster surface density, provided that the experimental conditions allow interaction between glycocluster and lectin at single‐molecule level (no surface cluster effect).     

Biophysical Features of Bacillithiol,the Glutathione Surrogate of Bacillus subtilis and other Firmicutes

Bacillithiol (BSH) is the major low‐molecular‐weight (LMW) thiol in many low‐G+C Gram‐positive bacteria (Firmicutes). Evidence now emerging suggests that BSH functions as an important LMW thiol in redox regulation and xenobiotic detoxification, analogous to what is already known for glutathione and mycothiol in other microorganisms. The biophysical properties and cellular concentrations of such LMW thiols are important determinants of their biochemical efficiency both as biochemical nucleophiles and as redox buffers. Here, BSH has been characterised and compared with other LMW thiols in terms of its thiol pK_a, redox potential and thiol–disulfide exchange reactivity. Both the thiol pK_a and the standard thiol redox potential of BSH are shown to be significantly lower than those of glutathione whereas the reactivities of the two compounds in thiol–disulfide reactions are comparable. The cellular concentration of BSH in Bacillus subtilis varied over different growth phases and reached up to 5 mM , which is significantly greater than previously observed from single measurements taken during mid‐exponential growth. These results demonstrate that the biophysical characteristics of BSH are distinctively different from those of GSH and that its cellular concentrations can reach levels much higher than previously reported.