Monitoring Binding of HIV‐1 Capsid Assembly Inhibitors Using 19F Ligand‐and 15N Protein‐Based NMR and X‐ray Crystallography: Early Hit Validation of a Benzodiazepine Series

The emergence of resistance to existing classes of antiretroviral drugs underlines the need to find novel human immunodeficiency virus (HIV)‐1 targets for drug discovery. The viral capsid protein (CA) represents one such potential target. Recently, a series of benzodiazepine inhibitors was identified via high‐throughput screening using an in vitro capsid assembly assay (CAA). Here, we demonstrate how a combination of NMR and X‐ray co‐crystallography allowed for the rapid characterization of the early hits from this inhibitor series. Ligand‐based ¹⁹F NMR was used to confirm inhibitor binding specificity and reversibility as well as to identify the N‐terminal domain of the capsid (CA_NTD) as its molecular target. Protein‐based NMR (¹H and ¹⁵N chemical shift perturbation analysis) identified key residues within the CA_NTD involved in inhibitor binding, while X‐ray co‐crystallography confirmed the inhibitor binding site and its binding mode. Based on these results, two conformationally restricted cyclic inhibitors were designed to further validate the possible binding modes. These studies were crucial to early hit confirmation and subsequent lead optimization.     

Fragmenting the S100B–p53 Interaction: Combined Virtual/Biophysical Screening Approaches to Identify Ligands

S100B contributes to cell proliferation by binding the C terminus of p53 and inhibiting its tumor suppressor function. The use of multiple computational approaches to screen fragment libraries targeting the human S100B–p53 interaction site is reported. This in silico screening led to the identification of 280 novel prospective ligands. NMR spectroscopic experiments revealed specific binding at the p53 interaction site for a set of these compounds and confirmed their potential for further rational optimization. The X‐ray crystal structure determined for one of the binders revealed key intermolecular interactions, thus paving the way for structure‐based ligand optimization.     

Microfocus small‐angle X‐ray scattering investigation of the skin–core microstructure of lyocell cellulose fibers

Skin–core microstructures in hydrated lyocell cellulose fibers were investigated using microfocus small‐angle X‐ray scattering (SAXS). Both single fibers and fiber cross sections were observed. In all the fibers studied, a thin skin layer was observed in which the voids were smaller in cross section and better oriented than those in the core. The division between skin and core may not be sharp. A draw ratio of 0.80 gave fibers with voids that were shorter, larger in cross section, and more misoriented than the fibers produced with a draw ratio of 1.68. In contrast, there was little difference in void structure between samples coagulated in water and in 25% amine oxide aqueous solution. The results are explained in the context of the spinodal decomposition, which is thought to occur during coagulation. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 83: 2799–2816, 2002; DOI 10.1002/app.10256     

Palladium N‐Heterocyclic Carbene Catalysts for the Ultrasound‐Promoted Suzuki–Miyaura Reaction in Glycerol

Novel palladium N‐heterocyclic‐carbene (NHC)‐based complexes with 3,4,5‐trimethoxybenzyl, alkyl and sulfonate N‐substituents were obtained and fully characterized. The new complexes were used as pre‐catalysts in the Suzuki–Miyaura coupling of various aryl halides/boron sources in glycerol under pulsed‐ultrasound (P‐US) activation. High yields were obtained under mild reaction conditions, without formation of undesired by‐products. The pure final cross‐coupling products were easily recovered without column chromatography and the catalytic/solvent system could be recycled. TEM (transmission electron microscopy) and XPS (X‐ray photoelectron spectroscopy) were used to characterize the nanoparticles and to investigate the fate of the catalysts.     

On the Metabolically Active Form of Metaglidasen: Improved Synthesis and Investigation of Its Peculiar Activity on Peroxisome Proliferator‐Activated Receptors and Skeletal Muscles

Metaglidasen is a fibrate‐like drug reported as a selective modulator of peroxisome proliferator‐activated receptor γ (PPARγ), able to lower plasma glucose levels in the absence of the side effects typically observed with thiazolidinedione antidiabetic agents in current use. Herein we report an improved synthesis of metaglidasen′s metabolically active form halofenic acid (R)‐ 2 and that of its enantiomer (S)‐ 2 . The activity of the two stereoisomers was carefully examined on PPARα and PPARγ subtypes. As expected, both showed partial agonist activity toward PPARγ; the investigation of PPARα activity, however, led to unexpected results. In particular, (S)‐ 2 was found to act as a partial agonist, whereas (R)‐ 2 behaved as an antagonist. X‐ray crystallographic studies with PPARγ were carried out to gain more insight on the molecular‐level interactions and to propose a binding mode. Given the adverse effects provoked by fibrate drugs on skeletal muscle function, we also investigated the capacity of (R)‐ 2 and (S)‐ 2 to block conductance of the skeletal muscle membrane chloride channel. The results showed a more beneficial profile for (R)‐ 2 , the activity of which on skeletal muscle function, however, should not be overlooked in the ongoing clinical trials studying its long‐term effects.     

Stereospecific Effects of Oxygen‐to‐Sulfur Substitution in DNA Phosphate on Ion Pair Dynamics and Protein–DNA Affinity

Oxygen‐to‐sulfur substitutions in DNA phosphate often enhance affinity for DNA‐binding proteins. Our previous studies have suggested that this effect of sulfur substitution of both O_P1 and O_P2 atoms is due to an entropic gain associated with enhanced ion pair dynamics. In this work, we studied stereospecific effects of single sulfur substitution of either the O_P1 or O_P2 atom in DNA phosphate at the Lys57 interaction site of the Antennapedia homeodomain–DNA complex. Using crystallography, we obtained structural information on the R_P and S_P diastereomers of the phosphoromonothioate and their interaction with Lys57. Using fluorescence‐based assays, we found significant affinity enhancement upon sulfur substitution of the O_P2 atom. Using NMR spectroscopy, we found significant mobilization of the Lys57 side‐chain NH₃⁺ group upon sulfur substitution of the O_P2 atom. These data provide further mechanistic insights into the affinity enhancement by oxygen‐to‐sulfur substitution in DNA phosphate.     

Inhibition of Leishmania infantum Trypanothione Reductase by Azole‐Based Compounds: a Comparative Analysis with Its Physiological Substrate by X‐ray Crystallography

Herein we report a study aimed at discovering a new class of compounds that are able to inhibit Leishmania donovani cell growth. Evaluation of an in‐house library of compounds in a whole‐cell screening assay highlighted 4‐((1‐(4‐ethylphenyl)‐2‐methyl‐5‐(4‐(methylthio)phenyl)‐1H‐pyrrol‐3‐yl)methyl)thiomorpholine (compound 1 ) as the most active. Enzymatic assays on Leishmania infantum trypanothione reductase (LiTR, belonging to the Leishmania donovani complex) shed light on both the interaction with, and the nature of inhibition by, compound 1 . A molecular modeling approach based on docking studies and on the estimation of the binding free energy aided our rationalization of the biological data. Moreover, X‐ray crystal structure determination of LiTR in complex with compound 1 confirmed all our results: compound 1 binds to the T(SH)₂ binding site, lined by hydrophobic residues such as Trp21 and Met113, as well as residues Glu18 and Tyr110. Analysis of the structure of LiTR in complex with trypanothione shows that Glu18 and Tyr110 are also involved in substrate binding, according to a competitive inhibition mechanism.     

The Tyrosine Gate of the Bacterial Lectin FimH: A Conformational Analysis by NMR Spectroscopy and X‐ray Crystallography

Urinary tract infections caused by uropathogenic E. coli are among the most prevalent infectious diseases. The mannose‐specific lectin FimH mediates the adhesion of the bacteria to the urothelium, thus enabling host cell invasion and recurrent infections. An attractive alternative to antibiotic treatment is the development of FimH antagonists that mimic the physiological ligand. A large variety of candidate drugs have been developed and characterized by means of in vitro studies and animal models. Here we present the X‐ray co‐crystal structures of FimH with members of four antagonist classes. In three of these cases no structural data had previously been available. We used NMR spectroscopy to characterize FimH–antagonist interactions further by chemical shift perturbation. The analysis allowed a clear determination of the conformation of the tyrosine gate motif that is crucial for the interaction with aglycone moieties and was not obvious from X‐ray structural data alone. Finally, ITC experiments provided insight into the thermodynamics of antagonist binding. In conjunction with the structural information from X‐ray and NMR experiments the results provide a mechanism for the often‐observed enthalpy–entropy compensation of FimH antagonists that plays a role in fine‐tuning of the interaction.     

Directed Divergent Evolution of a Thermostable D‐Tagatose Epimerase towards Improved Activity for Two Hexose Substrates

Functional promiscuity of enzymes can often be harnessed as the starting point for the directed evolution of novel biocatalysts. Here we describe the divergent morphing of an engineered thermostable variant (Var8) of a promiscuous D ‐tagatose epimerase (DTE) into two efficient catalysts for the C3 epimerization of D ‐fructose to D ‐psicose and of L ‐sorbose to L ‐tagatose. Iterative single‐site randomization and screening of 48 residues in the first and second shells around the substrate‐binding site of Var8 yielded the eight‐site mutant IDF8 (ninefold improved k_cat for the epimerization of D ‐fructose) and the six‐site mutant ILS6 (14‐fold improved epimerization of L ‐sorbose), compared to Var8. Structure analysis of IDF8 revealed a charged patch at the entrance of its active site; this presumably facilitates entry of the polar substrate. The improvement in catalytic activity of variant ILS6 is thought to relate to subtle changes in the hydration of the bound substrate. The structures can now be used to select additional sites for further directed evolution of the ketohexose epimerase.     

Assessing Carbohydrate–Carbohydrate Interactions by NMR Spectroscopy: The Trisaccharide Epitope from the Marine Sponge Microciona prolifera

Weak recognition processes : Weak calcium‐mediated carbohydrate–carbohydrate interactions have been detected by DOSY and TRNOESY NMR methods by employing a gold glyconanoparticle as a multivalent system. In addition, 3D models of trisaccharide‐Ca^II‐trisaccharide complexes based on results from molecular dynamics simulations are proposed.

     

Bicyclic Peptide Inhibitor of Urokinase‐Type Plasminogen Activator: Mode of Action

The development of protease inhibitors for pharmacological intervention has taken a new turn with the use of peptide‐based inhibitors. Here, we report the rational design of bicyclic peptide inhibitors of the serine protease urokinase‐type plasminogen activator (uPA), based on the established monocyclic peptide, upain‐2. It was successfully converted to a bicyclic peptide, without loss of inhibitory properties. The aim was to produce a peptide cyclised by an amide bond with an additional stabilising across‐the‐ring covalent bond. We expected this bicyclic peptide to exhibit a lower entropic burden upon binding. Two bicyclic peptides were synthesised with affinities similar to that of upain‐2, and their binding energetics were evaluated by isothermal titration calorimetry. Indeed, compared to upain‐2, the bicyclic peptides showed reduced loss of entropy upon binding to uPA. We also investigated the solution structures of the bicyclic peptide by NMR spectroscopy to map possible conformations. An X‐ray structure of the bicyclic‐peptide–uPA complex confirmed an interaction similar to that for the previous upain‐1/upain‐2–uPA complexes. These physical studies of the peptide–protease interactions will aid future designs of bicyclic peptide protease inhibitors.     

Biocatalysis with Thermostable Enzymes: Structure and Properties of a Thermophilic ‘ene’‐Reductase related to Old Yellow Enzyme

We report the crystal structure of a thermophilic “ene” reductase (TOYE) isolated from Thermoanaerobacter pseudethanolicus E39. The crystal structure reveals a tetrameric enzyme and an active site that is relatively large compared to most other structurally determined and related Old Yellow Enzymes. The enzyme adopts higher order oligomeric states (octamers and dodecamers) in solution, as revealed by sedimentation velocity and multiangle laser light scattering. Bead modelling indicates that the solution structure is consistent with the basic tetrameric structure observed in crystallographic studies and electron microscopy. TOYE is stable at high temperatures (T_m>70 °C) and shows increased resistance to denaturation in water‐miscible organic solvents compared to the mesophilic Old Yellow Enzyme family member, pentaerythritol tetranitrate reductase. TOYE has typical ene‐reductase properties of the Old Yellow Enzyme family. There is currently major interest in using Old Yellow Enzyme family members in the preparative biocatalysis of a number of activated alkenes. The increased stability of TOYE in organic solvents is advantageous for biotransformations in which water‐miscible organic solvents and biphasic reaction conditions are required to both deliver novel substrates and minimize product racemisation.     

A Murine Monoclonal Antibody to Glycogen: Characterization of Epitope‐Fine Specificity by Saturation Transfer Difference (STD) NMR Spectroscopy and Its Use in Mycobacterial Capsular α‐Glucan Research

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a major pathogen responsible for 1.5 million deaths annually. This bacterium is characterized by a highly unusual and impermeable cell envelope, which plays a key role in mycobacterial survival and virulence. Although many studies have focused on the composition and functioning of the mycobacterial cell envelope, the capsular α‐glucan has received relatively minor attention. Here we show that a murine monoclonal antibody (Mab) directed against glycogen cross‐reacts with mycobacterial α‐glucans, polymers of α(1–4)‐linked glucose residues with α(1–6)‐branch points. We identified the Mab epitope specificity by saturation transfer difference NMR and show that the α(1–4)‐linked glucose residues are important in glucan–Mab interaction. The minimal epitope is formed by (linear) maltotriose. Notably, a Mycobacterium mutant lacking the branching enzyme GlgB does not react with the Mab; this suggests that the α(1–6)‐branches form part of the epitope. These seemingly conflicting data can be explained by the fact that in the mutant the linear form of the α‐glucan (amylose) is insoluble. This Mab was subsequently used to develop several techniques helpful in capsular α‐glucan research. By using a capsular glucan‐screening methodology based on this Mab we were able to identify several unknown genes involved in capsular α‐glucan biogenesis. Additionally, we developed two methods for the detection of capsular α‐glucan levels. This study therefore opens new ways to study capsular α‐glucan and to identify possible targets for further research.     

Identification of Low‐Molecular‐Weight Compounds Inhibiting Growth of Corynebacteria: Potential Lead Compounds for Antibiotics

The bacterial genus Corynebacteria contains several pathogenic species that cause diseases such as diphtheria in humans and “cheesy gland” in goats and sheep. Thus, identifying new therapeutic targets to treat Corynebacteria infections is both medically and economically important. CG2496, a functionally uncharacterized protein from Corynebacterium glutamicum, was evaluated using an NMR ligand‐affinity screen. A total of 11 compounds from a library of 460 biologically active compounds were shown to selectively bind CG2496 in a highly conserved region of the protein. The best binder was identified to be methiothepin (K_D=54±19 µM ), an FDA‐approved serotonin receptor antagonist. Methiothepin was also shown to inhibit the growth of C. glutamicum, but not bacteria that lack CG2496 homologs. Our results suggest that CG2496 is a novel therapeutic target and methiothepin is a potential lead compound or structural scaffold for developing new antibiotics specifically targeting Corynebacteria.