Bernard Gsell

Systematic Investigation of Halogen Bonding in Protein-Ligand Interactions.

Halogen bonding (XB) refers to the noncovalent interaction of general structure DX···A between halogen-bearing compounds (DX: XB donor, where X=Cl, Br, I) and nucleophiles (A: XB acceptor). Since the first observation in cocrystal structures of 1,4-dioxane and Br2 by Hassel and Hvoslef in 1954, XB has been widely used in crystal engineering and solid-state supramolecular chemistry. The nature of the interaction and the underlying electronic prerequisite, the s hole in the XB donor, have been the subject of extensive theoretical studies. 7–9] Most recently, the attractive nature of XB between 1-iodoperfluoroalkanes and various donors has also been demonstrated and quantified in solution studies. Novel inhibitors of human Cathepsin L (hCatL) were discovered which bind covalently to the side chain of the catalytic Cys25 residue in the S1 pocket under formation of thioimidates, which are stabilized by the oxyanion hole of the protease. These ligands form hydrogen bonds to the backbone NH and C=O groups of Gly68 and Asp162, respectively, and fill the S2 and S3 pockets, thereby interacting with the enzyme through multiple lipophilic contacts. During the course of this research, we obtained an indication of an XB contact between a 4-chlorophenyl moiety of a ligand, whose binding affinity was enhanced by a factor of 13 compared to the unsubstituted phenyl derivative, and the backbone C=O group of Gly61 in the S3 pocket (Figure 1). This finding stimulated the prepa-

Halogen Bonding at the Active Sites of Human Cathepsin L and MEK1 Kinase: Efficient Interactions in Different Environments

In two series of small‐molecule ligands, one inhibiting human cathepsin L (hcatL) and the other MEK1 kinase, biological affinities were found to strongly increase when an aryl ring of the inhibitors is substituted with the larger halogens Cl, Br, and I, but to decrease upon F substitution. X‐ray co‐crystal structure analyses revealed that the higher halides engage in halogen bonding (XB) with a backbone CO in the S3 pocket of hcatL and in a back pocket of MEK1. While the S3 pocket is located at the surface of the enzyme, which provides a polar environment, the back pocket in MEK1 is deeply buried in the protein and is of pronounced apolar character. This study analyzes environmental effects on XB in protein–ligand complexes. It is hypothesized that energetic gains by XB are predominantly not due to water replacements but originate from direct interactions between the XB donor (CarylX) and the XB acceptor (CO) in the correct geometry. New X‐ray co‐crystal structures in the same crystal form (space group P212121) were obtained for aryl chloride, bromide, and iodide ligands bound to hcatL. These high‐resolution structures reveal that the backbone CO group of Gly61 in most hcatL co‐crystal structures maintains water solvation while engaging in XB. An arylCF3‐substituted ligand of hcatL with an unexpectedly high affinity was found to adopt the same binding geometry as the aryl halides, with the CF3 group pointing to the CO group of Gly61 in the S3 pocket. In this case, a repulsive F2CF⋅⋅⋅OC contact apparently is energetically overcompensated by other favorable protein–ligand contacts established by the CF3 group.

Structure of the Human Fatty Acid Synthase KS–MAT Didomain as a Framework for Inhibitor Design

The human fatty acid synthase (FAS) is a key enzyme in the metabolism of fatty acids and a target for antineoplastic and antiobesity drug development. Due to its size and flexibility, structural studies of mammalian FAS have been limited to individual domains or intermediate-resolution studies of the complete porcine FAS. We describe the high-resolution crystal structure of a large part of human FAS that encompasses the tandem domain of beta-ketoacyl synthase (KS) connected by a linker domain to the malonyltransferase (MAT) domain. Hinge regions that allow for substantial flexibility of the subdomains are defined. The KS domain forms the canonical dimer, and its substrate-binding site geometry differs markedly from that of bacterial homologues but is similar to that of the porcine orthologue. The didomain structure reveals a possible way to generate a small and compact KS domain by omitting a large part of the linker and MAT domains, which could greatly aid in rapid screening of KS inhibitors. In the crystal, the MAT domain exhibits two closed conformations that differ significantly by rigid-body plasticity. This flexibility may be important for catalysis and extends the conformational space previously known for type I FAS and 6-deoxyerythronolide B synthase.

Mapping the conformational space accessible to BACE2 using surface mutants and cocrystals with Fab fragments, Fynomers and Xaperones

The aspartic protease BACE2 is responsible for the shedding of the transmembrane protein Tmem27 from the surface of pancreatic β-cells, which leads to inactivation of the β-cell proliferating activity of Tmem27. This role of BACE2 in the control of β-cell maintenance suggests BACE2 as a drug target for diabetes. Inhibition of BACE2 has recently been shown to lead to improved control of glucose homeostasis and to increased insulin levels in insulin-resistant mice. BACE2 has 52% sequence identity to the well studied Alzheimers disease target enzyme β-secretase (BACE1). High-resolution BACE2 structures would contribute significantly to the investigation of this enzyme as either a drug target or anti-target. Surface mutagenesis, BACE2-binding antibody Fab fragments, single-domain camelid antibody VHH fragments (Xaperones) and Fyn-kinase-derived SH3 domains (Fynomers) were used as crystallization helpers to obtain the first high-resolution structures of BACE2. Eight crystal structures in six different packing environments define an ensemble of low-energy conformations available to the enzyme. Here, the different strategies used for raising and selecting BACE2 binders for cocrystallization are described and the crystallization success, crystal quality and the time and resources needed to obtain suitable crystals are compared.

Crystal engineering: deletion mutagenesis of the 24 kDa fragment of the DNA gyrase B subunit from Staphylococcus aureus

The 24 kDa fragment of DNA gyrase B from Staphylococcus aureus was expressed in Escherichia coli and purified for crystallization. Crystals of the wild-type protein grew in the presence of cyclothialidine but proved difficult to reproduce. In order to improve the crystallization, the flexible regions of the protein were deleted by mutagenesis. The mutant proteins were analyzed by differential scanning calorimetry and the most stable mutants produced crystals. It was possible to reproducibly grow single well defined crystals in the microbatch system which belonged to the space group C2 and diffracted isotropically to approximately 2 A resolution.

Structural basis for the cyclophilin A binding affinity and immunosuppressive potency of E-ISA247 (voclosporin)

X-ray crystal structures of the cyclosporin A analogue E-ISA247 (voclosporin) and its stereoisomer Z-ISA247 bound to cyclophilin A suggest the molecular basis for the differences in their binding affinities and immunosuppressive efficacies.

Identification of Potent and Selective Cathepsin S Inhibitors Containing Different Central Cyclic Scaffolds

Starting from the weakly active dual CatS/K inhibitor 5, structure-based design supported by X-ray analysis led to the discovery of the potent and selective (>50,000-fold vs CatK) cyclopentane derivative 22 by exploiting specific ligand-receptor interactions in the S2 pocket of CatS. Changing the central cyclopentane scaffold to the analogous pyrrolidine derivative 57 decreased the enzyme as well as the cell-based activity significantly by 24- and 69-fold, respectively. The most promising scaffold identified was the readily accessible proline derivative (e.g., 79). This compound, with an appealing ligand efficiency (LE) of 0.47, included additional structural modifications binding in the S1 and S3 pockets of CatS, leading to favorable in vitro and in vivo properties. Compound 79 reduced IL-2 production in a transgenic DO10.11 mouse model of antigen presentation in a dose-dependent manner with an ED50 of 5 mg/kg.

HMQC and HSQC experiments with water flip-back optimized for large proteins

An HMQC experiment is proposed, dubbed FHMQC, where water flip-back is achieved by a single water-selective pulse preceding the basic HMQC pulse sequence. The scheme is demonstrated with a 15N, 1H-HMQC spectrum of uniformly 15N/2H-labelled S. aureus DNA gyrase B with a molecular weight of 45 kDa for the unlabelled protein. The sensitivity of the experiment is improved compared to that of an FHSQC spectrum. It is further shown that the original FHSQC experiment can be shortened by the use of bipolar gradients. Relaxation times of different 15N magnetizations and coherences were measured. The new FHMQC scheme is implemented in 3D NOESY-15N-HMQC and 3D15 N-HMQC-NOESY-15N-HMQC pulse sequences which are demonstrated with a 24 kDa fragment of uniformly 15N/13C/2H-labelled S. aureus DNA gyrase B.

Structure of the malaria vaccine candidate antigen CyRPA and its complex with a parasite invasion inhibitory antibody

Invasion of erythrocytes by Plasmodial merozoites is a composite process involving the interplay of several proteins. Among them, the Plasmodium falciparum Cysteine-Rich Protective Antigen (PfCyRPA) is a crucial component of a ternary complex, including Reticulocyte binding-like Homologous protein 5 (PfRH5) and the RH5-interacting protein (PfRipr), essential for erythrocyte invasion. Here, we present the crystal structures of PfCyRPA and its complex with the antigen-binding fragment of a parasite growth inhibitory antibody. PfCyRPA adopts a 6-bladed β-propeller structure with similarity to the classic sialidase fold, but it has no sialidase activity and fulfills a purely non-enzymatic function. Characterization of the epitope recognized by protective antibodies may facilitate design of peptidomimetics to focus vaccine responses on protective epitopes. Both in vitro and in vivo anti-PfCyRPA and anti-PfRH5 antibodies showed more potent parasite growth inhibitory activity in combination than on their own, supporting a combined delivery of PfCyRPA and PfRH5 in vaccines. DOI: http://dx.doi.org/10.7554/eLife.20383.001

Generation of an antibody toolbox to characterize hERG

The human ether-a-go-go related gene (hERG) potassium channel plays a major role in the repolarization of the cardiac action potential. Inhibition of the hERG function by mutations or a wide variety of pharmaceutical compounds cause long QT syndrome and lead to potentially lethal arrhythmias. For detailed insights into the structural and biochemical background of hERG function and drug binding, the purification of recombinant protein is essential. Because the hERG channel is a challenging protein to purify, fast and easy techniques to evaluate different expression, solubilization and purification conditions are of primary importance. Here, we describe the generation of a set of 12 monoclonal antibodies against hERG. Beside their suitability in western blot, immunoprecipitation and immunostaining, these antibodies were used to establish a sandwich ELISA for the detection and relative quantification of hERG in different expression systems. Furthermore, a Fab fragment was used in fluorescence size exclusion chromatography to determine the oligomeric state of hERG after solubilization. These new tools can be used for a fast and efficient screening of expression, solubilization and purification conditions.