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The endocannabinoid system (ECS) is a critical regulatory network composed of endogenous cannabinoids (eCBs), their synthesizing and degrading enzymes, and associated receptors. It is integral to maintaining homeostasis and orchestrating key functions within the central nervous and immune systems. Given its therapeutic significance, we have launched a series of drug discovery endeavors aimed at ECS targets, including peroxisome proliferator-activated receptors (PPARs), cannabinoid receptors types 1 (CB1R) and 2 (CB2R), and monoacylglycerol lipase (MAGL), addressing a wide array of medical needs. The pursuit of new therapeutic agents has been enhanced by the creation of specialized labeled chemical probes, which aid in target localization, mechanistic studies, assay development, and the establishment of biomarkers for target engagement. By fusing medicinal chemistry with chemical biology in a comprehensive, translational end-to-end drug discovery strategy, we have expedited the development of novel therapeutics. Additionally, this strategy promises to foster highly productive partnerships between industry and academia, as will be illustrated through various examples., (Copyright 2024 Johannes Aebi, Kenneth Atz, Simon M. Ametamey, Jörg Benz, Julie Blaising, Stefania Butini, Giuseppe Campiani, Erick M. Carreira, Ludovic Collin, Eva de Lago, Thais Gazzi, Jürg Gertsch, Luca Gobbi, Wolfgang Guba, Javier Fernández-Ruiz, Jürgen Fingerle, Ahmed Haider, Yingfang He, Laura H. Heitman, Michael Honer, Daniel Hunziker, Bernd Kuhn, Mauro Maccarrone, Hans Peter Märki, Rainer E. Martin, Peter Mohr, Linjing Mu, Marc Nazaré, David F. Nippa, Sergio Oddi, Fionn O’Hara, Pal Pacher, Julian Romero, Stephan Röver, Arne C. Rufer, Roger Schibli, Gisbert Schneider, Antonia F. Stepan, David A. Sykes, Christoph Ullmer, Mario van der Stelt, Dmitry B. Veprintsev, Matthias B. Wittwer, Uwe Grether. License: This work is licensed under a Creative Commons Attribution 4.0 International License.)

The pretargeting approach separates the biological half-life of an antibody from the physical half-life of the radioisotope label, providing a strategy for reducing the radiation burden. A widely explored pretargeting approach makes use of the bioorthogonal click reaction between tetrazines (Tzs) and trans -cyclooctenes (TCOs), combining the targeting specificity of monoclonal antibodies (mAbs) with the rapid clearance and precise reaction of Tzs and TCOs. Such a strategy can allow for the targeting and imaging (e.g., by positron emission tomography (PET)) of molecular markers, which cannot be addressed by solely relying on small molecules. Tz derivatives that undergo inverse electron-demand Diels-Alder (IEDDA) reactions with an antibody bearing TCO moieties have been investigated. This study describes the synthesis and characterization of 11 cold Tz imaging agent candidates. These molecules have the potential to be radiolabeled with 18 F or 3 H, and with the former label, they could be of use as imaging tracers for positron emission tomography studies. Selection was made using a multiparameter optimization score for the central nervous system (CNS) PET tracers. Novel tetrazines were tested for their pH-dependent chemical stability. Those which turned out to be stable in a pH range of 6.5-8 were further characterized in in vitro assays with regard to their passive permeability, microsomal stability, and P-glycoprotein transport. Furthermore, selected Tzs were examined for their systemic clearance and CNS penetration in a single-dose pharmacokinetic study in rats. Two tetrazines were successfully labeled with 18 F, one of which showed brain penetration in a biodistribution study in mice. Another Tz was successfully tritium-labeled and used to demonstrate a bioorthogonal click reaction on a TCO-modified antibody. As a result, we identified one Tz as a potential fluorine-18-labeled CNS-PET agent and a second as a 3 H-radioligand for an IEDDA-based reaction with a modified brain-penetrating antibody.

Introduction: The cannabinoid receptor (CBR) subtypes 1 (CB 1 R) and 2 (CB 2 R) are key components of the endocannabinoid system (ECS), playing a central role in the control of peripheral pain, inflammation and the immune response, with further roles in the endocrine regulation of food intake and energy balance. So far, few medicines targeting these receptors have reached the clinic, suggesting that a better understanding of the receptor signalling properties of existing tool compounds and clinical candidates may open the door to the development of more effective and safer treatments. Both CB 1 R and CB 2 R are Gα i protein-coupled receptors but detecting Gα i protein signalling activity reliably and reproducibly is challenging. This is due to the inherent variability in live cell-based assays and restrictions around the use of radioactive [ 35 S]-GTPγS, a favoured technology for developing higher-throughput membrane-based Gα i protein activity assays. Methods: Here, we describe the development of a membrane-based Gα i signalling system, produced from membrane preparations of HEK293TR cells, stably overexpressing CB 1 R or CB 2 R, and components of the Gα i -CASE biosensor. This BRET-based system allows direct detection of Gα i signalling in both cells and membranes by monitoring bioluminescence resonance energy transfer (BRET) between the α and the βγ subunits. Cells and membranes were subject to increasing concentrations of reference cannabinoid compounds, with 10 μM furimazine added to generate RET signals, which were detected on a PHERAstar FSX plate reader, then processed using MARS software and analysed in GraphPad PRISM 9.2. Results: In membranes expressing the Gi-CASE biosensor, the cannabinoid ligands profiled were found to show agonist and inverse agonist activity. Agonist activity elicited a decrease in the BRET signal, indicative of receptor activation and G protein dissociation. Inverse agonist activity caused an increase in BRET signal, indicative of receptor inactivation, and the accumulation of inactive G protein. Our membrane-based Gi-CASE NanoBRET system successfully characterised the potency (pEC 50 ) and efficacy (E max ) of CBR agonists and inverse agonists in a 384-well screening format. Values obtained were in-line with whole-cell Gi-CASE assays and consistent with literature values obtained in the GTPγS screening format. Discussion: This novel, membrane-based Gα i protein activation assay is applicable to other Gα i -coupled GPCRs, including orphan receptors, allowing real-time higher-throughput measurements of receptor activation., Competing Interests: Authors WG, AR, and UG was/were employed by F Hoffmann-La Roche Ltd. Authors DV and DS was/were employed by Z7 Biotech Limited. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Scott-Dennis, Rafani, Yi, Perera, Harwood, Guba, Rufer, Grether, Veprintsev and Sykes.)

Ground-breaking research in disease biology and continuous efforts in method development have uncovered a range of potential new drug targets. Increasingly, the drug discovery process is informed by technologies involving chemical probes as tools. Applications for chemical probes comprise target identification and assessment, as well as the qualification of small molecules as chemical starting points and drug candidates. Progress in probe chemistry has opened the way to novel assay formats and pharmaceutical compound classes. The European Federation of Medicinal Chemistry and Chemical Biology (EFMC) has launched the Chemical Biology Initiative to advance science in the field of medicinal chemistry and chemical biology, while representing all members of this extended scientific community. This review provides an overview of the many important developments in the field of chemical biology that have happened at the lively interface of academic and industrial research., (© 2023 Wiley-VCH GmbH.)

Despite its essential role in the (patho)physiology of several diseases, CB 2 R tissue expression profiles and signaling mechanisms are not yet fully understood. We report the development of a highly potent, fluorescent CB 2 R agonist probe employing structure-based reverse design. It commences with a highly potent, preclinically validated ligand, which is conjugated to a silicon-rhodamine fluorophore, enabling cell permeability. The probe is the first to preserve interspecies affinity and selectivity for both mouse and human CB 2 R. Extensive cross-validation (FACS, TR-FRET and confocal microscopy) set the stage for CB 2 R detection in endogenously expressing living cells along with zebrafish larvae. Together, these findings will benefit clinical translatability of CB 2 R based drugs., Competing Interests: The authors have no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Enzymes are essential, physiological catalysts involved in all processes of life, including metabolism, cellular signaling and motility, as well as cell growth and division. They are attractive drug targets because of the presence of defined substrate-binding pockets, which can be exploited as binding sites for pharmaceutical enzyme inhibitors. Understanding the reaction mechanisms of enzymes and the molecular mode of action of enzyme inhibitors is indispensable for the discovery and development of potent, efficacious, and safe novel drugs. The combination of classical concepts of enzymology with new experimental and data analysis methods opens new routes for drug discovery., (Copyright © 2021 The Author. Published by Elsevier Ltd.. All rights reserved.)

Genetic, preclinical and clinical data link Parkinson's disease and Gaucher's disease and provide a rational entry point to disease modification therapy via enhancement of β-Glucocerebrosidase (GCase) activity. We discovered a new class of pyrrolo[2,3-b]pyrazine activators effecting both Vmax and Km. They bind to human GCase and increase substrate metabolism in the lysosome in a cellular assay. We obtained the first crystal structure for an activator and identified a novel non-inhibitory binding mode at the interface of a dimer, rationalizing the observed structure-activity relationship (SAR). The compound binds GCase inducing formation of a dimeric state at both endoplasmic reticulum (ER) and lysosomal pHs, as confirmed by analytical ultracentrifugation. Importantly, the pyrrolo[2,3-b]pyrazines have central nervous system (CNS) drug-like properties. Our findings are important for future drug discovery efforts in the field of GCase activation and provide a deeper mechanistic understanding of the requirements for enzymatic activation, pointing to the relevance of dimerization., (© 2020 Wiley-VCH GmbH.)

Pharmacological modulation of cannabinoid type 2 receptor (CB 2 R) holds promise for the treatment of numerous conditions, including inflammatory diseases, autoimmune disorders, pain, and cancer. Despite the significance of this receptor, researchers lack reliable tools to address questions concerning the expression and complex mechanism of CB 2 R signaling, especially in cell-type and tissue-dependent contexts. Herein, we report for the first time a versatile ligand platform for the modular design of a collection of highly specific CB 2 R fluorescent probes, used successfully across applications, species, and cell types. These include flow cytometry of endogenously expressing cells, real-time confocal microscopy of mouse splenocytes and human macrophages, as well as FRET-based kinetic and equilibrium binding assays. High CB 2 R specificity was demonstrated by competition experiments in living cells expressing CB 2 R at native levels. The probes were effectively applied to FACS analysis of microglial cells derived from a mouse model relevant to Alzheimer's disease.

Activation of the T cell receptor (TCR) results in binding of the adapter protein Nck (noncatalytic region of tyrosine kinase) to the CD3ϵ subunit of the TCR. The interaction was suggested to be important for the amplification of TCR signals and is governed by a proline-rich sequence (PRS) in CD3ϵ that binds to the first Src homology 3 (SH3) domain of Nck (Nck-SH3.1). Inhibition of this protein/protein interaction ameliorated inflammatory symptoms in mouse models of multiple sclerosis, psoriasis, and asthma. A small molecule, AX-024, was reported to inhibit the Nck/CD3ϵ interaction by physically binding to the Nck1-SH3.1 domain, suggesting a route to develop an inhibitor of the Nck1/CD3ϵ interaction for modulating TCR activity in autoimmune and inflammatory diseases. We show here that AX-024 reduces T cell proliferation upon weak TCR stimulation but does not significantly affect phosphorylation of Zap70 (ζ chain of T cell receptor-associated protein kinase 70). We also find that AX-024 is likely not involved in modulating the Nck/TCR interaction but probably has other targets in T cells. An array of biophysical techniques did not detect a direct interaction between AX-024 and Nck-SH3.1 in vitro Crystal structures of the Nck-SH3.1 domain revealed its binding mode to the PRS in CD3ϵ. The SH3 domain tends to generate homodimers through a domain swap. Domain swaps observed previously in other SH3 domains indicate a general propensity of this protein fold to exchange structural elements. The swapped form of Nck-SH3.1 is unable to bind CD3ϵ, possibly representing an inactive form of Nck in cells., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Richter et al.)

Purpose: Immunogenicity against biotherapeutics can lead to the formation of drug/anti-drug-antibody (ADA) immune complexes (ICs) with potential impact on safety and drug pharmacokinetics (PK). This work aimed to generate defined drug/ADA ICs, characterized by quantitative (bio) analytical methods for dedicated determination of IC sizes and IC profile changes in serum to facilitate future in vivo studies., Methods: Defined ICs were generated and extensively characterized with chromatographic, biophysical and imaging methods. Quantification of drug fully complexed with ADAs (drug in ICs) was performed with an acid dissociation ELISA. Sequential coupling of SEC and ELISA enabled the reconstruction of IC patterns and thus analysis of IC species in serum., Results: Characterization of generated ICs identified cyclic dimers, tetramers, hexamers, and larger ICs of drug and ADA as main IC species. The developed acid dissociation ELISA enabled a total quantification of drug fully complexed with ADAs. Multiplexing of SEC and ELISA allowed unbiased reconstruction of IC oligomeric states in serum., Conclusions: The developed in vitro IC model system has been properly characterized by biophysical and bioanalytical methods. The specificity of the developed methods enable discrimination between different oligomeric states of ICs and can be bench marking for future in vivo studies with ICs.

Doublecortin, a microtubule-associated protein that is only produced during neurogenesis, cooperatively binds to microtubules and stimulates microtubule polymerization and cross-linking by unknown mechanisms. A domain swap is observed in the crystal structure of the C-terminal domain of doublecortin. As determined by analytical ultracentrifugation, an open conformation is also present in solution. At higher concentrations, higher-order oligomers of the domain are formed. The domain swap and additional interfaces observed in the crystal lattice can explain the formation of doublecortin tetramers or multimers, in line with the analytical ultracentrifugation data. Taken together, the domain swap offers a mechanism for the observed cooperative binding of doublecortin to microtubules. Doublecortin-induced cross-linking of microtubules can be explained by the same mechanism. The effect of several mutations leading to lissencephaly and double-cortex syndrome can be traced to the domain swap and the proposed self-association of doublecortin.

The endocannabinoid system, and in particular the cannabinoid type 2 receptor (CB2R), raised the interest of many medicinal chemistry programs for its therapeutic relevance in several (patho)physiologic processes. However, the physico-chemical properties of tool compounds for CB2R (e.g., the radioligand [ 3 H]CP55,940) are not optimal, despite the research efforts in developing effective drugs to target this system. At the same time, the importance of drug-target binding kinetics is growing since the kinetic binding profile of a ligand may provide important insights for the resulting in vivo efficacy. In this context we synthesized and characterized [ 3 H]RO6957022, a highly selective CB2R inverse agonist, as a radiolabeled tool compound. In equilibrium and kinetic binding experiments [ 3 H]RO6957022 showed high affinity for human CB2R with fast association ( k on ) and moderate dissociation ( k off ) kinetics. To demonstrate the robustness of [ 3 H]RO6957022 binding, affinity studies were carried out for a wide range of CB2R reference ligands, spanning the range of full, partial, and inverse agonists. Finally, we used [ 3 H]RO6957022 to study the kinetic binding profiles (i.e., k on and k off values) of selected synthetic and endogenous (i.e., 2-arachidonoylglycerol, anandamide, and noladin ether) CB2R ligands by competition association experiments. All tested ligands, and in particular the endocannabinoids, displayed distinct kinetic profiles, shedding more light on their mechanism of action and the importance of association rates in the determination of CB2R affinity. Altogether, this study shows that the use of a novel tool compound, i.e., [ 3 H]RO6957022, can support the development of novel ligands with a repertoire of kinetic binding profiles for CB2R., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

Therapeutic approaches to fight Alzheimer's disease include anti-Amyloidβ (Aβ) antibodies and secretase inhibitors. However, the blood-brain barrier (BBB) limits the brain exposure of biologics and the chemical space for small molecules to be BBB permeable. The Brain Shuttle (BS) technology is capable of shuttling large molecules into the brain. This allows for new types of therapeutic modalities engineered for optimal efficacy on the molecular target in the brain independent of brain penetrating properties. To this end, we designed BACE1 peptide inhibitors with varying lipid modifications with single-digit picomolar cellular potency. Secondly, we generated active-exosite peptides with structurally confirmed dual binding mode and improved potency. When fused to the BS via sortase coupling, these BACE1 inhibitors significantly reduced brain Aβ levels in mice after intravenous administration. In plasma, both BS and non-BS BACE1 inhibitor peptides induced a significant time- and dose-dependent decrease of Aβ. Our results demonstrate that the BS is essential for BACE1 peptide inhibitors to be efficacious in the brain and active-exosite design of BACE1 peptide inhibitors together with lipid modification may be of therapeutic relevance., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Cathepsin(Cat)-S processing of the invariant chain-MHC-II complex inside antigen presenting cells is a central pathomechanism of autoimmune-diseases. Additionally, Cat-S is released by activated-myeloid cells and was recently described to activate protease-activated-receptor-(PAR)-2 in extracellular compartments. We hypothesized that Cat-S blockade targets both mechanisms and elicits synergistic therapeutic effects on autoimmune tissue injury. MRL-(Fas)lpr mice with spontaneous autoimmune tissue injury were treated with different doses of Cat-S inhibitor RO5459072, mycophenolate mofetil or vehicle. Further, female MRL-(Fas)lpr mice were injected with recombinant Cat-S with/without concomitant Cat-S or PAR-2 blockade. Cat-S blockade dose-dependently reversed aberrant systemic autoimmunity, e.g. plasma cytokines, activation of myeloid cells and hypergammaglobulinemia. Especially IgG autoantibody production was suppressed. Of note (MHC-II-independent) IgM were unaffected by Cat-S blockade while they were suppressed by MMF. Cat-S blockade dose-dependently suppressed immune-complex glomerulonephritis together with a profound and early effect on proteinuria, which was not shared by MMF. In fact, intravenous Cat-S injection induced severe glomerular endothelial injury and albuminuria, which was entirely prevented by Cat-S or PAR-2 blockade. In-vitro studies confirm that Cat-S induces endothelial activation and injury via PAR-2. Therapeutic Cat-S blockade suppresses systemic and peripheral pathomechanisms of autoimmune tissue injury, hence, Cat-S is a promising therapeutic target in lupus nephritis.

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.

Doublecortin is a microtubule-associated protein produced during neurogenesis. The protein stabilizes microtubules and stimulates their polymerization, which allows migration of immature neurons to their designated location in the brain. Mutations in the gene that impair doublecortin function and cause severe brain formation disorders are located on a tandem repeat of two doublecortin domains. The molecular mechanism of action of doublecortin is only incompletely understood. Anti-doublecortin antibodies, such as the rabbit polyclonal Abcam 18732, are widely used as neurogenesis markers. Here, we report the generation and characterization of antibodies that bind to single doublecortin domains. The antibodies were used as tools to obtain structures of both domains. Four independent crystal structures of the N-terminal domain reveal several distinct open and closed conformations of the peptide linking N- and C-terminal domains, which can be related to doublecortin function. An NMR assignment and a crystal structure in complex with a camelid antibody fragment show that the doublecortin C-terminal domain adopts the same well defined ubiquitin-like fold as the N-terminal domain, despite its reported aggregation and molten globule-like properties. The antibodies' unique domain specificity also renders them ideal research tools to better understand the role of individual domains in doublecortin function. A single chain camelid antibody fragment specific for the C-terminal doublecortin domain affected microtubule binding, whereas a monoclonal mouse antibody specific for the N-terminal domain did not. Together with steric considerations, this suggests that the microtubule-interacting doublecortin domain observed in cryo-electron micrographs is the C-terminal domain rather than the N-terminal one., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The formation of undesired high molecular weight species such as dimers is an important quality attribute for therapeutic monoclonal antibody formulations. Therefore, the thorough understanding of mAb dimerization and the detailed characterization mAb dimers is of great interest for future pharmaceutical development of therapeutic antibodies. In this work, we focused on the analyses of different mAb dimers regarding size, surface properties, chemical identity, overall structure and localization of possible dimerization sites. Dimer fractions of different mAbs were isolated to a satisfactory purity from bulk material and revealed 2 predominant overall structures, namely elongated and compact dimer forms. The elongated dimers displayed one dimerization site involving the tip of the Fab domain. Depending on the stress applied, these elongated dimers are connected either covalently or non-covalently. In contrast, the compact dimers exhibited non-covalent association. Several interaction points were detected for the compact dimers involving the hinge region or the base of the Fab domain. These results indicate that mAb dimer fractions are rather complex and may contain more than one kind of dimer. Nevertheless, the overall appearance of mAb dimers suggests the existence of 2 predominant dimeric structures, elongated and compact, which are commonly present in preparations of therapeutic mAbs.

The cholesteryl ester transfer protein (CETP) enables the transfer of cholesteryl ester (CE) from high-density lipoproteins (HDL) to low-density lipoproteins (LDL) in the plasma compartment. CETP inhibition raises plasma levels of HDL cholesterol; a ternary tunnel complex with CETP bridging HDL and LDL was suggested as a mechanism. Here, we test whether the inhibition of CETP tunnel complex formation is a promising approach to suppress CE transfer from HDL to LDL, for potential treatment of cardio-vascular disease (CVD). Three monoclonal antibodies against different epitopes of CETP are assayed for their potential to interfere with CE transfer between HDL and/or LDL. Surprisingly, antibodies that target the tips of the elongated CETP molecule, interaction sites sterically required to form the suggested transfer complexes, do not interfere with CETP activity, but an antibody binding to the central region does. We show that CETP interacts with HDL, but not with LDL. Our findings demonstrate that a ternary tunnel complex is not the mechanistic prerequisite to transfer CE among lipoproteins., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.

Membrane proteins (MPs) are prevalent drug discovery targets involved in many cell processes. Despite their high potential as drug targets, the study of MPs has been hindered by limitations in expression, purification and stabilization in order to acquire thermodynamic and kinetic parameters of small molecules binding. These bottlenecks are grounded on the mandatory use of detergents to isolate and extract MPs from the cell plasma membrane and the coexistence of multiple conformations, which reflects biochemical versatility and intrinsic instability of MPs. In this work ,we set out to define a new strategy to enable surface plasmon resonance (SPR) measurements on a thermostabilized and truncated version of the human adenosine (A2A) G-protein-coupled receptor (GPCR) inserted in a lipid bilayer nanodisc in a label- and detergent-free manner by using a combination of affinity tags and GFP-based fluorescence techniques. We were able to detect and characterize small molecules binding kinetics on a GPCR fully embedded in a lipid environment. By providing a comparison between different binding assays in membranes, nanodiscs and detergent micelles, we show that nanodiscs can be used for small molecule binding studies by SPR to enhance the MP stability and to trigger a more native-like behaviour when compared to kinetics on A2A receptors isolated in detergent. This work provides thus a new methodology in drug discovery to characterize the binding kinetics of small molecule ligands for MPs targets in a lipid environment., (Copyright © 2015. Published by Elsevier B.V.)

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 Alzheimer's 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.

Carnitine palmitoyl transferase 2 (CPT-2) is a key enzyme in the mitochondrial fatty acid metabolism. The active site is comprised of a Y-shaped tunnel with distinct binding sites for the substrate acylcarnitine and the cofactor CoA. We investigated the thermodynamics of binding of four inhibitors directed against either the CoA or the acylcarnitine binding sites using isothermal titration calorimetry (ITC). CPT-2 is a monotopic membrane protein and was solubilized by β-octylglucoside (β-OG) above its critical micellar concentration (CMC) to perform inhibitor titrations in solutions containing detergent micelles. The CMC of β-OG in the presence of inhibitors was measured with ITC and small variations were observed. The inhibitors bound to rat CPT-2 (rCPT-2) with 1:1 stoichiometry and the dissociation constants were in the range of K D = 2-20 μM. New X-ray structures and docking models of rCPT-2 in complex with inhibitors enable an analysis of the thermodynamic data in the context of the interaction observed for the individual binding sites of the ligands. For all ligands the binding enthalpy was exothermic, and enthalpy as well as entropy contributed to the binding reaction, with the exception of ST1326 for which binding was solely enthalpy-driven. The substrate analog ST1326 binds to the acylcarnitine binding site and a heat capacity change close to zero suggests a balance of electrostatic and hydrophobic interactions. An excellent correlation of the thermodynamic (ITC) and structural (X-ray crystallography, models) data was observed suggesting that ITC measurements provide valuable information for optimizing inhibitor binding in drug discovery.

Purpose: To investigate structure and function of different monoclonal antibody (MAb) dimers., Methods: MAb dimers were induced by process-related, low pH and UV light stress. Dimers were isolated and purified by chromatography and extensively characterized by biochemical, structural and functional methods., Results: Highly purified dimer forms were obtained which enabled detailed characterization. Dimers induced by process stress were associated by a single non-covalent interaction site between two Fab domains in a characteristic "bone-like" structure observed in Transmission Electron Microscopy (TEM). These dimers showed reduced potency and antigen binding affinity. Low pH stress generated more stable but also non-covalently associated dimers without chemical alterations in a typical "closed" conformation according to TEM. These dimer species were more compact and more hydrophobic as dimers induced by process stress. They showed bioactivity and antigen binding affinity similar to the native monomer. Light-induced dimers, exhibiting various different conformations, were the most stable dimers with various chemical modifications leading to a broad range in size, charge and hydrophobicity. These dimers fully lost bioactivity and antigen binding affinity., Conclusion: The use of highly purified MAb dimers and a panel of characterizations methods enabled to obtain a clear picture about molecular architecture and function of dimers.

Antibody-mediated cellular cytotoxicity (ADCC), a key immune effector mechanism, relies on the binding of antigen-antibody complexes to Fcγ receptors expressed on immune cells. Antibodies lacking core fucosylation show a large increase in affinity for FcγRIIIa leading to an improved receptor-mediated effector function. Although afucosylated IgGs exist naturally, a next generation of recombinant therapeutic, glycoenginereed antibodies is currently being developed to exploit this finding. In this study, the crystal structures of a glycosylated Fcγ receptor complexed with either afucosylated or fucosylated Fc were determined allowing a detailed, molecular understanding of the regulatory role of Fc-oligosaccharide core fucosylation in improving ADCC. The structures reveal a unique type of interface consisting of carbohydrate-carbohydrate interactions between glycans of the receptor and the afucosylated Fc. In contrast, in the complex structure with fucosylated Fc, these contacts are weakened or nonexistent, explaining the decreased affinity for the receptor. These findings allow us to understand the higher efficacy of therapeutic antibodies lacking the core fucose and also suggest a unique mechanism by which the immune system can regulate antibody-mediated effector functions.

The control of fatty acid translocation across the mitochondrial membrane is mediated by the carnitine palmitoyltransferase (CPT) system. Modulation of its functionality has simultaneous effects on fatty acid and glucose metabolism. This encourages use of the CPT system as drug target for reduction of gluconeogenesis and restoration of lipid homeostasis, which are beneficial in the treatment of type 2 diabetes mellitus and obesity. Recently, crystal structures of CPT-2 were determined in uninhibited forms and in complexes with inhibitory substrate-analogs with anti-diabetic properties in animal models and in clinical studies. The CPT-2 crystal structures have advanced understanding of CPT structure-function relationships and will facilitate discovery of novel inhibitors by structure-based drug design. However, a number of unresolved questions regarding the biochemistry and pharmacology of CPT enzymes remain and are addressed in this review.

The adaptor protein Tks5/FISH (tyrosine kinase substrate 5/five SH3 domains, hereafter termed Tks5) is a crucial component of a protein network that controls the invasiveness of cancer cells and progression of Alzheimer's disease. Tks5 consists of an amino-terminal PX domain that is followed by five SH3 domains (SH3A-E), and two different splice variants are expressed. We identified son of sevenless-1 (Sos1) as a novel binding partner of Tks5 and found colocalization of Tks5 with Sos1 in human epithelial lung carcinoma (A549) cells and in podosomes of Src-transformed NIH 3T3 cells. We observe synergistic binding of SH3A and SH3B to Sos1 when peptide arrays are used, indicating that the tandem SH3A and SH3B domains of Tks5 can potentially bind in a superSH3 binding mode, as was described for the homologous protein p47phox. These results are further corroborated by pull-down assays and isothermal titration calorimetry showing that both intact SH3 domains are required for efficient binding to the entire proline-rich domain of Sos1. The presence of a basic insertion between the SH3A and SH3B domains in the long splice variant of Tks5 decreases the affinity to Sos1 isoforms about 10-fold as determined by analytical ultracentrifugation. Furthermore, it leads to an alteration in the recognition of binding motifs for the interaction with Sos1: While the insertion abrogates the interaction with the majority of peptides derived from the proline-rich domains of Sos1 and dynamin that are recognized by the short splice isoform, it enables binding to a different set of peptides including a sequence comprising the splice insertion in the long isoform of Sos1 (Sos1_2). In the absence of the basic insertion, Tks5 was found to bind a range of Sos1 and dynamin peptides including conventional proline-rich motifs and atypical recognition sequences. Hereby, the tandem SH3 domains in Tks5 employ two distinct types of binding modes: One class of peptides is recognized by single SH3 domains, whereas a second class of peptides requires the presence of both domains to bind synergistically. We conclude that the tandem SH3A and SH3B domains of Tks5 constitute a versatile module for the implementation of isoform-specific protein-protein interactions.

The mitochondrial membrane-associated carnitine palmitoyltransferase system is a validated target for the treatment of type 2 diabetes mellitus. To further facilitate structure-based drug discovery, we determined the crystal structure of rat CPT-2 (rCPT-2) in complex with the substrate analogue palmitoyl-aminocarnitine at 1.8A resolution. Biochemical analyses revealed a strong effect of this compound on rCPT-2 activity and stability. Using a computational approach we examined the membrane association of rCPT-2. The protein interacts with the membrane as a functional monomer and the calculations confirm the presence of a membrane association domain that consists of layers of hydrophobic and positively charged residues.

Carnitine palmitoyltransferases 1 and 2 (CPTs) facilitate the import of long-chain fatty acids into mitochondria. Modulation of the catalytic activity of the CPT system is currently under investigation for the development of novel drugs against diabetes mellitus. We report here the 1.6 A resolution structure of the full-length mitochondrial membrane protein CPT-2. The structure of CPT-2 in complex with the generic CPT inhibitor ST1326 ([R]-N-[tetradecylcarbamoyl]-aminocarnitine), a substrate analog mimicking palmitoylcarnitine and currently in clinical trials for diabetes mellitus treatment, was solved at 2.5 A resolution. These structures of CPT-2 provide insight into the function of residues involved in substrate binding and determination of substrate specificity, thereby facilitating the rational design of antidiabetic drugs. We identify a sequence insertion found in CPT-2 that mediates membrane localization. Mapping of mutations described for CPT-2 deficiency, a hereditary disorder of lipid metabolism, implies effects on substrate recognition and structural integrity of CPT-2.

The crystal structure of the 26 kDa glutathione S-transferase from Schistosoma japonicum (SjGST) was determined at 3 A resolution in the new space group P2(1)2(1)2(1). The structure of orthorhombic SjGST reveals unique features of the ligand-binding site and dimer interface when compared with previously reported structures. SjGST is recognized as the major detoxification enzyme of S. japonicum, a pathogenic helminth causing schistosomiasis. As resistance against the established inhibitor of SjGST, praziquantel, has been reported these results might prove to be valuable for the development of novel drugs.