The active form of lactate dehydrogenase B (LDHB) is a homotetramer, and disruption of this quaternary structure represents a promising strategy for therapeutic intervention, particularly in cancer metabolism. However, accurate characterization of LDHB oligomerization is challenging due to the weak, reversible, and environmentally sensitive nature of subunit interactions. Here, we establish a robust size exclusion chromatography coupled with multi angle light scattering (SEC–MALS) workflow for analysis of LDHB oligomerization under physiologically relevant conditions.Wild type LDHB and three single point interface mutants (I9A, L71A, and F72A), covering oligomeric states from tetramers to monomers, were analyzed. SEC–MALS enabled clear separation and absolute molar mass determination of coexisting oligomeric species at micromolar protein concentrations, capturing concentration dependent equilibria inaccessible to techniques requiring nanomolar levels. The wild type enzyme remained exclusively tetrameric and remarkably stable, whereas the mutants displayed heterogeneous, dynamic oligomeric distributions.Systematic evaluation of experimental parameters demonstrated that LDHB oligomerization is highly sensitive to buffer composition. While the wild type protein was largely unaffected, variations in salt nature, ionic strength, pH, organic solvent, and arginine selectively destabilized mutant assemblies. Sodium chloride emerged as the most appropriate salt for preserving native oligomeric states while minimizing ion specific perturbations.Together, these results establish SEC–MALS as a powerful and essential method for characterizing dynamic protein oligomerization equilibria under biologically relevant conditions and provide critical insights into the structural determinants governing LDHB assembly and stability.

Intracellular membrane proteins represent a vast and pharmacologically essential class of therapeutic targets, yet their characterization within native pathological environments remains experimentally challenging. Here, we report what is, to the best of our knowledge, the first direct affinity measurements performed at the surface of Golgi microsomes isolated from cancer cells. By integrating affinity capillary electrophoresis (ACE) with a refined mathematical framework, we overcome long-standing obstacles associated with membrane protein analysis, including the need for target modification, intrinsic enzymatic activity, labeled probe, or large protein excess. The microfluidic format further minimizes sample consumption while enabling both direct and indirect detection modalities, thereby expanding applicability to impure compounds and ligands lacking charge or UV absorbance. Applying this strategy to UGCG, a Golgi-resident membrane protein of oncological relevance, we demonstrate quantitative ligand-binding measurements on fairly monodisperse vesicular fractions (∼150 nm) that preserve native intracellular architecture. Collectively, this work establishes a robust, broadly applicable platform for target engagement studies in complex biological systems and highlights the potential of ACE to accelerate drug discovery efforts involving intracellular or extracellular membrane-bound targets.

The direct and precise assessment of ligand-protein interactions under nearly physiological conditions is the core of drug discovery. In this context, affinity capillary electrophoresis (ACE) has become an emerging and reliable approach. The hyphenation of ACE with mass spectrometry (MS) is even more powerful than the classical ACE-UV methodology. It reduces compound identification errors and increases throughput by facilitating the analysis of the mixtures. However, buffers and capillary coatings compatible with mass spectrometry and operating under physiological conditions are very limited. In this paper, n-methylmorpholine acetate buffer and polydopamine-based coating were highlighted as major assets for CE-MS studies involving native proteins. Thanks to its protein desorption property, n-methylmorpholine improved the peak shape of proteins during CE analysis at physiological pH. The polydopamine-based neutral coating developed in this study is simple to prepare and demonstrated high stability at pH 7.4, enabling its use with an MS detector. The combination of these two key elements enabled us to successfully convert our ACE-UV method for coagulation factor XIIa into an ACE-MS approach operating at physiological pH. This study extends the scope of ACE for medicinal chemistry projects.

Previously, we described weak coumarin inhibitors of factor XIIa, a promising target for artificial surface-induced thrombosis and various inflammatory diseases. In this work, we used fragment-based drug discovery approach to improve our coumarin series. First, we screened about 200 fragments for the S1 pocket. The S1 pocket of trypsin-like serine proteases, such as factor XIIa, is highly conserved and is known to drive a major part of the association energy. From the screening, we selected fragments displaying a micromolar activity and studied their selectivity on other serine proteases. Then, these fragments were merged to our coumarin templates, leading to the generation of nanomolar inhibitors. The mechanism of inhibition was further studied by mass spectrometry demonstrating the covalent binding through the formation of an acyl enzyme complex. The most potent compound was tested in plasma to evaluate its stability and efficacy on coagulation assays. It exhibited a plasmatic half-life of 1.9 h and a good selectivity for the intrinsic coagulation pathway over the extrinsic one.

Detection and characterization of biomolecular interactions are the backbone of the drug discovery process. One of the most widely used approaches to developing new drugs is the fragment-based drug discovery (FBDD) strategy. The FBDD approach begins with the discovery of low molecular weight chemical fragments that bind weakly to the target of interest. The identified fragments are then combined or optimized into potent drug-like compounds. Despite its advantages over the high-throughput screening approach, its execution can be challenging. The reason is that discovering weak binders and determining how to grow or bind them are difficult. Therefore, intensive research is still underway to develop analytical technologies to detect and characterize weak, non-covalent interactions. The purpose of this article is to comprehensively review the emerging analytical technologies used in FBDD compared with the conventional ones. Particularly, we summarize their principle, advantages, limitations, and potential artifacts. For each emerging technique, we provide practical examples. Accurate detection and characterization of weak interactions are critical for the success of a FBDD project. Hence, knowledge of the features of the different techniques can support the selection and implementation of the project's analytical platform.

In this study, a partial-filling affinity capillary electrophoresis (pf-ACE) method was developed for the cross-validation of fragment hits revealed by chromogenic factor XIIa (FXIIa) assay. Chromogenic assay produces false positives, mainly due to spectrophotometric interferences and sample purity issues. pf-ACE was selected as counter-screening technology because of its separative character and the fact that the target does not have to be attached or tagged. The effects of protein plug length, applied voltage and composition of the running buffer were examined and optimized. Detection limit in terms of dissociation constant was estimated at 400 mu M. The affinity evaluation was performed close to physiological conditions (pH 7.4, ionic strength 0.13 mol L-1) in a poly (ethylene oxide)-coated capillary of 75 mu m internal diameter x 33 cm length with an applied voltage of 3 kV. This method uncovered chromogenic assay's false positives due to zinc contamination. Moreover, pf-ACE supported the evaluation of compounds absorbing at 405 nm.

Fragment-based lead discovery is a usual strategy in drug discovery to identify innovative lead compounds. The success of this approach strongly relies on the capacity to detect weak binders and characterize their binding site. NMR and X-ray crystallography are the conventional technologies used to tackle this challenge. However, their large protein consumption and the cost of equipment reduce their accessibility. Here, an affinity capillary electrophoresis methodology was developed that enables the detection of mM binders, the determination of dissociation constants, and the characterization of the fragment binding site. On the basis of multiple equilibrium theory, dissociation constants in the μM-mM range were determined, and a new methodology is proposed to establish graphically if two fragments bind the same protein pocket. The applicability of this methodology was demonstrated experimentally on coagulation factor XIIa by evaluating pairs of fragments with expected behavior. This study reinforces the significance of using affinity capillary electrophoresis to gather valuable information for medicinal chemistry projects.

Coagulation factor XII (FXII), a S1A serine protease, was discovered more than fifty years ago. However, its in vivo functions and its three-dimensional structure started to be disclosed in the last decade. FXII was found at the crosstalk of several physiological pathways including the intrinsic coagulation pathway, the kallikrein-kinin system, and the immune response. The FXII inhibition emerges as a therapeutic strategy for the safe prevention of artificial surface-induced thrombosis and in patients suffering from hereditary angioedema. The anti-FXII antibody garadacimab discovered by phage-display library technology is actually under phase II clinical evaluation for the prophylactic treatment of hereditary angioedema. The implication of FXII in neuro-inflammatory and neurodegenerative disorders is also an emerging research field. The FXII or FXIIa inhibitors currently under development include peptides, proteins, antibodies, RNA-based technologies, and, to a lesser extent, small-molecular weight inhibitors. Most of them are proteins, mainly isolated from hematophagous arthropods and plants. The discovery and development of these FXII inhibitors and their potential indications are discussed in the review. (c) 2020 Elsevier Masson SAS. All rights reserved.