Microscale Thermophoresis to Evaluate the Functionality of Heterologously Overexpressed Membrane Proteins in Membrane Preparations.

Transmembrane proteins are challenging to express in heterologous systems and to purify, thus any technique enabling to evaluate the functionality of the protein produced prior purification provides a huge step forward. Furthermore, the membrane environment may be critical for the activity of the target protein and accessing information in the membrane fragments instead of solubilizing the target into a detergent that may be unsuitable for its function is key to study and evaluate its activity. Herein, we describe how microscale thermophoresis (MST) was used to evaluate the functionality of membrane proteins directly in host membrane preparation before purification. We give a protocol to measure the affinity between the human Hedgehog (Hh) receptor Ptch1 in yeast plasma membrane and the small molecule PAH, which was shown to inhibit its drug efflux activity.

An original approach to measure ligand/receptor binding affinity in non-purified samples.

Several biochemical and biophysical methods are available to determine ligand binding affinities between a biological target and its ligands, most of which require purification, labelling or surface immobilisation. These measurements, however, remain challenging in regards to membrane proteins, as purification processes require their extraction from their native lipid environment, which may in turn impact receptor conformation and functionality. In this study, we have developed a novel experimental procedure using microscale thermophoresis (MST) directly from cell membrane fragments, to determine different ligand binding affinities to a membrane protein, the dopamine D2 receptor (D2R). In order to achieve this, two main challenges had to be overcome: determining the concentration of dopamine D2R in the crude sample; finding ways to minimize or account for non-specific binding of the ligand to cell fragments. Using MST, we were able to determine the D2R concentration in cell membrane fragments to approximately 36.8 ± 2.6 pmol/mg. Next, the doses-responses curves allowed for the determination of KD, to approximately 5.3 ± 1.7 nM, which is very close to the reported value. Important details of the experimental procedure have been detailed in this paper to allow the application of this novel method to various membrane proteins.

Fast Mek1 Hit Identification with TRIC Technology Correlates Well with Other Biophysical Methods.

The variety and complexity of drug targets are expanding rapidly. At the same time, there is significant interest in exploring a larger chemical space to identify new candidates. Fragment-based screening (FBS) has emerged as a popular alternative to traditional high-throughput screening campaigns to identify such drug candidates. FBS identifies hit fragments that exhibit weak interactions with the target of interest, thereby enabling the rational design of small-molecule compounds from the identified hit fragments, which serve as building blocks. This strategy reduces the number of molecules to screen while also allowing the exploration of a greater chemical space.Here we use temperature-related intensity change (TRIC) technology to perform FBS against the target MAPK/ERK kinase-1 (Mek1). TRIC describes the change in fluorescence intensity of a fluorescently labeled molecule upon a change in temperature. This intensity variation is dependent on the physicochemical environment in the vicinity of the dye and strongly affected by binding events. Thus, the detection of binding events is independent of mass, making TRIC an ideal tool for FBS.Using only 150 pmol of labeled Mek1, the authors screened 193 fragments from a prescreened library in less than 1 h of measurement time, leading to 66 hits. Among those hits, they identified more than 80% of the published top hits found using orthogonal techniques. Furthermore, TRIC allowed the identification of fragments that were of poor solubility but could be mistaken as false-positive hits in other methods.

Macromolecular interactions in vitro, comparing classical and novel approaches.

Biophysical quantification of protein interactions is central to unveil the molecular mechanisms of cellular processes. Researchers can choose from a wide panel of biophysical methods that quantify molecular interactions in different ways, including both classical and more novel techniques. We report the outcome of an ARBRE-MOBIEU training school held in June 2019 in Gif-sur-Yvette, France ( https://mosbio.sciencesconf.org/ ). Twenty European students benefited from a week's training with theoretical and practical sessions in six complementary approaches: (1) analytical ultracentrifugation with or without a fluorescence detector system (AUC-FDS), (2) isothermal titration calorimetry (ITC), (3) size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS), (4) bio-layer interferometry (BLI), (5) microscale thermophoresis (MST) and, (6) switchSENSE. They implemented all these methods on two examples of macromolecular interactions with nanomolar affinity: first, a protein-protein interaction between an artificial alphaRep binder, and its target protein, also an alphaRep; second, a protein-DNA interaction between a DNA repair complex, Ku70/Ku80 (hereafter called Ku), and its cognate DNA ligand. We report the approaches used to analyze the two systems under study and thereby showcase application of each of the six techniques. The workshop provided students with improved understanding of the advantages and limitations of different methods, enabling future choices concerning approaches that are most relevant or informative for specific kinds of sample and interaction.

Discovery of first-in-class reversible dual small molecule inhibitors against G9a and DNMTs in hematological malignancies.

The indisputable role of epigenetics in cancer and the fact that epigenetic alterations can be reversed have favoured development of epigenetic drugs. In this study, we design and synthesize potent novel, selective and reversible chemical probes that simultaneously inhibit the G9a and DNMTs methyltransferase activity. In vitro treatment of haematological neoplasia (acute myeloid leukaemia-AML, acute lymphoblastic leukaemia-ALL and diffuse large B-cell lymphoma-DLBCL) with the lead compound CM-272, inhibits cell proliferation and promotes apoptosis, inducing interferon-stimulated genes and immunogenic cell death. CM-272 significantly prolongs survival of AML, ALL and DLBCL xenogeneic models. Our results represent the discovery of first-in-class dual inhibitors of G9a/DNMTs and establish this chemical series as a promising therapeutic tool for unmet needs in haematological tumours.

Binding of sperm protein Izumo1 and its egg receptor Juno drives Cd9 accumulation in the intercellular contact area prior to fusion during mammalian fertilization.

Little is known about the molecular mechanisms that induce gamete fusion during mammalian fertilization. After initial contact, adhesion between gametes only leads to fusion in the presence of three membrane proteins that are necessary, but insufficient, for fusion: Izumo1 on sperm, its receptor Juno on egg and Cd9 on egg. What happens during this adhesion phase is a crucial issue. Here, we demonstrate that the intercellular adhesion that Izumo1 creates with Juno is conserved in mouse and human eggs. We show that, along with Izumo1, egg Cd9 concomitantly accumulates in the adhesion area. Without egg Cd9, the recruitment kinetics of Izumo1 are accelerated. Our results suggest that this process is conserved across species, as the adhesion partners, Izumo1 and its receptor, are interchangeable between mouse and human. Our findings suggest that Cd9 is a partner of Juno, and these discoveries allow us to propose a new model of the molecular mechanisms leading to gamete fusion, in which the adhesion-induced membrane organization assembles all key players of the fusion machinery.

Fast multicolor 3D imaging using aberration-corrected multifocus microscopy.

Conventional acquisition of three-dimensional (3D) microscopy data requires sequential z scanning and is often too slow to capture biological events. We report an aberration-corrected multifocus microscopy method capable of producing an instant focal stack of nine 2D images. Appended to an epifluorescence microscope, the multifocus system enables high-resolution 3D imaging in multiple colors with single-molecule sensitivity, at speeds limited by the camera readout time of a single image.