scholarly journals Single-molecule Dynamic In-Solution Inhibition Assay: A Method for Full Kinetic Profiling of Drug Candidate Binding to GPCRs in Native Membranes

2021 ◽  
Author(s):  
Tim Patrick Kaminski ◽  
Vladimir P Zhdanov ◽  
Fredrik Hook

Kinetic profiling of drug-target interactions using surface-based label-free technologies is well established for water-soluble pharmaceutical targets but is difficult to execute for membrane proteins in general and G-protein-coupled receptors (GPCRs) in particular. That is because surface immobilization of GPCRs tends to alter their configuration and function, leading to low target coverage and non-specific binding. We here describe a novel assay for kinetic profiling of drug binding to the GPCR human beta 2 adrenergic receptor (β2AR). The assay involves temporally-resolved imaging of the binding of individual β2AR-containing cell membrane-derived liposomes to a surface-immobilized ligand in the presence of screened drugs. This approach allowed to determine association and dissociation constants of β2AR and suspended alprenolol (antagonist) and fenoterol (agonist). The setup combines a 384 well-plate sensor chip with automated liquid handling and the assay takes minutes to complete, making it well adapted for drug screening campaigns.

2020 ◽  
Vol 61 (8) ◽  
pp. 1244-1251 ◽  
Author(s):  
Manisha Ray ◽  
Kazufumi Nagai ◽  
Yasuyuki Kihara ◽  
Amanda Kussrow ◽  
Michael N. Kammer ◽  
...  

Native interactions between lysophospholipids (LPs) and their cognate LP receptors are difficult to measure because of lipophilicity and/or the adhesive properties of lipids, which contribute to high levels of nonspecific binding in cell membrane preparations. Here, we report development of a free-solution assay (FSA) where label-free LPs bind to their cognate G protein-coupled receptors (GPCRs), combined with a recently reported compensated interferometric reader (CIR) to quantify native binding interactions between receptors and ligands. As a test case, the binding parameters between lysophosphatidic acid (LPA) receptor 1 (LPA1; one of six cognate LPA GPCRs) and LPA were determined. FSA-CIR detected specific binding through the simultaneous real-time comparison of bound versus unbound species by measuring the change in the solution dipole moment produced by binding-induced conformational and/or hydration changes. FSA-CIR identified KD values for chemically distinct LPA species binding to human LPA1 and required only a few nanograms of protein: 1-oleoyl (18:1; KD = 2.08 ± 1.32 nM), 1-linoleoyl (18:2; KD = 2.83 ± 1.64 nM), 1-arachidonoyl (20:4; KD = 2.59 ± 0.481 nM), and 1-palmitoyl (16:0; KD = 1.69 ± 0.1 nM) LPA. These KD values compared favorably to those obtained using the previous generation back-scattering interferometry system, a chip-based technique with low-throughput and temperature sensitivity. In conclusion, FSA-CIR offers a new increased-throughput approach to assess quantitatively label-free lipid ligand-receptor binding, including nonactivating antagonist binding, under near-native conditions.


2019 ◽  
Author(s):  
Xiaoyi Wang ◽  
Mark D. Wilkinson ◽  
Xiaoyan Lin ◽  
Ren Ren ◽  
Keith Willison ◽  
...  

AbstractActin is a key protein in the dynamic processes within the eukaryotic cell. To date, methods exploring the molecular state of actin are limited to insights gained from structural approaches, providing a snapshot of protein folding, or methods that require chemical modifications compromising actin monomer thermostability. Nanopore sensing permits label-free investigation of native proteins and is ideally suited to study proteins such as actin that require specialised buffers and cofactors. Using nanopores we determined the state of actin at the macromolecular level (filamentous or globular) and in its monomeric form bound to inhibitors. We revealed urea-dependent and voltage-dependent transitional states and observed unfolding process within which sub-populations of transient actin oligomers are visible. We detected, in real-time, drug-binding and filament-growth events at the single-molecule level. This enabled us to calculate binding stoichiometries and to propose a model for protein dynamics using unmodified, native actin molecules, demostrating the promise of nanopores sensing for in-depth understanding of protein folding landscapes and for drug discovery.


2020 ◽  
Author(s):  
Petar M. Mitrasinovic

The infectious disease CoViD-19 is caused by a new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also referred to as hCoV-19. A possible infection mechanism includes dual host receptor recognitions by the SARS-CoV-2 transmembrane spike (S) glycoproteins. SARS-CoV-2 S contains two different domains, the receptor-binding domain (RBD) and the N-terminal domain (NTD), which interact with the angiotensin-converting enzyme 2 (ACE2) and the ganglioside-rich domain of the plasma membrane at the surface of respiratory cell, respectively. The NTD amino acid residues (111-162) form a functional ganglioside-binding domain (GBD) that is conserved in all clinical isolates. Herein, the single point mutations (SPMs) of the GBD residues to which the virus is prone during genetic adaptation are predicted using an in silico protein engineering approach. Consequently, their effects on the attachment of SARS-CoV-2 S to the ganglioside-linked 9-O-acetylated sialic acid (9-O-Ac-Sia) are explored using molecular docking simulations. Val120Tyr and Asn122Trp are found to be the most likely SPMs in the GBD of SARS-CoV-2 S being involved in very specific recognition with 9-O-Ac-Sia through electrostatic interactions. Val120Tyr and Asn122Trp are also found to be the most likely SPMs in the GBD of SARS-CoV-2 S that is involved in conspicuously hydrophobic recognition with hidroxychloroquine (Hcq), thereby indicating the ability of Hcq to competitively inhibit GBD interactions with lipid rafts. However, the considerably non-specific binding of Hcq and the micromolar range of the dissociation constants of the SARS-CoV-2 S/Hcq complexes do not support the proposal of treating Hcq as a drug candidate. Maintaining a clear resemblance of the structure of a potential drug candidate to a natural substrate, accompanied by essential functional group modifications, may be a usable guideline for the structure-based design of anti-CoViD-19 drugs.<br>


2013 ◽  
Vol 2013 ◽  
pp. 1-5 ◽  
Author(s):  
Hongliang Guan ◽  
Zhike He

Water-soluble fluorescent conjugated polymer is a promising material which could be used as an optical platform in highly sensitive molecular sensors. In this paper, a simple label-free DNA sensor, which consisted of a poly(3-alkoxy-4-methylthiophene) and an aptamer, was used to detect L-argininamide (L-Arm). Due to the specific binding reaction between L-Arm and its aptamer, the proposed method can easily determinate the L-Arm through the recovery of fluorescence without any modification. Other ions or similar molecules had little effect on the detection. Moreover, there was a linear relationship between fluorescence intensity and the concentration of L-Arm. The detection limit of L-Arm was as low as 4.7 nM.


2020 ◽  
Author(s):  
Petar M. Mitrasinovic

The infectious disease CoViD-19 is caused by a new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also referred to as hCoV-19. A possible infection mechanism includes dual host receptor recognitions by the SARS-CoV-2 transmembrane spike (S) glycoproteins. SARS-CoV-2 S contains two different domains, the receptor-binding domain (RBD) and the N-terminal domain (NTD), which interact with the angiotensin-converting enzyme 2 (ACE2) and the ganglioside-rich domain of the plasma membrane at the surface of respiratory cell, respectively. The NTD amino acid residues (111-162) form a functional ganglioside-binding domain (GBD) that is conserved in all clinical isolates. Herein, the single point mutations (SPMs) of the GBD residues to which the virus is prone during genetic adaptation are predicted using an in silico protein engineering approach. Consequently, their effects on the attachment of SARS-CoV-2 S to the ganglioside-linked 9-O-acetylated sialic acid (9-O-Ac-Sia) are explored using molecular docking simulations. Val120Tyr and Asn122Trp are found to be the most likely SPMs in the GBD of SARS-CoV-2 S being involved in very specific recognition with 9-O-Ac-Sia through electrostatic interactions. Val120Tyr and Asn122Trp are also found to be the most likely SPMs in the GBD of SARS-CoV-2 S that is involved in conspicuously hydrophobic recognition with hidroxychloroquine (Hcq), thereby indicating the ability of Hcq to competitively inhibit GBD interactions with lipid rafts. However, the considerably non-specific binding of Hcq and the micromolar range of the dissociation constants of the SARS-CoV-2 S/Hcq complexes do not support the proposal of treating Hcq as a drug candidate. Maintaining a clear resemblance of the structure of a potential drug candidate to a natural substrate, accompanied by essential functional group modifications, may be a usable guideline for the structure-based design of anti-CoViD-19 drugs.<br>


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Terry McAfee ◽  
Thomas Ferron ◽  
Isvar A. Cordova ◽  
Phillip D. Pickett ◽  
Charles L. McCormick ◽  
...  

AbstractSelf-assembled molecular nanostructures embody an enormous potential for new technologies, therapeutics, and understanding of molecular biofunctions. Their structure and function are dependent on local environments, necessitating in-situ/operando investigations for the biggest leaps in discovery and design. However, the most advanced of such investigations involve laborious labeling methods that can disrupt behavior or are not fast enough to capture stimuli-responsive phenomena. We utilize X-rays resonant with molecular bonds to demonstrate an in-situ nanoprobe that eliminates the need for labels and enables data collection times within seconds. Our analytical spectral model quantifies the structure, molecular composition, and dynamics of a copolymer micelle drug delivery platform using resonant soft X-rays. We additionally apply this technique to a hydrocarbon sequestrating polysoap micelle and discover that the critical organic-capturing domain does not coalesce upon aggregation but retains distinct single-molecule cores. This characteristic promotes its efficiency of hydrocarbon sequestration for applications like oil spill remediation and drug delivery. Such a technique enables operando, chemically sensitive investigations of any aqueous molecular nanostructure, label-free.


2018 ◽  
Vol 115 (37) ◽  
pp. E8652-E8659 ◽  
Author(s):  
Shuguang Zhang ◽  
Fei Tao ◽  
Rui Qing ◽  
Hongzhi Tang ◽  
Michael Skuhersky ◽  
...  

Structure and function studies of membrane proteins, particularly G protein-coupled receptors and multipass transmembrane proteins, require detergents. We have devised a simple tool, the QTY code (glutamine, threonine, and tyrosine), for designing hydrophobic domains to become water soluble without detergents. Here we report using the QTY code to systematically replace the hydrophobic amino acids leucine, valine, isoleucine, and phenylalanine in the seven transmembrane α-helices of CCR5, CXCR4, CCR10, and CXCR7. We show that QTY code-designed chemokine receptor variants retain their thermostabilities, α-helical structures, and ligand-binding activities in buffer and 50% human serum. CCR5QTY, CXCR4QTY, and CXCR7QTY also bind to HIV coat protein gp41-120. Despite substantial transmembrane domain changes, the detergent-free QTY variants maintain stable structures and retain their ligand-binding activities. We believe the QTY code will be useful for designing water-soluble variants of membrane proteins and other water-insoluble aggregated proteins.


2021 ◽  
Author(s):  
Gustavo Nieto-Alamilla ◽  
Juan Escamilla-Sánchez ◽  
José Antonio Arias-Montaño

In HEK-293T cells transiently transfected with the human histamine H3 receptor (hH3R), we studied the effect of over-expressing the human RGS9-2 protein on H3R-mediated stimulation of [35S]-GTPγS binding and inhibition of forskolin-induced cAMP formation. Maximal specific binding (Bmax) of [3H]-N-methyl-histamine to cell membranes was 468 ± 12 and 442 ± 38 fmol/mg protein for HEK-293T-hH3R and HEK-293T-hH3R/hRGS9-2 cells, respectively, with dissociation constants (Kd) 2.57 nM and 3.38 nM. The H3R agonist immepip stimulated [35S]-GTPγS binding with similar potency and efficacy (Emax 146.3 ± 4.4 % and 150.0 ± 5.3 % of basal, pEC50 8.57 ± 0.26 and 9.00 ± 0.33, respectively), but was significantly less efficacious to inhibit forskolin-induced cAMP accumulation in HEK-293T-hH3R/hRGS9 cells (-19.2 ± 5.3 versus -37.7 ± 5.1 % in HEK-293T-hH3R cells) with no significant difference in potency (pEC50 9.60 ± 0.14 and 9.07 ± 0.29, respectively). These results indicate that in HEK-293T cells hRGS9-2 regulates hH3R445 signaling downstream G protein activation


2020 ◽  
Author(s):  
Nikolas Hundt

Abstract Single-molecule imaging has mostly been restricted to the use of fluorescence labelling as a contrast mechanism due to its superior ability to visualise molecules of interest on top of an overwhelming background of other molecules. Recently, interferometric scattering (iSCAT) microscopy has demonstrated the detection and imaging of single biomolecules based on light scattering without the need for fluorescent labels. Significant improvements in measurement sensitivity combined with a dependence of scattering signal on object size have led to the development of mass photometry, a technique that measures the mass of individual molecules and thereby determines mass distributions of biomolecule samples in solution. The experimental simplicity of mass photometry makes it a powerful tool to analyse biomolecular equilibria quantitatively with low sample consumption within minutes. When used for label-free imaging of reconstituted or cellular systems, the strict size-dependence of the iSCAT signal enables quantitative measurements of processes at size scales reaching from single-molecule observations during complex assembly up to mesoscopic dynamics of cellular components and extracellular protrusions. In this review, I would like to introduce the principles of this emerging imaging technology and discuss examples that show how mass-sensitive iSCAT can be used as a strong complement to other routine techniques in biochemistry.


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