Identification of a Sialosides binding pocket in early-lineage SARS-CoV-2 via an optimized STD NMR method: a novel explanation for coronavirus virulence and zoonosis.
Identification of a Sialosides binding pocket in early-lineage SARS-CoV-2 via an optimized STD NMR method: a novel explanation for coronavirus virulence and zoonosis.
Project description:STD NMR spectroscopy is a powerful ligand-observed NMR tool for screening and characterizing the interactions of small molecules and low molecular weight fragments with a given macromolecule, identifying the main intermolecular contacts in the bound state. It is also a powerful analytical technique for the accurate determination of protein-ligand dissociation constants (KD) of medium-to-weak affinity, of interest in the pharmaceutical industry. However, accurate KD determination and epitope mapping requires a long series of experiments at increasing saturation times to carry out a full analysis using the so-called STD NMR build-up curve approach and apply the "initial slopes approximation". Here, we have developed a new protocol to bypass this important limitation, which allows us to obtain initial slopes by using just two saturation times and, hence, to very quickly determine precise protein-ligand dissociation constants by STD NMR.
Project description:In recent years, Saturation Transfer Difference NMR (STD NMR) has been proven to be a powerful and versatile ligand-based NMR technique to elucidate crucial aspects in the investigation of protein-ligand complexes. Novel STD NMR approaches relying on "multi-frequency" irradiation have enabled us to even elucidate specific ligand-amino acid interactions and explore the binding of fragments in previously unknown binding subsites. Exploring multi-subsite protein binding pockets is especially important in Fragment Based Drug Discovery (FBDD) to design leads of increased specificity and efficacy. We hereby propose a novel multi-frequency STD NMR approach based on direct irradiation of one of the ligands in a multi-ligand binding process, to probe the vicinity and explore the relative orientation of fragments in adjacent binding sub-sites, which we called Inter-Ligand STD NMR (IL-STD NMR). We proved its applicability on (i) a standard protein-ligand system commonly used for ligand-observed NMR benchmarking: Naproxen as bound to Bovine Serum Albumin, and (ii) the biologically relevant system of Cholera Toxin Subunit B and two inhibitors adjacently bound within the GM1 binding site. Relative to Inter-Ligand NOE (ILOE), the current state-of-the-art methodology to probe relative orientations of adjacent ligands, IL-STD NMR requires about one tenth of the experimental time and protein consumption, making it a competitive methodology with the potential to be applied in the pharmaceutical industries.
Project description:Fucosyltransferase 2 (FUT2) catalyzes the biosynthesis of A, B, and H antigens and other important glycans, such as (Sialyl Lewisx) sLex, and (Sialyl Lewisy) sLey. The production of these glycans is increased in various cancers, hence to design and develop specific inhibitors of FUT2 is a therapeutic strategy. The current study was designed to identify the inhibitors for FUT2. In silico screening of 300 synthetic compounds was performed. Molecular docking studies highlighted the interactions of ligands with critical amino acid residues, present in the active site of FUT2. The epitope mapping in ligands was performed using the STD-NMR experiments to identify the interactions between ligands, and receptor protein. Finally, we have identified 5 lead compounds 4, 5, 26, 27, and 28 that can be studied for further development as cancer therapeutic agents.
Project description:Ligand-based NMR techniques to study protein-ligand interactions are potent tools in drug design. Saturation transfer difference (STD) NMR spectroscopy stands out as one of the most versatile techniques, allowing screening of fragments libraries and providing structural information on binding modes. Recently, it has been shown that a multi-frequency STD NMR approach, differential epitope mapping (DEEP)-STD NMR, can provide additional information on the orientation of small ligands within the binding pocket. Here, the approach is extended to a so-called DEEP-STD NMR fingerprinting technique to explore the binding subsites of cholera toxin subunit B (CTB). To that aim, the synthesis of a set of new ligands is presented, which have been subject to a thorough study of their interactions with CTB by weak affinity chromatography (WAC) and NMR spectroscopy. Remarkably, the combination of DEEP-STD NMR fingerprinting and Hamiltonian replica exchange molecular dynamics has proved to be an excellent approach to explore the geometry, flexibility, and ligand occupancy of multi-subsite binding pockets. In the particular case of CTB, it allowed the existence of a hitherto unknown binding subsite adjacent to the GM1 binding pocket to be revealed, paving the way to the design of novel leads for inhibition of this relevant toxin.
Project description:We have combined saturation transfer difference NMR (STD NMR) with chemical shift imaging (CSI) and controlled concentration gradients of small molecule ligands to develop imaging STD NMR, a new tool for the assessment of protein-ligand interactions. Our methodology allows the determination of protein-ligand dissociation constants (KD) and assessment of the binding specificity in a single NMR tube, avoiding time-consuming titrations. We demonstrate the formation of suitable and reproducible concentration gradients of ligand along the vertical axis of the tube, against homogeneous protein concentration, and present a CSI pulse sequence for the acquisition of STD NMR experiments at different positions along the sample tube. Compared to the conventional methodology in which the [ligand]/[protein] ratio is increased manually, we can perform STD NMR experiments at a greater number of ratios and construct binding epitopes in a fraction (∼20%) of the experimental time. Second, imaging STD NMR also allows us to screen for non-specific binders, by monitoring any variation of the binding epitope map at increasing [ligand]/[protein] ratios. Hence, the proposed method does carry the potential to speed up and smooth out the drug discovery process.
Project description:Aberrant fucosylation is the hallmark of malignant cell transformation, leading to many cellular events, such as uncontrolled cell proliferation, angiogenesis, tumor cell invasion, and metastasis. This increased fucosylation is caused due to the over-expression of fucosyltransferases (FUTs) that catalyzes the transfer of the fucose (Fuc) residue from GDP-fucose (donor substrate) to various oligosaccharides, glycoproteins, and glycolipids (acceptor substrates). Hence, fucosyltransferases (FUTs) are considered as validated target for the drug discovery against on cancers. In the current study, a drug repurposing approach was deployed to identify new hits against fucosyltransferase 2 (FUT2), using computational and biophysical techniques. A library of 500 US-FDA approved drugs were screened in-silico against fucosyltransferase 2 (FUT2) donor and acceptor sites. Five drugs were predicted as hits, based on their significant docking scores (-5.8 to -8.2), and binding energies (-43 to -51.19 Kcal/mol). Furthermore, STD-NMR highlighted the epitope of these drugs in the binding site of fucosyltransferase 2 (FUT2). Simulation studies provided insights about the binding site of these drugs, and 4 of them, acarbose, ascorbic acid, ibuprofen, and enalaprilat dihydrate, were found as significant binders at the donor binding site of fucosyltransferase 2 (FUT2). Hence, the current study reports the repurposed drugs as potential hits against fucosyltransferase 2 (FUT2). These may be further studied through in-vitro and in-vivo inhibitory and mechanistic studies.
Project description:The recent coronavirus disease (COVID-19) outbreak is one of its kind in the history of public health that has created a major global threat. The causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a zoonotic source and hence, reverse zoonosis (disease transmission from humans to animals) increases the risk and rate of SARS-CoV-2 infection. Serological and molecular analyses and experimental infection studies have identified SARS-CoV-2 infection in several animal species in various countries. Different domestic and wild animals, including cats, dogs, tigers, lions, puma, snow leopard, minks, and pet ferrets, are infected naturally with SARS-CoV-2, mostly through suspected human to animal transmission. In addition, in vivo experimental inoculation studies have reported the susceptibility of cats, ferrets, hamsters, Egyptian fruit bats, and non-human primates to the virus. These experimentally infected species are found to be capable of virus transmission to co-housed animals of the same species. However, SARS-CoV-2 showed poor replication in livestock species such as pigs, chickens, and ducks with no detection of viral RNA after the animals were deliberately inoculated with the virus or exposed to the infected animals. As the pets/companion animals are more susceptible to COVID-19, the infection in animals needs an in-depth and careful study to avoid any future transmissions. The one health approach is the best inter-disciplinary method to understand the consequences of viral spread and prevention in novel host populations for the betterment of public health. Further in this review, we will explain in detail the different natural and experimentally induced cases of human to animal SARS-CoV-2 infection.
Project description:Structural studies of integral membrane proteins have been limited by the intrinsic conformational flexibility and the need to stabilize the proteins in solution. Stabilization by mutagenesis was very successful for structural biology of G protein-coupled receptors (GPCRs). However, it requires heavy protein engineering and may introduce structural deviations. Here we describe the use of specific calixarenes-based detergents for native GPCR stabilization. Wild type, full length human adenosine A2A receptor was used to exemplify the approach. We could stabilize native, glycosylated, non-aggregated and homogenous A2AR that maintained its ligand binding capacity. The benefit of the preparation for fragment screening, using the Saturation-Transfer Difference nuclear magnetic resonance (STD-NMR) experiment is reported. The binding of the agonist adenosine and the antagonist caffeine were observed and competition experiments with CGS-21680 and ZM241385 were performed, demonstrating the feasibility of the STD-based fragment screening on the native A2A receptor. Interestingly, adenosine was shown to bind a second binding site in the presence of the agonist CGS-21680 which corroborates published results obtained with molecular dynamics simulation. Fragment-like compounds identified using STD-NMR showed antagonistic effects on A2AR in the cAMP cellular assay. Taken together, our study shows that stabilization of native GPCRs represents an attractive approach for STD-based fragment screening and drug design.
Project description:Biofilms confine bacterial cells within self-produced matrices, offering advantages such as protection from antibiotics and entrapment of nutrients. Polysaccharides are major components in these macromolecular assemblies, and their interactions with other chemicals are of high relevance for the benefits provided by the biofilm 3D molecular matrix. NMR is a powerful technique for the study and characterization of the interactions between molecules of biological relevance. In this study, we have applied multifrequency saturation transfer difference (STD) NMR and DOSY NMR approaches to elucidate the interactions between the exopolysaccharide produced by Burkholderia multivorans C1576 (EpolC1576) and the antibiotics kanamycin and ceftadizime. The NMR strategies presented here allowed for an extensive characterization at an atomic level of the mechanisms behind the implication of the EpolC1576 in the recalcitrance phenomena, which is the ability of bacteria in biofilms to survive in the presence of antibiotics. Our results suggest an active role for EpolC1576 in the recalcitrance mechanisms toward kanamycin and ceftadizime, though through two different mechanisms.
Project description:Saturation transfer difference (STD) NMR spectroscopy is extensively used to obtain epitope maps of ligands binding to protein receptors, thereby revealing structural details of the interaction, which is key to direct lead optimization efforts in drug discovery. However, it does not give information about the nature of the amino acids surrounding the ligand in the binding pocket. Herein, we report the development of the novel method differential epitope mapping by STD NMR (DEEP-STD NMR) for identifying the type of protein residues contacting the ligand. The method produces differential epitope maps through 1) differential frequency STD NMR and/or 2) differential solvent (D2 O/H2 O) STD NMR experiments. The two approaches provide different complementary information on the binding pocket. We demonstrate that DEEP-STD NMR can be used to readily obtain pharmacophore information on the protein. Furthermore, if the 3D structure of the protein is known, this information also helps in orienting the ligand in the binding pocket.