The Interaction of the Transmembrane Domain of SARS-CoV-2 E-Protein with Glycyrrhizic Acid in Lipid Bilayer.

The interaction of the transmembrane domain of SARS-CoV-2 E-protein with glycyrrhizic acid in a model lipid bilayer (small isotropic bicelles) is demonstrated using various NMR techniques. Glycyrrhizic acid (GA) is the main active component of licorice root, and it shows antiviral activity against various enveloped viruses, including coronavirus. It is suggested that GA can influence the stage of fusion between the viral particle and the host cell by incorporating into the membrane. Using NMR spectroscopy, it was shown that the GA molecule penetrates into the lipid bilayer in a protonated state, but localizes on the bilayer surface in a deprotonated state. The transmembrane domain of SARS-CoV-2 E-protein facilitates deeper GA penetration into the hydrophobic region of bicelles at both acidic and neutral pH and promotes the self-association of GA at neutral pH. Phenylalanine residues of the E-protein interact with GA molecules inside the lipid bilayer at neutral pH. Furthermore, GA influences the mobility of the transmembrane domain of SARS-CoV-2 E-protein in the bilayer. These data provide deeper insight into the molecular mechanism of antiviral activity of glycyrrhizic acid.

Conformational Screening of Arbidol Solvates: Investigation via 2D NOESY.

Understanding of the nucleation process's fundamental principles in saturated solutions is an urgent task. To do this task, it is necessary to control the formation of polymorphic forms of biologically active compounds. In certain cases, a compound can exist in a single polymorphic form, but have several solvates which can appear in different crystal forms, depending on the medium and conditions of formation, and show different pharmaceutical activity. In the present paper, we report on the analysis of Arbidol conformational preferences in two solvents of different polarities-deuterated chloroform and dimethyl sulfoxide-at 25 °C, using the 2D NOESY method. The Arbidol molecule has various solvate forms depending on the molecular conformation. The method based on the nuclear Overhauser effect spectroscopy was shown to be efficient in the analysis of complex heterocyclic compounds possessing conformation-dependent pseudo-polymorphism. It is one of the types of polymorphism observed in compounds forming crystal solvates. Combined use of NMR methods and X-ray data allowed determining of conformer populations of Arbidol in CDCl3 and DMSO-d6 which were found to be 8/92% and 37/63%, respectively. The preferred conformation in solution is the same that appears in stable crystal solvates of Arbidol.

Conformational ensemble of the TNF-derived peptide solnatide in solution.

Tumor necrosis factor (TNF) is a homotrimer that has two spatially distinct binding regions, three lectin-like domains (LLD) at the TIP of the protein and three basolaterally located receptor-binding sites, the latter of which are responsible for the inflammatory and cell death-inducing properties of the cytokine. Solnatide (a.k.a. TIP peptide, AP301) is a 17-mer cyclic peptide that mimics the LLD of human TNF which activates the amiloride-sensitive epithelial sodium channel (ENaC) and, as such, recapitulates the capacity of TNF to enhance alveolar fluid clearance, as demonstrated in numerous preclinical studies. TNF and solnatide interact with glycoproteins and these interactions are necessary for their trypanolytic and ENaC-activating activities. In view of the crucial role of ENaC in lung liquid clearance, solnatide is currently being evaluated as a novel therapeutic agent to treat pulmonary edema in patients with moderate-to-severe acute respiratory distress syndrome (ARDS), as well as severe COVID-19 patients with ARDS. To facilitate the description of the functional properties of solnatide in detail, as well as to further target-docking studies, we have analyzed its folding properties by NMR. In solution, solnatide populates a set of conformations characterized by a small hydrophobic core and two electrostatically charged poles. Using the structural information determined here and also that available for the ENaC protein, we propose a model to describe solnatide interaction with the C-terminal domain of the ENaCα subunit. This model may serve to guide future experiments to validate specific interactions with ENaCα and the design of new solnatide analogs with unexplored functionalities.

Cover Feature: The Extended Hadamard Transform: Sensitivity-Enhanced NMR Experiments Among Labile and Non-Labile 1Hs of SARS-CoV-2-derived RNAs (ChemPhysChem 4/2022)

The Cover Feature illustrates how artifact-free 2D NOE correlations between labile protons can be obtained from an extended Hadamard encoding/decoding matrix, which supersedes problems in conventional Hadamard schemes. The sensitivity-enhancing abilities of extended Hadamard encoding operating in conjunction with solvent repolarization mechanisms are demonstrated on GHz NMR studies on SARS-CoV-2 RNA fragments. More information can be found in the Article by Lucio Frydman and co-workers.

The Extended Hadamard Transform: Sensitivity-Enhanced NMR Experiments Among Labile and Non-Labile 1 Hs of SARS-CoV-2-derived RNAs.

Hadamard encoded saturation transfer can significantly improve the efficiency of NOE-based NMR correlations from labile protons in proteins, glycans and RNAs, increasing the sensitivity of cross-peaks by an order of magnitude and shortening experimental times by ≥100-fold. These schemes, however, fail when tackling correlations within a pool of labile protons - for instance imino-imino correlations in RNAs or amide-amide correlations in proteins. Here we analyze the origin of the artifacts appearing in these experiments and propose a way to obtain artifact-free correlations both within the labile pool as well as between labile and non-labile 1 Hs, while still enjoying the gains arising from Hadamard encoding and solvent repolarizations. The principles required for implementing what we define as the extended Hadamard scheme are derived, and its clean, artifact-free, sensitivity-enhancing performance is demonstrated on RNA fragments derived from the SARS-CoV-2 genome. Sensitivity gains per unit time approaching an order of magnitude are then achieved in both imino-imino and imino-amino/aromatic protons 2D correlations; similar artifact-free sensitivity gains can be observed when carrying out extended Hadamard encodings of 3D NOESY/HSQC-type experiments. The resulting spectra reveal significantly more correlations than their conventionally acquired counterparts, which can support the spectral assignment and secondary structure determination of structured RNA elements.

Magnetization Transfer to Enhance NOE Cross-Peaks among Labile Protons: Applications to Imino-Imino Sequential Walks in SARS-CoV-2-Derived RNAs.

2D NOESY plays a central role in structural NMR spectroscopy. We have recently discussed methods that rely on solvent-driven exchanges to enhance NOE correlations between exchangeable and non-exchangeable protons in nucleic acids. Such methods, however, fail when trying to establish connectivities within pools of labile protons. This study introduces an alternative that also enhances NOEs between such labile sites, based on encoding a priori selected peaks by selective saturations. The resulting selective magnetization transfer (SMT) experiment proves particularly useful for enhancing the imino-imino cross-peaks in RNAs, which is a first step in the NMR resolution of these structures. The origins of these enhancements are discussed, and their potential is demonstrated on RNA fragments derived from the genome of SARS-CoV-2, recorded with better sensitivity and an order of magnitude faster than conventional 2D counterparts.

Magnetization Transfer to Enhance NOE Cross-Peaks among Labile Protons: Applications to Imino-Imino Sequential Walks in SARS-CoV-2-Derived RNAs.

2D NOESY plays a central role in structural NMR spectroscopy. We have recently discussed methods that rely on solvent-driven exchanges to enhance NOE correlations between exchangeable and non-exchangeable protons in nucleic acids. Such methods, however, fail when trying to establish connectivities within pools of labile protons. This study introduces an alternative that also enhances NOEs between such labile sites, based on encoding a priori selected peaks by selective saturations. The resulting selective magnetization transfer (SMT) experiment proves particularly useful for enhancing the imino-imino cross-peaks in RNAs, which is a first step in the NMR resolution of these structures. The origins of these enhancements are discussed, and their potential is demonstrated on RNA fragments derived from the genome of SARS-CoV-2, recorded with better sensitivity and an order of magnitude faster than conventional 2D counterparts.