Molecular basis of N-glycan recognition by pradimicin a and its potential as a SARS-CoV-2 entry inhibitor.

Virus entry inhibitors are emerging as an attractive class of therapeutics for the suppression of viral transmission. Naturally occurring pradimicin A (PRM-A) has received particular attention as the first-in-class entry inhibitor that targets N-glycans present on viral surface. Despite the uniqueness of its glycan-targeted antiviral activity, there is still limited knowledge regarding how PRM-A binds to viral N-glycans. Therefore, in this study, we performed binding analysis of PRM-A with synthetic oligosaccharides that reflect the structural motifs characteristic of viral N-glycans. Binding assays and molecular modeling collectively suggest that PRM-A preferentially binds to branched oligomannose motifs of N-glycans via simultaneous recognition of two mannose residues at the non-reducing ends. We also demonstrated, for the first time, that PRM-A can effectively inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in vitro. Significantly, the anti-SARS-CoV-2 effect of PRM-A is attenuated in the presence of the synthetic branched oligomannose, suggesting that the inhibition of SARS-CoV-2 infection is due to the interaction of PRM-A with the branched oligomannose-containing N-glycans. These data provide essential information needed to understand the antiviral mechanism of PRM-A and suggest that PRM-A could serve as a candidate SARS-CoV-2 entry inhibitor targeting N-glycans.

siRNA Seed Region Is Divided into Two Functionally Different Domains in RNA Interference in Response to 2'-OMe Modifications.

In RNA interference (RNAi), small interfering RNA (siRNA) functions to suppress the expression of its target mRNA with perfect sequence complementarity. In a mechanism different from above, siRNA also suppresses unintended mRNAs with partial sequence complementarities, mainly to the siRNA seed region (nucleotides 2-8). This mechanism is largely utilized by microRNAs (miRNAs) and results in siRNA-mediated off-target effects. Thus, the siRNA seed region is considered to be involved in both RNAi and off-target effects. In this study, we revealed that the impact of 2'-O-methyl (2'-OMe) modification is different according to the nucleotide positions. The 2'-OMe modifications of nucleotides 2-5 inhibited off-target effects without affecting on-target RNAi activities. In contrast, 2'-OMe modifications of nucleotides 6-8 increased both RNAi and off-target activities. The computational simulation revealed that the structural change induced by 2'-OMe modifications interrupts base pairing between siRNA and target/off-target mRNAs at nucleotides 2-5 but enhances at nucleotides 6-8. Thus, our results suggest that siRNA seed region consists of two functionally different domains in response to 2'-OMe modifications: nucleotides 2-5 are essential for avoiding off-target effects, and nucleotides 6-8 are involved in the enhancement of both RNAi and off-target activities.

d-Mannose binding, aggregation property, and antifungal activity of amide derivatives of pradimicin A.

Pradimicin A (PRM-A) and its derivatives comprise a unique family of antibiotics that show antifungal, antiviral, and antiparasitic activities through binding to d-mannose (Man)-containing glycans of pathogenic species. Despite their great potential as drug leads with an exceptional antipathogenic action, therapeutic application of PRMs has been severely limited by their tendency to form water-insoluble aggregates. Recently, we found that attachment of 2-aminoethanol to the carboxy group of PRM-A via amide linkage significantly suppressed the aggregation. Here, we prepared additional amide derivatives (2-8) of PRM-A to examine the possibility that the amide formation of PRM-A could suppress its aggregation propensity. Sedimentation assay and isothermal titration calorimetry experiment confirmed that all amide derivatives can bind Man without significant aggregation. Among them, hydroxamic acid derivative (4) showed the most potent Man-binding activity, which was suggested to be derived from the anion formation of the hydroxamic acid moiety by molecular modeling. Derivative 4 also exhibited significant antifungal activity comparable to that of PRM-A. These results collectively indicate that amide formation of PRM-A is the promising strategy to develop less aggregative derivatives, and 4 could serve as a lead compound for exploring the therapeutic application of PRM-A.

Photo-Induced Ring-Opening Reaction of Flav-3-en-2-ol Monitored by Time-Resolved Infrared Spectroscopy.

Photo-induced ring-opening reaction from flav-3-en-2-ol to 2-hydroxychalcone has been studied by time-resolved infrared (TR-IR) spectroscopy and quantum chemical calculations. A vibrational band due to the C═O stretching modes for intermediate species, enol forms of 2-hydroxychalcone in the electronic ground state, was observed at 1632 cm-1 in the TR-IR spectra after photoexcitation of flav-3-en-2-ol. We also found that the C═O stretching modes of the keto forms of 2-hydroxychalcone at 1664 cm-1 appeared immediately after photoexcitation and increased in intensity in synchronization with the depletion of the 1632 cm-1 band. Because the decay of the 1632 cm-1 band and the rise of the 1664 cm-1 band were fitted with bi-exponential model functions with common rate constants 0.5 and 11 µs-1, we propose that two kinds of enol form, single bond cis- (s-cis-) and trans- (s-trans-) enols, transformed into keto forms, cis-2-hydroxychalcone (Cc) and trans-2-hydroxychalcone (Ct), respectively. Quantum chemically calculated IR spectra of related species are consistent with the proposal. The observed temporal behavior of the TR-IR spectra indicates that there were reaction paths to the photogeneration of Cc and Ct within the time resolution of the TR-IR spectrometer (∼0.1 µs) in addition to the reaction paths via the enol forms of 2-hydroxychalcone.

Molecular Basis of Mannose Recognition by Pradimicins and their Application to Microbial Cell Surface Imaging.

Naturally occurring pradimicins (PRMs) show specific recognition of d-mannose (d-Man) in aqueous media, which has never been achieved by artificial small molecules. Although the Ca2+-mediated dimerization of PRMs is essential for their d-Man binding, the dimeric structure has yet to be elucidated, leaving the question open as to how PRMs recognize d-Man. Thus, we herein report the structural elucidation of the dimer by a combination of X-ray crystallography and solid-state NMR spectroscopy. Coupled with our previous knowledge regarding the d-Man binding geometry of PRMs, elucidation of the dimer allowed reliable estimation of the mode of d-Man binding. Based on the binding model, we further developed an azide-functionalized PRM derivative (PRM-Azide) with d-Man binding specificity. Notably, PRM-Azide stained Candida rugosa cells having mannans on their cell surface through conjugation with the tetramethylrhodamine fluorophore. The present study provides the practical demonstration that PRMs can serve as lectin mimics for use in glycobiological studies.

Structural Effects of Fusicoccin upon Upregulation of 14-3-3-Phospholigand Interaction and Cytotoxic Activity.

Fusicoccins (FCs) exhibit various cellular activities in mammalian cells, but details of the mechanism of action are not fully understood. In this study, we synthesized two pairs of model derivatives of FCs differing only in the presence and absence of a 12-hydroxyl group and evaluated their binding to a 14-3-3 protein together with various mode 1 and mode 3 phosphopeptide ligands. Our results demonstrate that the 12-hydroxyl group hampers binding to 14-3-3 with mode 1 phospholigands, presumably due to steric repulsion with the i+2 residue. Furthermore, cell-based evaluations showed that only non-substituted FCs exhibit significant cytotoxicity and all 12-hydroxyl derivatives were inactive, demonstrating a clear correlation with their ability to form ternary complexes with 14-3-3 and a mode 1 ligand. These results suggest that binding to 14-3-3 and a partner protein(s) possessing a mode 1 sequence plays a role in the mechanism of action of 12-non-substituted FCs.

Electronic origin of the dependence of hydrogen bond strengths on nearest-neighbor and next-nearest-neighbor hydrogen bonds in polyhedral water clusters (H2O)n, n = 8, 20 and 24.

The influence of the nearest neighbor and next-nearest neighbor water molecules on the strength of the hydrogen (H) bonds was examined for the polyhedral clusters of cubic (H2O)8, dodecahedral (H2O)20 and tetrakaidecahedral (H2O)24 cages. The relative stability and the characteristics of the H bond networks are also studied. The charge-transfer (CT) and dispersion interaction terms of every pair of H bonds are evaluated using perturbation theory based on the locally-projected molecular orbitals (LPMO PT). Every water molecule and every H-bonded pair in these polyhedral clusters are classified by the types of the neighbor molecules and H bonds. The relative binding energies among the polyhedral clusters are grouped by these classifications. The optimized OO distances, which are strongly correlated with the calculated pairwise CT terms, are dependent on the 49 sub-groups of the H bonds determined by the type of the neighbor molecules. The electronic origin of this dependence is analyzed using Mulliken's charge-transfer theory, and employing a few assumptions, the analytical formulas for the contribution of the CT terms to the H bond energy are derived.

Universal scaling of potential energy functions describing intermolecular interactions. II. The halide-water and alkali metal-water interactions.

The scaled versions of the newly introduced [S. S. Xantheas and J. C. Werhahn, J. Chem. Phys. 141, 064117 (2014)] generalized forms of some popular potential energy functions (PEFs) describing intermolecular interactions--Mie, Lennard-Jones, Morse, and Buckingham exponential-6--have been used to fit the ab initio relaxed approach paths and fixed approach paths for the halide-water, X(-)(H2O), X = F, Cl, Br, I, and alkali metal-water, M(+)(H2O), M = Li, Na, K, Rb, Cs, interactions. The generalized forms of those PEFs have an additional parameter with respect to the original forms and produce fits to the ab initio data that are between one and two orders of magnitude better in the χ(2) than the original PEFs. They were found to describe both the long-range, minimum and repulsive wall of the respective potential energy surfaces quite accurately. Overall the 4-parameter extended Morse (eM) and generalized Buckingham exponential-6 (gBe-6) potentials were found to best fit the ab initio data for these two classes of ion-water interactions. The fitted values of the parameter of the (eM) and (gBe-6) PEFs that control the repulsive wall of the potential correlate remarkably well with the ionic radii of the halide and alkali metal ions.

Distribution of topologically distinct isomers of water clusters and dipole moments of constituent water molecules at finite atmospheric temperatures.

Hydrogen-bonding (HB) patterns correspond to topologically distinct isomers of a water cluster and can be expressed by digraphs. We make use of the HB pattern to divide the configuration space of a water cluster ((H2O)n, n = 3-8) at a finite temperature. Each configuration of a water cluster generated in Monte Carlo (MC) simulation is classified into an HB pattern. The number of observed HB patterns increases exponentially with the cluster size, whereas the population of the most abundant HB pattern decreases. The populations of the HB patterns are transformed into the relative Helmholtz energies. At a finite temperature, it can be observed that isomers other than local minimum structures on the potential energy surface are highly populated. The dipole moment of a constituent molecule in a water cluster is enhanced, depending on the molecular circumstance. The change is rationalized by the difference in the local HB type of the water molecule in the HB network.