Potential of in vitro nuclear magnetic resonance of biofluids and tissues in clinical research.

Body fluids, cells, and tissues contain a wide variety of metabolites that consist of a mixture of various low-molecular-weight compounds, including amino acids, peptides, lipids, nucleic acids, and organic acids, which makes comprehensive analysis more difficult. Quantitative nuclear magnetic resonance (NMR) spectroscopy is a well-established analytical technique for analyzing the metabolic profiles of body fluids, cells, and tissues. It enables fast and comprehensive detection, characterization, a high level of experimental reproducibility, minimal sample preparation, and quantification of various endogenous metabolites. In recent times, NMR-based metabolomics has been appreciably utilized in diverse branches of medicine, including microbiology, toxicology, pathophysiology, pharmacology, nutritional intervention, and disease diagnosis/prognosis. In this review, the utility of NMR-based metabolomics in clinical studies is discussed. The significance of in vitro NMR-based metabolomics as an effective tool for detecting metabolites and their variations in different diseases are discussed, together with the possibility of identifying specific biomarkers that can contribute to early detection and diagnosis of disease.

Repurposing of US-FDA approved drugs against SARS-CoV-2 main protease (Mpro) by using STD-NMR spectroscopy, in silico studies and antiviral assays.

SARS-CoV-2 Main protease (Mpro) is a well-known drug target against SARS-CoV-2 infection. Identification of Mpro inhibitors is vigorously pursued due to its crucial role in viral replication. The present study was aimed to identify Mpro inhibitors via repurposing of US-FDA approved drugs by STD-NMR spectroscopy. In this study, 156 drugs and natural compounds were evaluated against Mpro. Among them, 10 drugs were found to be interacting with Mpro, including diltiazem HCl (1), mefenamic acid (2), losartan potassium (3), mexiletine HCl (4), glaucine HBr (5), trimebutine maleate (6), flurbiprofen (7), amantadine HCl (8), dextromethorphan (9), and lobeline HCl (10) in STD-NMR spectroscopy. Their interactions were compared with three standards (Repurposed anti-viral drugs), dexamethasone, chloroquine phosphate, and remdesivir. Thermal stability of Mpro and dissociation constant (Kd) of six interacting drugs were also determined using DSF. RMSD plots in MD simulation studies showed the formation of stable protein-ligand complexes. They were further examined for their antiviral activity by plaque reduction assay against SARS-CoV-2, which showed 55-100% reduction in viral plaques. This study demonstrates the importance of drug repurposing against emerging and neglected diseases. This study also exhibits successful application of STD-NMR spectroscopy combined with plaque reduction assay in rapid identification of potential anti-viral agents.

Low-Dimensional Compounds Containing Bioactive Ligands. Part XX: Crystal Structures, Cytotoxic, Antimicrobial Activities and DNA/BSA Binding of Oligonuclear Zinc Complexes with Halogen Derivatives of 8-Hydroxyquinoline

Two tetranuclear [Zn4Cl2(ClQ)6]·2DMF (1) and [Zn4Cl2(ClQ)6(H2O)2]·4DMF (2), as well as three dinuclear [Zn2(ClQ)3(HClQ)3]I3 (3), [Zn2(dClQ)2(H2O)6(SO4)] (4) and [Zn2(dBrQ)2(H2O)6(SO4)] (5), complexes (HClQ = 5-chloro-8-hydroxyquinoline, HdClQ = 5,7-dichloro-8-hydroxyquinoline and HdBrQ = 5,7-dibromo-8-hydroxyquinoline) were prepared as possible anticancer or antimicrobial agents and characterized by IR spectroscopy, elemental analysis and single crystal X-ray structure analysis. The stability of the complexes in solution was verified by NMR spectroscopy. Antiproliferative activity and selectivity of the prepared complexes were studied using in vitro MTT assay against the HeLa, A549, MCF-7, MDA-MB-231, HCT116 and Caco-2 cancer cell lines and on the Cos-7 non-cancerous cell line. The most sensitive to the tested complexes was Caco-2 cell line. Among the tested complexes, complex 3 showed the highest cytotoxicity against all cell lines. Unfortunately, all complexes showed only poor selectivity to normal cells, except for complex 5, which showed a certain level of selectivity. Antibacterial potential was observed for complex 5 only. Moreover, the DNA/BSA binding potential of complexes 1–3 was investigated by UV-vis and fluorescence spectroscopic methods.

The 5'UTR of HCoV-OC43 adopts a topologically constrained structure to intrinsically repress translation.

The emergence of SARS-CoV-2, which is responsible for the COVID-19 pandemic, has highlighted the need for rapid characterization of viral mechanisms associated with cellular pathogenesis. Viral UTRs represent conserved genomic elements that contribute to such mechanisms. Structural details of most CoV UTRs are not available, however. Experimental approaches are needed to allow for the facile generation of high-quality viral RNA tertiary structural models, which can facilitate comparative mechanistic efforts. By integrating experimental and computational techniques, we herein report the efficient characterization of conserved RNA structures within the 5'UTR of the HCoV-OC43 genome, a lab-tractable model coronavirus. We provide evidence that the 5'UTR folds into a structure with well-defined stem-loops (SLs) as determined by chemical probing and direct detection of hydrogen bonds by NMR. We combine experimental base-pair restraints with global structural information from SAXS to generate a 3D model that reveals that SL1-4 adopts a topologically constrained structure wherein SLs 3 and 4 coaxially stack. Coaxial stacking is mediated by short linker nucleotides and allows SLs 1 to 2 to sample different cojoint orientations by pivoting about the SL3,4 helical axis. To evaluate the functional relevance of the SL3,4 coaxial helix, we engineered luciferase reporter constructs harboring the HCoV-OC43 5'UTR with mutations designed to abrogate coaxial stacking. Our results reveal that the SL3,4 helix intrinsically represses translation efficiency since the destabilizing mutations correlate with increased luciferase expression relative to wildtype without affecting reporter mRNA levels, thus highlighting how the 5'UTR structure contributes to the viral mechanism.

Evidence for multiple binding modes in the initial contact between SARS-CoV-2 spike S1 protein and cell surface glycans.

Infection of host cells by SARS-CoV-2 begins with recognition by the virus S (spike) protein of cell surface heparan sulfate (HS), tethering the virus to the extracellular matrix environment, and causing the subunit S1-RBD to undergo a conformational change into the 'open' conformation. These two events promote the binding of S1-RBD to the angiotensin converting enzyme 2 (ACE2) receptor, a preliminary step toward viral-cell membrane fusion. Combining ligand-based NMR spectroscopy with molecular dynamics, oligosaccharide analogues were used to explore the interactions between S1-RBD of SARS CoV-2 and HS, revealing several low-specificity binding modes and previously unidentified potential sites for the binding of extended HS polysaccharide chains. The evidence for multiple binding modes also suggest that highly specific inhibitors will not be optimal against protein S but, rather, diverse HS-based structures, characterized by high affinity and including multi-valent compounds, may be required.

The COVID19-NMR Consortium: A Public Report on the Impact of this New Global Collaboration.

The outbreak of COVID-19 in December 2019 required the formation of international consortia for a coordinated scientific effort to understand and combat the virus. In this Viewpoint Article, we discuss how the NMR community has gathered to investigate the genome and proteome of SARS-CoV-2 and tested them for binding to low-molecular-weight binders. External factors including extended lockdowns due to the global pandemic character of the viral infection triggered the transition from locally focused collaborative research conducted within individual research groups to digital exchange formats for immediate discussion of unpublished results and data analysis, sample sharing, and coordinated research between more than 50 groups from 18 countries simultaneously. We discuss key lessons that might pertain after the end of the pandemic and challenges that we need to address.

Structural insights into the activity regulation of full-length non-structural protein 1 from SARS-CoV-2.

Non-structural protein 1 (Nsp1) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a major virulence factor and thus an attractive drug target. The last 33 amino acids of Nsp1 have been shown to bind within the mRNA entry tunnel of the 40S ribosomal subunit, shutting off host gene expression. Here, we report the solution-state structure of full-length Nsp1, which features an α/ß fold formed by a six-stranded, capped ß-barrel-like globular domain (N-terminal domain [NTD]), flanked by short N-terminal and long C-terminal flexible tails. The NTD has been found to be critical for 40S-mediated viral mRNA recognition and promotion of viral gene expression. We find that in free Nsp1, the NTD mRNA-binding surface is occluded by interactions with the acidic C-terminal tail, suggesting a mechanism of activity regulation based on the interplay between the folded NTD and the disordered C-terminal region. These results are relevant for drug-design efforts targeting Nsp1.

Binding of Glycans to the SARS CoV-2 Spike Protein, an Open Question: NMR Data on Binding Site Localization, Affinity, and Selectivity.

We have used NMR experiments to explore the binding of selected glycans and glycomimetics to the SARS CoV-2 spike glycoprotein (S-protein) and to its receptor binding domain (RBD). STD NMR experiments confirm the binding of sialoglycans to the S-protein of the prototypic Wuhan strain virus and yield dissociation constants in the millimolar range. The absence of STD effects for sialoglycans in the presence of the Omicron/BA.1 S-protein reflects a loss of binding as a result of S-protein evolution. Likewise, no STD effects are observed for the deletion mutant Δ143-145 of the Wuhan S-protein, thus supporting localization of the binding site in the N-terminal domain (NTD). The glycomimetics Oseltamivir and Zanamivir bind weakly to the S-protein of both virus strains. Binding of blood group antigens to the Wuhan S-protein cannot be confirmed by STD NMR. Using 1 H,15 N TROSY HSQC-based chemical shift perturbation (CSP) experiments, we excluded binding of any of the ligands studied to the RBD of the Wuhan S-protein. Our results put reported data on glycan binding into perspective and shed new light on the potential role of glycan-binding to the S-protein.

Comprehensive Fragment Screening of the SARS-CoV-2 Proteome Explores Novel Chemical Space for Drug Development.

SARS-CoV-2 (SCoV2) and its variants of concern pose serious challenges to the public health. The variants increased challenges to vaccines, thus necessitating for development of new intervention strategies including anti-virals. Within the international Covid19-NMR consortium, we have identified binders targeting the RNA genome of SCoV2. We established protocols for the production and NMR characterization of more than 80 % of all SCoV2 proteins. Here, we performed an NMR screening using a fragment library for binding to 25 SCoV2 proteins and identified hits also against previously unexplored SCoV2 proteins. Computational mapping was used to predict binding sites and identify functional moieties (chemotypes) of the ligands occupying these pockets. Striking consensus was observed between NMR-detected binding sites of the main protease and the computational procedure. Our investigation provides novel structural and chemical space for structure-based drug design against the SCoV2 proteome.

NMR study of human macroPARPs domains: 1H, 15N and 13C resonance assignment of hPARP14 macro domain 2 in the free and the ADPr bound state.

hPARP14 is a human ADP-ribosyl-transferase (ART) that belongs to the macroPARPs family, together with hPARP9 and hPARP15. It contains a tandem of three macro domains (MD) while each of them has different properties. The first one, namely MD1, has not been reported to exhibit a high binding affinity for ADP-ribose (ADPr) in contrast to the following two (MD2 and MD3). All three MDs exhibit an α/ß/α sandwich-like fold as reported by the deposited crystallographic structures. MD2 and MD3 recognize mono-ADP-ribosylated (MARylated) but not poly-ADP-ribosylated (PARylated) substrates and thus they allow hPARP14 to bind its targets, which can be potentially MARylated by its catalytic domain (CD). hPARP14 participates in DNA damage repair process and immune response against viruses like SARS-CoV-2, which also harbors an MD fold. Furthermore, hPARP14 like the other two macroPARPs (hPARP9 and hPARP15), is implicated in numerous types of cancer, such as B-aggressive lymphoma and sarcoma, rendering its MDs as potential important drug targets. Herein, we report the complete NMR backbone and side chain assignment (1H, 13C, 15N) of hPARP14 MD2 in the free and ADPr bound states and the NMR chemical shift-based prediction of its secondary structure elements. This is the first reported NMR study of a hPARP macro domain, paving the way to screen by NMR chemical compounds which may alter the ability of hPARP14 to interact with its substrates affecting its function.

The PROVIT Study-Effects of Multispecies Probiotic Add-on Treatment on Metabolomics in Major Depressive Disorder-A Randomized, Placebo-Controlled Trial.

The gut-brain axis plays a role in major depressive disorder (MDD). Gut-bacterial metabolites are suspected to reduce low-grade inflammation and influence brain function. Nevertheless, randomized, placebo-controlled probiotic intervention studies investigating metabolomic changes in patients with MDD are scarce. The PROVIT study (registered at clinicaltrials.com NCT03300440) aims to close this scientific gap. PROVIT was conducted as a randomized, single-center, double-blind, placebo-controlled multispecies probiotic intervention study in individuals with MDD (n = 57). In addition to clinical assessments, metabolomics analyses (1H Nuclear Magnetic Resonance Spectroscopy) of stool and serum, and microbiome analyses (16S rRNA sequencing) were performed. After 4 weeks of probiotic add-on therapy, no significant changes in serum samples were observed, whereas the probiotic groups' (n = 28) stool metabolome shifted towards significantly higher concentrations of butyrate, alanine, valine, isoleucine, sarcosine, methylamine, and lysine. Gallic acid was significantly decreased in the probiotic group. In contrast, and as expected, no significant changes resulted in the stool metabolome of the placebo group. Strong correlations between bacterial species and significantly altered stool metabolites were obtained. In summary, the treatment with multispecies probiotics affects the stool metabolomic profile in patients with MDD, which sets the foundation for further elucidation of the mechanistic impact of probiotics on depression.

NMR Observation of Sulfhydryl Signals in SARS-CoV-2 Main Protease Aids Structural Studies.

The 68-kDa homodimeric 3C-like protease of SARS-CoV-2, Mpro (3CLpro /Nsp5), is a key antiviral drug target. NMR spectroscopy of this large system proved challenging and resonance assignments have remained incomplete. Here we present the near-complete (>97 %) backbone assignments of a C145A variant of Mpro (Mpro C145A ) both with, and without, the N-terminal auto-cleavage substrate sequence, in its native homodimeric state. We also present SILLY (Selective Inversion of thioL and Ligand for NOESY), a simple yet effective pseudo-3D NMR experiment that utilizes NOEs to identify interactions between Cys-thiol or aliphatic protons, and their spatially proximate backbone amides in a perdeuterated protein background. High protection against hydrogen exchange is observed for 10 of the 11 thiol groups in Mpro C145A , even those that are partially accessible to solvent. A combination of SILLY methods and high-resolution triple-resonance NMR experiments reveals site-specific interactions between Mpro , its substrate peptides, and other ligands, which present opportunities for competitive binding studies in future drug design efforts.

A review of the effects of ATP and hydroxychloroquine on the phase separation of the SARS-CoV-2 nucleocapsid protein.

SARS-CoV-2 is the coronavirus causing the ongoing pandemic with > 460 millions of infections and > 6 millions of deaths. SARS-CoV-2 nucleocapsid (N) is the only structural protein which plays essential roles in almost all key steps of the viral life cycle with its diverse functions depending on liquid-liquid phase separation (LLPS) driven by interacting with various nucleic acids. The 419-residue N protein is highly conserved in all variants including delta and omicron, and composed of both folded N-/C-terminal domains (NTD/CTD) as well as three long intrinsically disordered regions (IDRs). Recent results have suggested that its CTD and IDRs are also cryptic nucleic acid-binding domains. In this context, any small molecules capable of interfering in its interaction with nucleic acids are anticipated to modulate its LLPS and associated functions. Indeed, ATP, the energy currency existing at very high concentrations (2-12 mM) in all living cells but absent in viruses, modulates LLPS of N protein, and consequently appears to be evolutionarily hijacked by SARS-CoV-2 to promote its life cycle. Hydroxychloroquine (HCQ) has been also shown to specifically bind NTD and CTD to inhibit their interactions with nucleic acids, as well as to disrupt LLPS. Particularly, the unique structure of the HCQ-CTD complex offers a promising strategy for further design of anti-SARS-CoV-2 drugs with better affinity and specificity. The finding may indicate that LLPS is indeed druggable by small molecules, thus opening up a promising direction for drug discovery/design by targeting LLPS in general.

High-Resolution Magic-Angle Spinning NMR Spectroscopy for Evaluation of Cell Shielding by Virucidal Composites Based on Biogenic Silver Nanoparticles, Flexible Cellulose Nanofibers and Graphene Oxide.

Antiviral and non-toxic effects of silver nanoparticles onto in vitro cells infected with coronavirus were evaluated in this study using High-Resolution Magic-Angle Spinning Nuclear Magnetic Resonance (HR-MAS NMR) spectroscopy. Silver nanoparticles were designed and synthesized using an orange flavonoid-hesperetin (HST)-for reduction of silver(I) and stabilization of as obtained nanoparticles. The bio-inspired process is a simple, clean, and sustainable way to synthesize biogenic silver nanoparticles (AgNP@HST) with diameters of ∼20 nm and low zeta potential (-40 mV), with great colloidal stability monitored for 2 years. The nanoparticles were used for the fabrication of two types of antiviral materials: colloids (AgNP@HST spray) and 3D flexible nanostructured composites. The composites, decorated with AgNP@HST (0.05 mmol L-1), were made using cellulose nanofibers (CNF) obtained from orange peel and graphene oxide (GO), being denominated CNF@GO@AgNP@HST. Both materials showed high virucidal activity against coronaviruses in cell infection in vitro models and successfully inhibited the viral activity in cells. HR-MAS 1H-NMR technique was used for determining nanomaterials' effects on living cells and their influences on metabolic pathways, as well as to study viral effects on cells. It was proven that none of the manufactured materials showed toxicity towards the intact cells used. Furthermore, viral infection was reverted when cells, infected with the coronavirus, were treated using the as-fabricated nanomaterials. These significant results open possibilities for antiviral application of 3D flexible nanostructured composite such as packaging papers and filters for facial masks, while the colloidal AgNP@HST spray can be used for disinfecting surfaces, as well as a nasal, mouth, and eye spray.

Persistence of Metabolomic Changes in Patients during Post-COVID Phase: A Prospective, Observational Study.

Several relatively recently published studies have shown changes in plasma metabolites in various viral diseases such as Zika, Dengue, RSV or SARS-CoV-1. The aim of this study was to analyze the metabolome profile of patients during acute COVID-19 approximately one month after the acute infection and to compare these results with healthy (SARS-CoV-2-negative) controls. The metabolome analysis was performed by NMR spectroscopy from the peripheral blood of patients and controls. The blood samples were collected on 3 different occasions (at admission, during hospitalization and on control visit after discharge from the hospital). When comparing sample groups (based on the date of acquisition) to controls, there is an indicative shift in metabolomics features based on the time passed after the first sample was taken towards controls. Based on the random forest algorithm, there is a strong discriminatory predictive value between controls and different sample groups (AUC equals 1 for controls versus samples taken at admission, Mathew correlation coefficient equals 1). Significant metabolomic changes persist in patients more than a month after acute SARS-CoV-2 infection. The random forest algorithm shows very strong discrimination (almost ideal) when comparing metabolite levels of patients in two various stages of disease and during the recovery period compared to SARS-CoV-2-negative controls.

In Silico and Experimental Investigation of the Biological Potential of Some Recently Developed Carprofen Derivatives

The efficient regioselective bromination and iodination of the nonsteroidal anti-inflammatory drug (NSAID) carprofen were achieved by using bromine and iodine monochloride in glacial acetic acid. The novel halogenated carprofen derivatives were functionalized at the carboxylic group by esterification. The regioselectivity of the halogenation reaction was evidenced by NMR spectroscopy and confirmed by X-ray analysis. The compounds were screened for their in vitro antibacterial activity against planktonic cells and also for their anti-biofilm effect, using Gram-positive bacteria (Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212) and Gram-negative bacteria (Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853). The cytotoxic activity of the novel compounds was tested against HeLa cells. The pharmacokinetic and pharmacodynamic profiles of carprofen derivatives, as well as their toxicity, were established by in silico analyses.

Applications and Challenges for Metabolomics via Nuclear Magnetic Resonance Spectroscopy

Introduction Even though metabolomics is about 20 years old, the interest in this “-omic” science is still growing, and high expectations remain in the scientific community for new practical applications in biomedicine and in the agricultural field. [...]far, biomedical metabolomic studies have produced great advancements in biomarker discovery, identification of novel metabolites, and more detailed characterization of biological pathways involved in the manifestation and progression of diseases. The EBC and blood plasma samples from 20 workers exposed to NPs were collected pre-shift (i.e., before 2.5 h of exposure to NPs) and post-shift (i.e., after NP exposure). [...]20 controls (not exposed to NPs) were enrolled for this study. [...]the current Special Issue of Applied Sciences offers a variety of examples on how NMR-based metabolomics can potentially be used in several and varied settings.

1H, 13C and 15N chemical shift assignment of the stem-loops 5b + c from the 5'-UTR of SARS-CoV-2.

The ongoing pandemic of the respiratory disease COVID-19 is caused by the SARS-CoV-2 (SCoV2) virus. SCoV2 is a member of the Betacoronavirus genus. The 30 kb positive sense, single stranded RNA genome of SCoV2 features 5'- and 3'-genomic ends that are highly conserved among Betacoronaviruses. These genomic ends contain structured cis-acting RNA elements, which are involved in the regulation of viral replication and translation. Structural information about these potential antiviral drug targets supports the development of novel classes of therapeutics against COVID-19. The highly conserved branched stem-loop 5 (SL5) found within the 5'-untranslated region (5'-UTR) consists of a basal stem and three stem-loops, namely SL5a, SL5b and SL5c. Both, SL5a and SL5b feature a 5'-UUUCGU-3' hexaloop that is also found among Alphacoronaviruses. Here, we report the extensive 1H, 13C and 15N resonance assignment of the 37 nucleotides (nts) long sequence spanning SL5b and SL5c (SL5b + c), as basis for further in-depth structural studies by solution NMR spectroscopy.

Bi(III) I-Complexes of Porphyrins for Biomedicine: Synthesis and Spectral-Optical Properties

Bismuth complexes of porphyrins are of interest for IR luminescence diagnostics of cancer, since rather intense emission bands in the range of 800–920 nm have been found. In connection with the COVID-19 pandemic, bismuth compounds are also of interest in the treatment of coronavirus infection. Bismuth complexes of porphyrins of various spatial configurations have been synthesized, and several spectral-optical properties have been investigated. The influence of various substituents on the spectral characteristics was evaluated by methods of studying electronic absorption spectra, luminescence spectra, IR-, and 1H NMR spectroscopy.

The SARS-CoV-2 Spike Glycoprotein Directly Binds Exogeneous Sialic Acids: A NMR View.

The interaction of the SARS CoV2 spike glycoprotein with two sialic acid-containing trisaccharides (α2,3 and α2,6 sialyl N-acetyllactosamine) has been demonstrated by NMR. The NMR-based distinction between the signals of those sialic acids in the glycans covalently attached to the spike protein and those belonging to the exogenous α2,3 and α2,6 sialyl N-acetyllactosamine ligands has been achieved by synthesizing uniformly 13 C-labelled trisaccharides at the sialic acid and galactose moieties. STD-1 H,13 C-HSQC NMR experiments elegantly demonstrate the direct interaction of the sialic acid residues of both trisaccharides with additional participation of the galactose moieties, especially for the α2,3-linked analogue. Additional experiments with the spike protein in the presence of a specific antibody for the N-terminal domain and with the isolated receptor binding and N-terminal domains of the spike protein unambiguously show that the sialic acid binding site is located at the N-terminal domain.