Secondary structure determination of conserved SARS-CoV-2 RNA elements by NMR spectroscopy

The current pandemic situation caused by the Betacoronavirus SARS-CoV-2 (SCoV2) highlights the need for coordinated research to combat COVID-19. A particularly important aspect is the development of medication. In addition to viral proteins, structured RNA elements represent a potent alternative as drug targets. The search for drugs that target RNA requires their high-resolution structural characterization. Using nuclear magnetic resonance (NMR) spectroscopy, a worldwide consortium of NMR researchers aims to characterize potential RNA drug targets of SCoV2. Here, we report the characterization of 15 conserved RNA elements located at the 5′ end, the ribosomal frameshift segment and the 3′-untranslated region (3′-UTR) of the SCoV2 genome, their large-scale production and NMR-based secondary structure determination. The NMR data are corroborated with secondary structure probing by DMS footprinting experiments. The close agreement of NMR secondary structure determination of isolated RNA elements with DMS footprinting and NMR performed on larger RNA regions shows that the secondary structure elements fold independently. The NMR data reported here provide the basis for NMR investigations of RNA function, RNA interactions with viral and host proteins and screening campaigns to identify potential RNA binders for pharmaceutical intervention.     

Non-peptide entry inhibitors of HIV-1 that target the gp41 coiled coil pocket

The ectodomain of HIV-1 gp41 mediates the fusion of viral and host cellular membranes. The peptide-based drug Enfuvirtide¹ is precedent that antagonists of this fusion activity may act as anti HIV-agents. Here, NMR screening was used to discover non-peptide leads against this target and resulted in the discovery of a new benzamide 1 series. This series is non-peptide, low molecular weight, and analogs have activity in a cell fusion assay with EC50 values ranging 3–41 μM. Structural work on the gp41/benzamide 1 complex was determined by NMR spectroscopy using a designed model peptide system that mimics an open pocket of the fusogenic form of the protein.     

Inhibiting HIV-1 entry: discovery of D-peptide inhibitors that target the gp41 coiled-coil pocket.

The HIV-1 gp41 protein promotes viral entry by mediating the fusion of viral and cellular membranes. A prominent pocket on the surface of a central trimeric coiled coil within gp41 was previously identified as a potential target for drugs that inhibit HIV-1 entry. We designed a peptide, IQN17, which properly presents this pocket. Utilizing IQN17 and mirror-image phage display, we identified cyclic, D-peptide inhibitors of HIV-1 infection that share a sequence motif. A 1.5 A cocrystal structure of IQN17 in complex with a D-peptide, and NMR studies, show that conserved residues of these inhibitors make intimate contact with the gp41 pocket. Our studies validate the pocket per se as a target for drug development. IQN17 and these D-peptide inhibitors are likely to be useful for development and identification of a new class of orally bioavailable anti-HIV drugs.     

NMR structure and dynamics of a designed water-soluble transmembrane domain of nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor (nAChR) is an important therapeutic target for a wide range of pathophysiological conditions, for which rational drug designs often require receptor structures at atomic resolution. Recent proof-of-concept studies demonstrated a water-solubilization approach to structure determination of membrane proteins by NMR (Slovic et al., PNAS, 101: 1828-1833, 2004; Ma et al., PNAS, 105: 16537-42, 2008). We report here the computational design and experimental characterization of WSA, a water-soluble protein with ~83% sequence identity to the transmembrane (TM) domain of the nAChR α1 subunit. Although the design was based on a low-resolution structural template, the resulting high-resolution NMR structure agrees remarkably well with the recent crystal structure of the TM domains of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC), demonstrating the robustness and general applicability of the approach. NMR T(2) dispersion measurements showed that the TM2 domain of the designed protein was dynamic, undergoing conformational exchange on the NMR timescale. Photoaffinity labeling with isoflurane and propofol photolabels identified a common binding site in the immediate proximity of the anesthetic binding site found in the crystal structure of the anesthetic-GLIC complex. Our results illustrate the usefulness of high-resolution NMR analyses of water-solubilized channel proteins for the discovery of potential drug binding sites.     

iVS analysis to evaluate the impact of scaffold diversity in the binding to cellular targets relevant in cancer

This study reports the application of inverse virtual screening (iVS) methodologies to identify cellular proteins as suitable targets for a library of heterocyclic small-molecules, with potential pharmacological implications. Standard synthetic procedures allow facile generation of these ligands showing a high degree of core scaffold diversity. Specifically, we have computationally investigated the binding efficacy of the new series for target proteins which are involved in cancer pathogenesis. As a result, nine macromolecules demonstrated efficient binding interactions for the molecular dataset, in comparison to the co-crystallised ligand for each target. Moreover, the iVS analysis led us to confirm that 27 analogues have high affinity for one or more examined cellular proteins. The additional evaluation of ADME and drug score for selected hits also highlights their capability as drug candidates, demonstrating valuable leads for further structure optimisation and biological studies.     

Drug repurposing and rediscovery: Design,synthesis and preliminary biological evaluation of 1-arylamino-3-aryloxypropan-2-ols as anti-melanoma agents

Malignant melanoma (MM) presents as the highest morbidity and mortality type in skin cancer. Herein, inspired by the previously reported anti-melanoma effect of propranolol, a widely applied β adrenergic receptor antagonist as cardiovascular drug, we set out to exploit its potential as anti-melanoma therapy based on the drug repurposing strategy. Structural optimization of propranolol yielded 5m, which exhibits dramatically improved potency on human melanoma cell growth (1.98–3.70 μM), compared to propranolol (59.5–75.8 μM). Further investigation demonstrated that 5m could inhibit colony formation of melanoma cell line (completely abolished at 2 μM for 5m, partially inhibited at 50 μM for propranolol), induce cell apoptosis and cell cycle arrest in the G₂/M phase (both observed at 1 μM). Preliminary mechanism study indicated that 5m could disrupt the cellular microtubule network, which suggested tubulin as a potential target. Docking study provided a structural insight into the interaction between 5m and tubulin. In summary, our study presents a drug repurposing case that redirects a cardiovascular agent to an anti-melanoma agent.     

In situ structural biology using in-cell NMR

BackgroundAccumulating evidence from the experimental and computational studies indicated that the functional properties of proteins are different between in vitro and living cells, raising the necessity to examine the protein structure under the native intracellular milieu. To gain structural information of the proteins inside the living cells at an atomic resolution, in-cell NMR method has been developed for the past two decades.Scope of reviewIn this review, we will overview the recent progress in the methodological developments and the biological applications of in-cell NMR, and discuss the advances and challenges in this filed.Major conclusionsA number of methods were developed to enrich the isotope-labeled proteins inside the cells, enabling the in-cell NMR observation of bacterial cells as well as eukaryotic cells. In-cell NMR has been applied to various biological systems, including de novo structure determinations, protein/protein or protein/drug interactions, and monitoring of chemical reactions exerted by the endogenous enzymes. The bioreactor system, in which the cells in the NMR tube are perfused by fresh culture medium, enabled the long-term in-cell NMR measurements, and the real-time observations of intracellular responses upon external stimuli.General significanceIn-cell NMR has become a unique technology for its ability to obtain the function-related structural information of the target proteins under the physiological or pathological cellular environments, which cannot be reconstituted in vitro.     

Nuclear magnetic resonance spectroscopy reveals the functional state of the signalling protein CheY in vivo in Escherichia coli

Two-component signal transduction (TCST) pathways are regulatory systems that are highly homologous throughout the bacterial kingdom. Their established role in virulence and absence in vertebrates has made TCST an attractive target for therapeutic intervention. However, such systems have yet to yield success in the development of novel antibiotics. CheY serves as a prototype for the analysis of response regulator function. The protein structure exhibits several conformations by both X-ray and nuclear magnetic resonance (NMR) analyses. Knowledge of which structures are relevant in vivo would be valuable in a rational drug design project. Our aim was to probe the in vivo conformation and ligand binding of CheY in Escherichia coli under resting conditions by in-cell NMR methods. CheY was selectively labelled with 15N by the control of growth and expression conditions. NMR spectra obtained in vivo demonstrated that the Mg2+ complex was the predominant form even though cells were resuspended in metal-free buffers and the intracellular free Mg2+ was low. In-cell NMR also confirmed the uptake and in vivo binding mode to CheY of small-molecular-weight compounds identified in vitro. This paper reports the first observation of the structure and interactions with a potential drug of a regulator protein in its native host in vivo using NMR spectroscopy.     

Examination of the specificity of tumor cell derived exosomes with tumor cells in vitro

Small endogenous vesicles called exosomes are beginning to be explored as drug delivery vehicles. The in vivo targets of exosomes are poorly understood; however, they are believed to be important in cell-to-cell communication and may play a prominent role in cancer metastasis. We aimed to elucidate whether cancer derived exosomes can be used as drug delivery vehicles that innately target tumors over normal tissue. Our in vitro results suggest that while there is some specificity towards cancer cells over “immortalized” cells, it is unclear if the difference is sufficient to achieve precise in vivo targeting. Additionally, we found that exosomes associate with their cellular targets to a significantly greater extent (> 10-fold) than liposomes of a similar size. Studies on the association of liposomes mimicking the unique lipid content of exosomes revealed that the lipid composition contributes significantly to cellular adherence/internalization. Cleavage of exosome surface proteins yielded exosomes exhibiting reduced association with their cellular targets, demonstrating the importance of proteins in binding/internalization. Furthermore, although acidic conditions are known to augment the metastatic potential of tumors, we found that cells cultured at low pH released exosomes with significantly less potential for cellular association than cells cultured at physiological pH.     

Biochemical and structural characterization of alanine racemase from Bacillus anthracis (Ames)

Background  

Bacillus anthracis is the causative agent of anthrax and a potential bioterrorism threat. Here we report the biochemical and structural characterization of B. anthracis (Ames) alanine racemase (Alr _Bax ), an essential enzyme in prokaryotes and a target for antimicrobial drug development. We also compare the native Alr _Bax structure to a recently reported structure of the same enzyme obtained through reductive lysine methylation.     

Monitoring of the ethionamide pro-drug activation in mycobacteria by (1)H high resolution magic angle spinning NMR

In this study, we use HRMAS NMR as a non-invasive technique to monitor the in vivo metabolism of a xenobiotic. The antituberculosis Ethionamide is a pro-drug that has to be activated in mycobacteria before inhibiting its cellular target. The use of (1)H HRMAS NMR has allowed to detect a metabolite (ETH*) of the drug directly in living bacteria, even with a spectrometer operating at the relatively low magnetic field of 300MHz. We show that metabolism monitoring of an unlabelled drug at a therapeutically relevant concentration as low as 5mug/ml is within reach of the technique. (1)H HRMAS NMR in combination with diffusion filtering leads to the conclusion that the metabolite is located inside the intact cells. The comparison of the metabolite NMR signature with that of synthetic molecules proves the non-identity of ETH* with the ETH derivatives described previously in the literature.     

Determination of strongly overlapping signaling activity from microarray data

Background  

As numerous diseases involve errors in signal transduction, modern therapeutics often target proteins involved in cellular signaling. Interpretation of the activity of signaling pathways during disease development or therapeutic intervention would assist in drug development, design of therapy, and target identification. Microarrays provide a global measure of cellular response, however linking these responses to signaling pathways requires an analytic approach tuned to the underlying biology. An ongoing issue in pattern recognition in microarrays has been how to determine the number of patterns (or clusters) to use for data interpretation, and this is a critical issue as measures of statistical significance in gene ontology or pathways rely on proper separation of genes into groups.     

Solution- and solid-state NMR studies of GPCRs and their ligands

G protein-coupled receptors (GPCRs) represent one of the major targets of new drugs on the market given their roles as key membrane receptors in many cellular signalling pathways. Structure-based drug design has potential to be the most reliable method for novel drug discovery. Unfortunately, GPCR-ligand crystallisation for X-ray diffraction studies is very difficult to achieve. However, solution- and solid-state NMR approaches have been developed and have provided new insights, particularly focussing on the study of protein-ligand interactions which are vital for drug discovery. This review provides an introduction for new investigators of GPCRs/ligand interactions using NMR spectroscopy. The guidelines for choosing a system for efficient isotope labelling of GPCRs and their ligands for NMR studies will be presented, along with an overview of the different sample environments suitable for generation of high resolution structural information from NMR spectra.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N chemical shift assignments of Saccharomyces cerevisiae type 1 thioredoxin in the oxidized state by solution NMR spectroscopy

Thioredoxins (Trx) are ubiquitous proteins that regulate several biochemical processes inside the cell. Trx is an important player, displaying oxidoreductase activity and helping to keep and regulate the oxidative state of the cellular environment. Trx also participates in the regulation of many cellular functions, such as DNA synthesis, protection against oxidative stress, cell cycle and signal transduction. The oxidized Trx is the target for another set of proteins, such as thioredoxin reductase (TrR), which used the reductive potential of NADPH. The oxidized state of Trx also plays important role in regulation of redox state in the cells. In this regard, the oxidized form of Trx is a putative conformer that contributes to the cellular redox environment. Here we report the chemical shift assignments (¹H, ¹³C and ¹⁵N) in solution at 15 °C. We also showed the secondary structure analysis of the oxidized form of yeast thioredoxin (yTrx1) as basis for future NMR studies of protein–target interactions and dynamics. The assignment was done at low concentration (200 µM) because it is important to keep intact the water cavity.     

Molecular hybridization conceded exceptionally potent quinolinyl-oxadiazole hybrids through phenyl linked thiosemicarbazide antileishmanial scaffolds: In silico validation and SAR studies

The high potential of quinoline containing natural products and their derivatives in medicinal chemistry led us to discover a novel series of compounds 6–23 based on the concept of molecular hybridization. Most of the synthesized analogues exhibited potent leishmanicidal potential. The most potent compound (23, IC₅₀ = 0.10 ± 0.001 μM) among the series was found ∼70 times more lethal than the standard drug. The current series 6–23 conceded in the development of fourteen (14) extraordinarily active compounds against leishmaniasis. In silico analysis were also performed to probe the mode of action while all the compounds structure were established by NMR and Mass spectral analysis.     

Properties and identification of antibiotic drug targets

Background  

We analysed 48 non-redundant antibiotic target proteins from all bacteria, 22 antibiotic target proteins from E. coli only and 4243 non-drug targets from E. coli to identify differences in their properties and to predict new potential drug targets.     

A refinement protocol to determine structure,topology, and depth of insertion of membrane proteins using hybrid solution and solid-state NMR restraints

To fully describe the fold space and ultimately the biological function of membrane proteins, it is necessary to determine the specific interactions of the protein with the membrane. This property of membrane proteins that we refer to as structural topology cannot be resolved using X-ray crystallography or solution NMR alone. In this article, we incorporate into XPLOR-NIH a hybrid objective function for membrane protein structure determination that utilizes solution and solid-state NMR restraints, simultaneously defining structure, topology, and depth of insertion. Distance and angular restraints obtained from solution NMR of membrane proteins solubilized in detergent micelles are combined with backbone orientational restraints (chemical shift anisotropy and dipolar couplings) derived from solid-state NMR in aligned lipid bilayers. In addition, a supplementary knowledge-based potential, E _z (insertion depth potential), is used to ensure the correct positioning of secondary structural elements with respect to a virtual membrane. The hybrid objective function is minimized using a simulated annealing protocol implemented into XPLOR-NIH software for general use. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

NMR solution structure and backbone dynamics of domain III of the E protein of tick-borne Langat flavivirus suggests a potential site for molecular recognition

Flaviviruses cause many human diseases, including dengue fever, yellow fever, West Nile viral encephalitis, and hemorrhagic fevers, and are transmitted to their vertebrate hosts by infected mosquitoes and ticks. Domain III of the envelope protein (E-D3) is considered to be the primary viral determinant involved in the virus-host-cell receptor interaction, and thus represents an excellent target for antiviral drug development. Langat (LGT) virus is a naturally attenuated BSL-2 TBE virus and is a model for the pathogenic BSL-3 and BSL-4 viruses in the serogroup. We have determined the solution structure of LGT-E-D3 using heteronuclear NMR spectroscopy. The backbone dynamics of LGT-E-D3 have been investigated using 15N relaxation measurements. A detailed analysis of the solution structure and dynamics of LGT-E-D3 suggests potential residues that could form a surface for molecular recognition, and thereby represent a target site for antiviral therapeutics design.