Solution Nuclear Magnetic Resonance Spectroscopy Research Articles

Exploring the dynamics and interactions of the N-myc transactivation domain through solution nuclear magnetic resonance spectroscopy.

Myc proteins are transcription factors crucial for cell proliferation. They have a C-terminal domain that mediates Max and DNA binding, and an N-terminal disordered region culminating in the transactivation domain (TAD). The TAD participates in many protein-protein interactions, notably with kinases that promote stability (Aurora-A) or degradation (ERK1, GSK3) via the ubiquitin-proteasome system. We probed the structure, dynamics and interactions of N-myc TAD using nuclear magnetic resonance (NMR) spectroscopy following its complete backbone assignment. Chemical shift analysis revealed that N-myc has two regions with clear helical propensity: Trp77-Glu86 and Ala122-Glu132. These regions also have more restricted ps-ns motions than the rest of the TAD, and, along with the phosphodegron, have comparatively high transverse (R2) 15N relaxation rates, indicative of slower timescale dynamics and/or chemical exchange. Collectively these features suggest differential propensities for structure and interaction, either internal or with binding partners, across the TAD. Solution studies on the interaction between N-myc and Aurora-A revealed a previously uncharacterised binding site. The specificity and kinetics of sequential phosphorylation of N-myc by ERK1 and GSK3 were characterised using NMR and resulted in no significant structural changes outside the phosphodegron. When the phosphodegron was doubly phosphorylated, N-myc formed a robust interaction with the Fbxw7-Skp1 complex, but mapping the interaction by NMR suggests a more extensive interface. Our study provides foundational insights into N-myc TAD dynamics and a backbone assignment that will underpin future work on the structure, dynamics, interactions and regulatory post-translational modifications of this key oncoprotein.

Characterizing the amyloid core region of the tumor suppressor protein p16INK4a using a limited proteolysis and peptide-based approach

The human tumor suppressor p16INK4a is a small monomeric protein that can form amyloid structures. Formation of p16INK4a amyloid fibrils is induced by oxidation which creates an intermolecular disulfide bond. The conversion into amyloid is associated with a change from an all α-helical structure into β-sheet fibrils. Currently, structural insights into p16INK4a amyloid fibrils are lacking. Here, we investigate the amyloid-forming regions of this tumor suppressor using isotope-labeling limited-digestion mass spectrometry analysis. We discover two key regions that likely form the structured core of the amyloid. Further investigations using thioflavin-T fluorescence assays, electron microscopy and solution nuclear magnetic resonance spectroscopy of shorter peptide regions confirm the self-assembly of the identified sequences that include methionine and leucine repeat regions. This work describes a simple approach for studying protein motifs involved in the conversion of monomeric species into aggregated fibril structures. It provides first insights into the polypeptide sequence underlying the core structure of amyloid p16INK4a formed after a unique oxidation-driven structural transition.

Solution structure, dynamics and tetrahedral assembly of Anti-TRAP, a homo-trimeric triskelion-shaped regulator of tryptophan biosynthesis in Bacillus subtilis

Cellular production of tryptophan is metabolically expensive and tightly regulated. The small Bacillus subtilis zinc binding Anti-TRAP protein (AT), which is the product of the yczA/rtpA gene, is upregulated in response to accumulating levels of uncharged tRNATrp through a T-box antitermination mechanism. AT binds to the undecameric axially symmetric ring-shaped protein TRAP (trp RNA Binding Attenuation Protein), thereby preventing it from binding to the trp leader RNA. This reverses the inhibitory effect of TRAP on transcription and translation of the trp operon. AT principally adopts two symmetric oligomeric states, a trimer (AT3) featuring three-fold axial symmetry or a dodecamer (AT12) comprising a tetrahedral assembly of trimers, whereas only the trimeric form binds and inhibits TRAP. We apply native mass spectrometry (nMS) and small-angle x-ray scattering (SAXS), together with analytical ultracentrifugation (AUC) to monitor the pH and concentration-dependent equilibrium between the trimeric and dodecameric structural forms of AT. In addition, we use solution nuclear magnetic resonance (NMR) spectroscopy to determine the solution structure of AT3, while heteronuclear 15N relaxation measurements on both oligomeric forms of AT provide insights into the dynamic properties of binding-active AT3 and binding-inactive AT12, with implications for TRAP binding and inhibition.

A simulation-guided “swapping” protocol for NMR titrations to study protein–protein interactions

Solution nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for characterizing protein–protein interactions. NMR-monitored titration proves to be effective in determining dissociation constants, particularly for Kd values in the micromolar to millimolar range. In conventional NMR titrations, planning for optimal titration conditions requires a prior estimate of Kd. In addition, a highly concentrated ligand stock solution is often required, posing challenges when the ligand exhibits limited solubility and stability at elevated concentrations. To overcome these constraints, we propose a simulation-guided “swapping” protocol for NMR titrations. Guided by simulations of the binding curves, two samples, one with zero and the other with maximum ligand concentration, but both containing identical protein concentrations, initiate the titration. Using a “swapping” strategy, intermediate ligand concentrations in between those of the two initial samples are generated without the need of a concentrated ligand stock, while maintaining constant protein concentration. More importantly, this protocol facilitates estimation of Kd by early titration points and allows on-the-fly optimization of the titration points. The proposed approach enhances the efficiency of NMR titrations and provides a straightforward means to optimize the experimental conditions for the titrations.

NMR characterization of RNA binding property of the DEAD-box RNA helicase DDX3X and its implications for helicase activity

The DEAD-box RNA helicase (DDX) plays a central role in many aspects of RNA metabolism by remodeling the defined structure of RNA molecules. While a number of structural studies have revealed the atomistic details of the interaction between DDX and RNA ligands, the molecular mechanism of how this molecule unwinds a structured RNA into an unstructured single-stranded RNA (ssRNA) has largely remained elusive. This is due to challenges in structurally characterizing the unwinding intermediate state and the lack of thermodynamic details underlying this process. In this study, we use solution nuclear magnetic resonance (NMR) spectroscopy to characterize the interaction of human DDX3X, a member of the DDX family, with various RNA ligands. Our results show that the inherent binding affinity of DDX3X for ssRNA is significantly higher than that for structured RNA elements. This preferential binding, accompanied by the formation of a domain-closed conformation in complex with ssRNA, effectively stabilizes the denatured ssRNA state and thus underlies the unwinding activity of DDX3X. Our results provide a thermodynamic and structural basis for the DDX function, whereby DDX can recognize and remodel a distinct set of structured RNAs to participate in a wide range of physiological processes.

Monitoring Drug-Protein Interactions in the Bacterial Periplasm by Solution Nuclear Magnetic Resonance Spectroscopy.

The rapid spread of antimicrobial resistance across bacterial pathogens poses a serious risk to the efficacy and sustainability of available treatments. This puts pressure on research concerning the development of new drugs. Here, we present an in-cell NMR-based research strategy to monitor the activity of the enzymes located in the periplasmic space delineated by the inner and outer membranes of Gram-negative bacteria. We demonstrate its unprecedented analytical power in monitoring in situ and in real time (i) the hydrolysis of β-lactams by β-lactamases, (ii) the interaction of drugs belonging to the β-lactam family with their essential targets, and (iii) the binding of inhibitors to these enzymes. We show that in-cell NMR provides a powerful analytical tool for investigating new drugs targeting the molecular components of the bacterial periplasm.

EGFR/MAPK signaling pathway acts as a potential therapeutic target for sulforaphane-rescued heart tube malformation induced by various concentrations of PhIP exposure

Background2-Amino-1-methyl-6-phenylimidazo [4,5-b] pyrimidine (PhIP) is a known carcinogen generated mainly from cooking meat and environmental pollutants. It is worth exploring the potential of natural small-molecule drugs to protect against adverse effects on embryonic development. PurposeIn this study, we investigated the potential toxicological effects of PhIP on embryonic heart tube formation and the effect of Sulforaphane (SFN) administration on the anti-toxicological effects of PhIP on embryonic cardiogenesis. Study design and methodsFirst, the chicken embryo model was used to investigate the different phenotypes of embryonic heart tubes induced by various concentrations of PhIP exposure. We also proved that SFN rescues PhIP-induced embryonic heart tube malformation. Second, immunofluorescence, western blot, Polymerase Chain Reaction (PCR) and flow cytometry experiments were employed to explore the mechanisms by which SFN protects cardiac cells from oxidative damage in the presence of PhIP. We used RNA-seq analysis, molecular docking, in situ hybridization, cellular thermal shift assay and solution nuclear magnetic resonance spectroscopy to explore whether SFN protects cardiogenesis through the EGFR/MAPK signaling pathway. ResultsThe study showed that PhIP might dose-dependently interfere with the C-looping heart tube (mild) or the fusion of a pair of bilateral endocardial tubes (severe) in chick embryos, while SFN administration prevented cardiac cells from oxidative damage in the presence of high-level PhIP. Furthermore, we found that excessive reactive oxygen species (ROS) production and subsequent apoptosis were not the principal mechanisms by which low-level PhIP induced malformation of heart tubes. This is due to PhIP-disturbed Mitogen-activated protein kinase (MAPK) signaling pathway could be corrected by SFN administration. ConclusionsThis study provided novel insight that PhIP exposure could increase the risk of abnormalities in early cardiogenesis and that SFN could partially rescue various concentrations of PhIP-induced abnormal heart tube formation by targeting EGFR and mediating EGFR/MAPK signaling pathways.

Modulation of Gold Nanoparticle Ligand Structure-Dynamic Relationships Probed Using Solution NMR.

Ligand dynamics plays a critical role in the chemical and biological properties of gold nanoparticles (AuNPs). In this study, ligands featuring hydrophobic alkanethiol interiors and hydrophilic shells were used to systematically examine the effects of ligand headgroups on the ligand dynamics. Solution nuclear magnetic resonance (NMR) spectroscopy provided quantitative insight into the monolayer ligand dynamics. Notably, the introduction of hydrophobic moieties to the cationic headgroups significantly decreased ligand conformational mobility; however, variations in hydrophobicity among these moieties had a limited effect on this reduction. Further examination of ligand dynamics under various physiological conditions, including ionic strength and temperature, showed that ligands bound to the AuNP surface become less conformationally mobile with an increase in ionic strength or decreasing temperature. This exploration of ligand dynamics provides insight into designing nanoparticles tailored to specific biological applications.

Chiral distinction between hydroxychloroquine enantiomers in binding to angiotensin-converting enzyme 2, the forward receptor of SARS-CoV-2

Soon after the outset of the Coronavirus Disease 2019 (COVID-19) pandemic (March-April 2020), formulations of the old antimalarial racemic drug hydroxychloroquine (HCQ) sulfate were authorized by the U.S. Food and Drug Administration (FDA) for emergency treatment of hospitalized patients with COVID-19. A call for the chiral switch of HCQ to the single enantiomer (S)-(+)-HCQ for treating the disease followed. The above authorizations were later withdrawn. Angiotensin-converting enzyme 2 (ACE2) has been recognized to be the forward receptor of SARS-CoV-2, the virus responsible for COVID-19. The objective of the present study was to evaluate the chiral distinction in the potential preferential binding of the HCQ enantiomers to ACE2, as a basis for its future drug repurposing, using high-field solution Nuclear Magnetic Resonance (NMR) spectroscopy. Proton selective spin-lattice relaxation rates were measured for selected diagnostic nuclei; in particular, protons belonging to the quinoline ring proved to be the most affected by the presence of the protein, for both (S)-(+)-HCQ and (R)-(−)-HCQ enantiomers. An increase in mono-selective relaxation rates was observed for both enantiomers. A significant difference in the magnitude of the increase was detected for all protons investigated, up to a 5-fold and an 8-fold increase in the case of (R)-(−)-HCQ and (S)-(+)-HCQ, respectively. Furthermore, comparison between the normalized mono-selective relaxation rates of the two HCQ enantiomers in their binary mixtures with ACE2 pointed out a certain preference for the (S)-(+)-HCQ enantiomer over (R)-(−)-HCQ in the interaction with ACE2. The findings form the basis for a future application of the drug repurposing/chiral-switch combination strategy to racemic HCQ in previously reported indications for hydroxychloroquine treatment and in the search for new indications in which ACE2 receptors are involved.

31P Nuclear Magnetic Resonance Spectroscopy as a Probe of Thorium-Phosphorus Bond Covalency: Correlating Phosphorus Chemical Shift to Metal-Phosphorus Bond Order.

We report the use of solution and solid-state 31P Nuclear Magnetic Resonance (NMR) spectroscopy combined with Density Functional Theory calculations to benchmark the covalency of actinide-phosphorus bonds, thus introducing 31P NMR spectroscopy to the investigation of molecular f-element chemical bond covalency. The 31P NMR data for [Th(PH2)(TrenTIPS)] (1, TrenTIPS = {N(CH2CH2NSiPri3)3}3-), [Th(PH)(TrenTIPS)][Na(12C4)2] (2, 12C4 = 12-crown-4 ether), [{Th(TrenTIPS)}2(μ-PH)] (3), and [{Th(TrenTIPS)}2(μ-P)][Na(12C4)2] (4) demonstrate a chemical shift anisotropy (CSA) ordering of (μ-P)3- > (═PH)2- > (μ-PH)2- > (-PH2)1- and for 4 the largest CSA for any bridging phosphido unit. The B3LYP functional with 50% Hartree-Fock mixing produced spin-orbit δiso values that closely match the experimental data, providing experimentally benchmarked quantification of the nature and extent of covalency in the Th-P linkages in 1-4 via Natural Bond Orbital and Natural Localized Molecular Orbital analyses. Shielding analysis revealed that the 31P δiso values are essentially only due to the nature of the Th-P bonds in 1-4, with largely invariant diamagnetic but variable paramagnetic and spin-orbit shieldings that reflect the Th-P bond multiplicities and s-orbital mediated transmission of spin-orbit effects from Th to P. This study has permitted correlation of Th-P δiso values to Mayer bond orders, revealing qualitative correlations generally, but which should be examined with respect to specific ancillary ligand families rather than generally to be quantitative, reflecting that 31P δiso values are a very sensitive reporter due to phosphorus being a soft donor that responds to the rest of the ligand field much more than stronger, harder donors like nitrogen.

Hepatitis B Virus Epsilon (ε) RNA Element: Dynamic Regulator of Viral Replication and Attractive Therapeutic Target.

Hepatitis B virus (HBV) chronically infects millions of people worldwide, which underscores the importance of discovering and designing novel anti-HBV therapeutics to complement current treatment strategies. An underexploited but attractive therapeutic target is ε, a cis-acting regulatory stem-loop RNA situated within the HBV pregenomic RNA (pgRNA). The binding of ε to the viral polymerase protein (P) is pivotal, as it triggers the packaging of pgRNA and P, as well as the reverse transcription of the viral genome. Consequently, small molecules capable of disrupting this interaction hold the potential to inhibit the early stages of HBV replication. The rational design of such ligands necessitates high-resolution structural information for the ε-P complex or its individual components. While these data are currently unavailable for P, our recent structural elucidation of ε through solution nuclear magnetic resonance spectroscopy marks a significant advancement in this area. In this review, we provide a brief overview of HBV replication and some of the therapeutic strategies to combat chronic HBV infection. These descriptions are intended to contextualize our recent experimental efforts to characterize ε and identify ε-targeting ligands, with the ultimate goal of developing novel anti-HBV therapeutics.

Chemical Diversity of Mo5S5 Clusters with Pyrazole: Synthesis, Redox and UV-vis-NIR Absorption Properties.

The chemistry of transition metal clusters has been intensively developed in the last decades, leading to the preparation of a number of compounds with promising and practically useful properties. In this context, the present work demonstrates the preparation and study of the reactivity, i.e., the possibility of varying the ligand environment, of new square pyramidal molybdenum chalcogenide clusters [{Mo5(μ3-S)i4(μ4-S)i(μ-pz)i4}(pzH)t5]1+/2+ (pzH = pyrazole, i = inner, t = terminal). The one-step synthesis starting from the octahedral Mo6Br12 cluster as well as the substitution of the apical pyrazole ligand or the selective bromination of the inner pyrazolate ligands were demonstrated. All the obtained compounds were characterized in detail using a series of physicochemical methods both in solid state (X-ray diffraction analysis, etc.) and in solution (nuclear magnetic resonance spectroscopy, mass spectrometry, etc.). In this work, redox properties and absorption in the ultraviolet-visible and near-infrared region of the obtained compounds were studied.

Excited-state observation of active K-Ras reveals differential structural dynamics of wild-type versus oncogenic G12D and G12C mutants

Despite the prominent role of the K-Ras protein in many different types of human cancer, major gaps in atomic-level information severely limit our understanding of its functions in health and disease. Here, we report the quantitative backbone structural dynamics of K-Ras by solution nuclear magnetic resonance spectroscopy of the active state of wild-type K-Ras bound to guanosine triphosphate (GTP) nucleotide and two of its oncogenic P-loop mutants, G12D and G12C, using a new nanoparticle-assisted spin relaxation method, relaxation dispersion and chemical exchange saturation transfer experiments covering the entire range of timescales from picoseconds to milliseconds. Our combined experiments allow detection and analysis of the functionally critical Switch I and Switch II regions, which have previously remained largely unobservable by X-ray crystallography and nuclear magnetic resonance spectroscopy. Our data reveal cooperative transitions of K-Ras·GTP to a highly dynamic excited state that closely resembles the partially disordered K-Ras·GDP state. These results advance our understanding of differential GTPase activities and signaling properties of the wild type versus mutants and may thus guide new strategies for the development of therapeutics.

Applications of Solution NMR Spectroscopy in Quality Assessment and Authentication of Bovine Milk.

Nuclear magnetic resonance (NMR) spectroscopy is emerging as a promising technique for the analysis of bovine milk, primarily due to its non-destructive nature, minimal sample preparation requirements, and comprehensive approach to untargeted milk analysis. These inherent strengths of NMR make it a formidable complementary tool to mass spectrometry-based techniques in milk metabolomic studies. This review aims to provide a comprehensive overview of the applications of NMR techniques in the quality assessment and authentication of bovine milk. It will focus on the experimental setup and data processing techniques that contribute to achieving accurate and highly reproducible results. The review will also highlight key studies that have utilized commonly used NMR methodologies in milk analysis, covering a wide range of application fields. These applications include determining milk animal species and feeding regimes, as well as assessing milk nutritional quality and authenticity. By providing an overview of the diverse applications of NMR in milk analysis, this review aims to demonstrate the versatility and significance of NMR spectroscopy as an invaluable tool for milk and dairy metabolomics research and hence, for assessing the quality and authenticity of bovine milk.

Unfolding under Pressure: An NMR Perspective.

This review aims to analyse the role of solution nuclear magnetic resonance spectroscopy in pressure-induced in vitro studies of protein unfolding. Although this transition has been neglected for many years because of technical difficulties, it provides important information about the forces that keep protein structure together. We first analyse what pressure unfolding is, then provide a critical overview of how NMR spectroscopy has contributed to the field and evaluate the observables used in these studies. Finally, we discuss the commonalities and differences between pressure-, cold- and heat-induced unfolding. We conclude that, despite specific peculiarities, in both cold and pressure denaturation the important contribution of the state of hydration of nonpolar side chains is a major factor that determines the pressure dependence of the conformational stability of proteins.

Production of isotopically enriched high molecular weight hyaluronic acid and characterization by solid-state NMR

Hyaluronic acid (HA) is a naturally occurring polysaccharide that is abundant in the extracellular matrix (ECM) of all vertebrate cells. HA-based hydrogels have attracted great interest for biomedical applications due to their high viscoelasticity and biocompatibility. In both ECM and hydrogel applications, high molecular weight (HMW)-HA can absorb a large amount of water to yield matrices with a high level of structural integrity. To understand the molecular underpinnings of structural and functional properties of HA-containing hydrogels, few techniques are available. Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for such studies, e.g. 13C NMR measurements can reveal the structural and dynamical features of (HMW) HA. However, a major obstacle to 13C NMR is the low natural abundance of 13C, necessitating the generation of HMW-HA that is enriched with 13C isotopes. Here we present a convenient method to obtain 13C- and 15N-enriched HMW-HA in good yield from Streptococcus equi subsp. zooepidemicus. The labeled HMW-HA has been characterized by solution and magic angle spinning (MAS) solid-state NMR spectroscopy, as well as other methods. These results will open new ways to study the structure and dynamics of HMW-HA-based hydrogels, and interactions of HMW-HA with proteins and other ECM components, using advanced NMR techniques.

Fast Dynamics of Difluprednate in Micelles or Swollen-Micelles Revealed by 19F Nuclear Magnetic Resonance Spin Relaxation Rates

Knowledge of molecular rotational dynamics is critical to interpret solution nuclear magnetic resonance (NMR) spectroscopy. The observation of sharp solute NMR signals in micelles contradicted the surfactant viscosity effects noted in the Stokes-Einstein-Debye (SED) equation. Herein, the 19F spin relaxation rates of difluprednate (DFPN) drug dissolved in polysorbate-80 (PS-80) micelles and castor oil swollen micelles (s-micelle) were measured and adequately fit using an isotropic diffusion model based spectral density function. Despite the high viscosity of PS-80 and castor oil, the fitting results revealed fast 4 and 12 ns dynamics of DFPN in both micelle globules. The observation of the fast ns motion in the viscous surfactant/oil micelle phase demonstrated motion decoupling between solute molecules inside micelles and the micelle itself in an aqueous solution. These observations support the role of intermolecular interaction in governing the rotational dynamics of small molecules, versus the viscosity of the solvent molecules as defined in the SED equation.

Cerebrospinal fluid lipoproteins inhibit α-synuclein aggregation by interacting with oligomeric species in seed amplification assays

BackgroundAggregation of α-synuclein (α-syn) is a prominent feature of Parkinson’s disease (PD) and other synucleinopathies. Currently, α-syn seed amplification assays (SAAs) using cerebrospinal fluid (CSF) represent the most promising diagnostic tools for synucleinopathies. However, CSF itself contains several compounds that can modulate the aggregation of α-syn in a patient-dependent manner, potentially undermining unoptimized α-syn SAAs and preventing seed quantification.MethodsIn this study, we characterized the inhibitory effect of CSF milieu on detection of α-syn aggregates by means of CSF fractionation, mass spectrometry, immunoassays, transmission electron microscopy, solution nuclear magnetic resonance spectroscopy, a highly accurate and standardized diagnostic SAA, and different in vitro aggregation conditions to evaluate spontaneous aggregation of α-syn.ResultsWe found the high-molecular weight fraction of CSF (> 100,000 Da) to be highly inhibitory on α-syn aggregation and identified lipoproteins to be the main drivers of this effect. Direct interaction between lipoproteins and monomeric α-syn was not detected by solution nuclear magnetic resonance spectroscopy, on the other hand we observed lipoprotein-α-syn complexes by transmission electron microscopy. These observations are compatible with hypothesizing an interaction between lipoproteins and oligomeric/proto-fibrillary α-syn intermediates. We observed significantly slower amplification of α-syn seeds in PD CSF when lipoproteins were added to the reaction mix of diagnostic SAA. Additionally, we observed a decreased inhibition capacity of CSF on α-syn aggregation after immunodepleting ApoA1 and ApoE. Finally, we observed that CSF ApoA1 and ApoE levels significantly correlated with SAA kinetic parameters in n = 31 SAA-negative control CSF samples spiked with preformed α-syn aggregates.ConclusionsOur results describe a novel interaction between lipoproteins and α-syn aggregates that inhibits the formation of α-syn fibrils and could have relevant implications. Indeed, the donor-specific inhibition of CSF on α-syn aggregation explains the lack of quantitative results from analysis of SAA-derived kinetic parameters to date. Furthermore, our data show that lipoproteins are the main inhibitory components of CSF, suggesting that lipoprotein concentration measurements could be incorporated into data analysis models to eliminate the confounding effects of CSF milieu on α-syn quantification efforts.

Robust automated backbone triple resonance NMR assignments of proteins using Bayesian-based simulated annealing

Assignment of resonances of nuclear magnetic resonance (NMR) spectra to specific atoms within a protein remains a labor-intensive and challenging task. Automation of the assignment process often remains a bottleneck in the exploitation of solution NMR spectroscopy for the study of protein structure-dynamics-function relationships. We present an approach to the assignment of backbone triple resonance spectra of proteins. A Bayesian statistical analysis of predicted and observed chemical shifts is used in conjunction with inter-spin connectivities provided by triple resonance spectroscopy to calculate a pseudo-energy potential that drives a simulated annealing search for the most optimal set of resonance assignments. Termed Bayesian Assisted Assignments by Simulated Annealing (BARASA), a C++ program implementation is tested against systems ranging in size to over 450 amino acids including examples of intrinsically disordered proteins. BARASA is fast, robust, accommodates incomplete and incorrect information, and outperforms current algorithms – especially in cases of sparse data and is sufficiently fast to allow for real-time evaluation during data acquisition.

Virtual Screening of Hepatitis B Virus Pre-Genomic RNA as a Novel Therapeutic Target.

The global burden imposed by hepatitis B virus (HBV) infection necessitates the discovery and design of novel antiviral drugs to complement existing treatments. One attractive and underexploited therapeutic target is ε, an ~85-nucleotide (nt) cis-acting regulatory stem-loop RNA located at the 3'- and 5'-ends of the pre-genomic RNA (pgRNA). Binding of the 5'-end ε to the viral polymerase protein (P) triggers two early events in HBV replication: pgRNA and P packaging and reverse transcription. Our recent solution nuclear magnetic resonance spectroscopy structure of ε permits structure-informed drug discovery efforts that are currently lacking for P. Here, we employ a virtual screen against ε using a Food and Drug Administration (FDA)-approved compound library, followed by in vitro binding assays. This approach revealed that the anti-hepatitis C virus drug Daclatasvir is a selective ε-targeting ligand. Additional molecular dynamics simulations demonstrated that Daclatasvir targets ε at its flexible 6-nt priming loop (PL) bulge and modulates its dynamics. Given the functional importance of the PL, our work supports the notion that targeting ε dynamics may be an effective anti-HBV therapeutic strategy.