Applications of paramagnetic NMR spectroscopy in drug discovery

NMR spectroscopy of proteins with paramagnetic metal ions, first performed with metalloproteins, is a unique technique to obtain long-range distance information for three-dimensional structure determinations. This thesis focuses on developing applications of paramagnetic NMR spectroscopy, particularly pseudocontact shifts, in drug discovery. The two-component dengue virus NS2B-NS3 protease (NS2B-NS3pro) from serotype 2 is a well-established drug target, but drug development has been hampered for many years by lack of structural information. In earlier work, pseudocontact shifts (PCSs) induced by lanthanide binding tags at multiple sites had successfully been used to determine the closed conformation of NS2B in the presence of a small inhibitor molecule. Subsequently, PCSs were used to prove that an unlinked construct of NS2B-NS3pro exists predominately in the closed conformation in solution, showing that the open conformation observed previously is an artefact generated by a covalent link between NS2B and NS3 (Paper 1). Next, PCSs generated for NS2B, NS3pro and bovine pancreatic trypsin inhibitor (BPTI) were used to show that the C-terminal segment of NS2B remains in the closed conformation in the presence of BPTI, correcting a crystallographic artefact (Paper 2). The work described in Papers 1 and 2 confirmed that the closed conformation of dengue virus NS2B-NS3pro is the best model for structure-guided drug design. As the sensitivity of NMR spectra of dynamic proteins, such as the dengue virus protease, is compromised by excessive line broadening, alternative NMR tags were sought. O-tert-butyltyrosine incorporated in proteins proved to be an outstanding NMR probe for conformational studies of high-molecular-weight systems and measurement of submicromolar ligand binding affinities in one-dimensional 1H-NMR spectra without any isotope labelling (Paper 3). A tert-butyl probe was also introduced into a tightly binding lead compound against the dengue virus protease. Measurement of ligand PCSs from intense intramolecular NOESY cross-peaks with the tert-butyl group allowed positioning of the ligand on the protein with respect to the paramagnetic centre, while strong intermolecular NOEs validated the structural model of the complex established with the use of PCSs (Paper 4). In summary, the paramagnetic NMR approach, demonstrated on the dengue virus NS2B-NS3 protease, presents a broadly applicable and elegant way for structure-guided drug design at atomic resolution

Binding mode of the activity-modulating C-terminal segment of NS2B to NS3 in the dengue virus NS2B–NS3 protease

The two-component dengue virus NS2B–NS3 protease (NS2B–NS3pro) is an established drug target but inhibitor design is hampered by uncertainties about its 3D structure in solution. Crystal structures reported very different conformations for the functionally important C-terminal segment of the NS2B cofactor (NS2Bc), indicating open and closed conformations in the absence and presence of inhibitors, respectively. An earlier NMR study in solution indicated that a closed state is the preferred conformation in the absence of an artificial linker engineered between NS2B and NS3pro. To obtain direct structural information on the fold of unlinked NS2B–NS3pro in solution, we tagged NS3pro with paramagnetic tags and measured pseudocontact shifts by NMR to position NS2Bc relative to NS3pro. NS2Bc was found to bind to NS3pro in the same way as reported in a previously published model and crystal structure of the closed state. The structure is destabilized, however, by high ionic strength and basic pH, showing the importance of electrostatic forces to tie NS2Bc to NS3pro. Narrow NMR signals previously thought to represent the open state are associated with protein degradation. In conclusion, the closed conformation of the NS2B–NS3 protease is the best model for structure-guided drug design

Survey on Online Adversarial Planning for Real-time Strategy Game

Real-time strategy game online adversarial planning is a challenging problem in the field of multi-agent learning.In the process of game confrontation,in the face of an uncertain threat environment and non-stationary opponents,the agent needs to reason about the opponent’s actions within a limited time according to the game situation,make your own action plan quickly and perform adversarial planning in the huge state space and action space.The real-time strategy game platform is an ideal testbed for studying online adversarial planning problems.This paper firstly uses a typical real-time strategy game model to elicit the real-time strategy game confrontation problems,and classifies them into three levels and two operation control methods,and sorts out the five challenges faced from five sub-directions.Secondly,the current online adversarial planning methods are comprehensively reviewed and analyzed from three perspectives of tactical adversarial planning,strategic adversarial planning and mixed adversarial planning.Finally,the key issues that need to be studied in the future are pointed out from three key aspects:opponent and player modeling,human-machine collaborative online ad hoc planning,and learning-based planning

Traumatic Neuroma around the Celiac Trunk after Gastrectomy Mimicking a Nodal Metastasis: A Case Report

Traumatic neuroma is a well-known disorder that occurs after trauma or surgery involving the peripheral nerve and develops from a nonneoplastic proliferation of the proximal end of a severed, partially transected, or injured nerve. We present a case of traumatic neuroma around the celiac trunk after gastrectomy in a 56-year-old man, which was confirmed by pathology. CT demonstrated the presence of a lobulated, homogeneous, hypoattenuating mass around the celiac trunk, mimicking a nodal metastasis

Identification and Functional Analysis of Variant Haplotypes in the 5′-Flanking Region of Protein Phosphatase 2A-Bδ Gene

Serine-threonine protein phosphatase 2A (PP2A) is a trimeric holoenzyme that plays an integral role in the regulation of cell growth, differentiation, and apoptosis. The substrate specificity and (sub)cellular localization of the PP2A holoenzymes are highly regulated by interaction with a family of regulatory B subunits (PP2A-Bs). The regulatory subunit PP2A-B/PR55δ (PP2A-Bδ) is involving in the dephosphorylation of PP2A substrates and is crucial for controlling entry into and exit from mitosis. The molecular mechanisms involved in the regulation of expression of PP2A-Bδ gene (PPP2R2D) remain largely unknown. To explore genetic variations in the 5′-flanking region of PPP2R2D gene as well as their frequent haplotypes in the Han Chinese population and determine whether such variations have an impact on transcriptional activity, DNA samples were collected from 70 healthy Chinese donors and sequenced for identifying genetic variants in the 5′-flanking region of PPP2R2D. Four genetic variants were identified in the 1836 bp 5′-flanking region of PPP2R2D. Linkage disequilibrium (LD) patterns and haplotype profiles were constructed for the genetic variants. Using serially truncated human PPP2R2D promoter luciferase constructs, we found that a 601 bp (−540 nt to +61 nt) fragment constitutes the core promoter region. The subcloning of individual 5′-flanking fragment revealed the existence of three haplotypes in the distal promoter of PPP2R2D. The luciferase reporter assay showed that different haplotypes exhibited distinct promoter activities. The EMSA revealed that the −462 G>A variant influences DNA-protein interactions involving the nuclear factor 1 (NF1). In vitro reporter gene assay indicated that cotransfection of NF1/B expression plasmid could positively regulate the activity of PPP2R2D proximal promoter. Introduction of exogenous NF1/B expression plasmid further confirmed that the NF1 involves in the regulation of PPP2R2D gene expression. Our findings suggest that functional genetic variants and their haplotypes in the 5′-flanking region of PPP2R2D are critical for transcriptional regulation of PP2A-Bδ

Ferroelectricity induced by interatomic magnetic exchange interaction

Multiferroics, where two or more ferroic order parameters coexist, is one of the hottest fields in condensed matter physics and materials science[1-9]. However, the coexistence of magnetism and conventional ferroelectricity is physically unfavoured[10]. Recently several remedies have been proposed, e.g., improper ferroelectricity induced by specific magnetic[6] or charge orders[2]. Guiding by these theories, currently most research is focused on frustrated magnets, which usually have complicated magnetic structure and low magnetic ordering temperature, consequently far from the practical application. Simple collinear magnets, which can have high magnetic transition temperature, have never been considered seriously as the candidates for multiferroics. Here, we argue that actually simple interatomic magnetic exchange interaction already contains a driving force for ferroelectricity, thus providing a new microscopic mechanism for the coexistence and strong coupling between ferroelectricity and magnetism. We demonstrate this mechanism by showing that even the simplest antiferromagnetic (AFM) insulator MnO, can display a magnetically induced ferroelectricity under a biaxial strain

Inhibitory Effects of Dopamine Receptor D1 Agonist on Mammary Tumor and Bone Metastasis

Dopaminergic signaling plays a critical role in the nervous system, but little is known about its potential role in breast cancer and bone metabolism. A screening of ~1,000 biologically active compounds revealed that a selective agonist of dopamine receptor D1 (DRD1), A77636, inhibited proliferation of 4T1.2 mammary tumor cells as well as MDA-MB-231 breast cancer cells. Herein, we examined the effect of A77636 on bone quality using a mouse model of bone metastasis from mammary tumor. A77636 inhibited migration of cancer cells in a DRD1-dependent fashion and suppressed development of bone-resorbing osteoclasts by downregulating NFATc1 through the elevation of phosphorylation of eIF2α. In the mouse model of bone metastasis, A77636 reduced osteolytic lesions and prevented mechanical weakening of the femur and tibia. Collectively, we expect that dopaminergic signaling might provide a novel therapeutic target for breast cancer and bone metastasis

The Ultrasmall Biocompatible CuS@BSA Nanoparticle and Its Photothermal Effects

Nanomaterials with localized surface plasmon resonance (LSPR) have exquisite optical properties, which allow a wide range of applications. Non-stoichiometric copper sulfides with active LSPR have drawn great attention, because its LSPR peak falls in the NIR region that is suitable for deep bioimaging and photothermal therapy (PTT). Despite numerous biomedical applications, the biocompatibility and toxicity of copper sulfides have not been studied systematically. In this contribution, we synthesized the ultrasmall biocompatible copper sulfide nanoparticle encapsulated within bovine serum albumin (BSA), CuS@BSA. The physical features of CuS@BSA were characterized. The MTT and flow cytometry assays were performed. The in vitro PTT was also investigated. The results indicated that such CuS@BSA nanoparticle had an average TEM size of 8 nm, and an average DLS size of 15 nm. A lower concentration of CuS@BSA was not toxic to HeLa cells, but the critical apoptotic events occurred in HeLa cells after co-incubation with 45 μg/mL CuS@BSA for 48 h. The photothermal effect of the CuS@BSA in aqueous medium were concentration-dependent and time-dependent, which were also verified by flow cytometry and microscopy, while the CuS@BSA were co-cultured with HeLa cells and treated with laser. This work designed an ultrasmall potential biocompatible nanoparticle, CuS@BSA, for cancer photothermal therapy, and provided the toxic information to safely guide its biomedical applications