Xun-Cheng Su

Small Molecules Based on Benzo(1,2-b:4,5-b')dithiophene Unit for High-Performance Solution-Processed Organic Solar Cells

Small molecules, namely, DCAO(3)TBDT and DR(3)TBDT, with 2-ethylhexoxy substituted BDT as the central building block and octyl cyanoacetate and 3-ethylrhodanine as different terminal units with the same linkage of dioctyltertthiophene, have been designed and synthesized. The photovoltaic properties of these two molecules as donors and fullerene derivatives as the acceptors in bulk heterojunction solar cells are studied. Among them, DR(3)TBDT shows excellent photovoltaic performance, and power conversion efficiency as high as 7.38% (certified 7.10%) under AM 1.5G irradiation (100 mW cm(-2)) has been achieved using the simple solution spin-coating fabrication process, which is the highest efficiency reported to date for any small-molecule-based solar cells. The results demonstrate that structure fine turning could cause significant performance difference and with that the performance of solution-processed small-molecule solar cells can indeed be comparable with or even surpass their polymer counterparts.

Paramagnetic labelling of proteins and oligonucleotides for NMR

Paramagnetic effects offer a rich source of long-range structural restraints. Here we review current methods for site-specific tagging of proteins and oligonucleotides with paramagnetic molecules. The paramagnetic tags include nitroxide radicals and metal chelators. Particular emphasis is placed on tags suitable for site-specific and rigid attachment of lanthanide ions to macromolecules.

Discovery of a Non-Peptidic Inhibitor of West Nile Virus NS3 Protease by High-Throughput Docking

Background The non-structural 3 protease (NS3pro) is an essential flaviviral enzyme and therefore one of the most promising targets for drug development against West Nile virus (WNV) and dengue infections. Methodology In this work, a small-molecule inhibitor of the WNV NS3pro has been identified by automatic fragment-based docking of about 12000 compounds and testing by nuclear magnetic resonance (NMR) spectroscopy of only 22 molecules. Specific binding of the inhibitor into the active site of NS3pro and its binding mode are confirmed by 15N-HSQC NMR spectra. The inhibitory activity is further validated by an enzymatic assay and a tryptophan fluorescence quenching assay. Conclusion The inhibitor [4-(carbamimidoylsulfanylmethyl)-2,5-dimethylphenyl]-methylsulfanylmethanimidamide has a good ratio of binding affinity versus molecular weight (ligand efficiency of 0.33 kcal/mol per non-hydrogen atom), and thus has good potential as lead compound for further development to combat West Nile virus infections.

A dipicolinic acid tag for rigid lanthanide tagging of proteins and paramagnetic NMR spectroscopy.

A new lanthanide tag was designed for site-specific labeling of proteins with paramagnetic lanthanide ions. The tag, 4-mercaptomethyl-dipicolinic acid, binds lanthanide ions with nanomolar affinity, is readily attached to proteins via a disulfide bond, and avoids the problems of diastereomer formation associated with most of the conventional lanthanide tags. The high lanthanide affinity of the tag opens the possibility to measure residual dipolar couplings in a single sample containing a mixture of paramagnetic and diamagnetic lanthanides. Using the DNA-binding domain of the E. coli arginine repressor as an example, it is demonstrated that the tag allows immobilization of the lanthanide ion in close proximity of the protein by additional coordination of the lanthanide by a carboxyl group of the protein. The close proximity of the lanthanide ion promotes accurate determinations of magnetic susceptibility anisotropy tensors. In addition, the small size of the tag makes it highly suitable for studies of intermolecular interactions.

A fluorescence quenching assay to discriminate between specific and nonspecific inhibitors of dengue virus protease

In drug discovery, the occurrence of false positives is a major hurdle in the search for lead compounds that can be developed into drugs. A small-molecular-weight compound that inhibits dengue virus protease at low micromolar levels was identified in a screening campaign. Binding to the enzyme was confirmed by isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR). However, a structure-activity relationship study that ensued did not yield more potent leads. To further characterize the parental compound and its analogues, we developed a high-speed, low-cost, quantitative fluorescence quenching assay. We observed that specific analogues quenched dengue protease fluorescence and showed variation in IC(50) values. In contrast, nonspecifically binding compounds did not quench its fluorescence and showed similar IC(50) values with steep dose-response curves. We validated the assay using single Trp-to-Ala protease mutants and the competitive protease inhibitor aprotinin. Specific compounds detected in the binding assay were further analyzed by competitive ITC, NMR, and surface plasmon resonance, and the assays utility in comparison with these biophysical methods is discussed. The sensitivity of this assay makes it highly useful for hit finding and validation in drug discovery. Furthermore, the technique can be readily adapted for studying other protein-ligand interactions.

Lanthanide-Binding Peptides for NMR Measurements of Residual Dipolar Couplings and Paramagnetic Effects from Multiple Angles

Lanthanide-binding peptide tags (LBTs) containing a single cysteine residue can be attached to proteins via a disulfide bond, presenting a flexible means of tagging proteins site-specifically with a lanthanide ion. Here we show that cysteine residues placed in different positions of the LBT can be used to expose the protein to different orientations of the magnetic susceptibility anisotropy (delta chi) tensor and to generate different molecular alignments in a magnetic field. Delta chi tensors determined by nuclear magnetic resonance (NMR) spectroscopy for LBT complexes with Yb3+, Tm3+, and Er3+ suggest a rational way of producing alignment tensors with different orientations. In addition, knowledge of the delta chi tensor of LBT allows modeling of the protein-LBT structures. Despite evidence for residual mobility of the LBTs with respect to the protein, the pseudocontact shifts and residual dipolar couplings displayed by proteins disulfide-bonded to LBTs are greater than those achievable with most other lanthanide binding tags.

Site-specific labelling of proteins with a rigid lanthanide-binding tag.

This paper describes a generic method for the site‐specific attachment of lanthanide complexes to proteins through a disulfide bond. The method is demonstrated by the attachment of a lanthanide‐binding peptide tag to the single cysteine residue present in the N‐terminal DNA‐binding domain of the Escherichia coli arginine repressor. Complexes with Y3+, Tb3+, Dy3+, Ho3+, Er3+, Tm3+ and Yb3+ ions were formed and analysed by NMR spectroscopy. Large pseudocontact shifts and residual dipolar couplings were induced by the lanthanide‐binding tag in the protein NMR spectrum, a result indicating that the tag was rigidly attached to the protein. The axial components of the magnetic susceptibility anisotropy tensors determined for the different lanthanide ions were similarly but not identically oriented. A single tag with a single protein attachment site can provide different pseudocontact shifts from different magnetic susceptibility tensors and thus provide valuable nondegenerate long‐range structure information in the determination of 3D protein structures by NMR spectroscopy.

Memory T cell RNA rearrangement programmed by heterogeneous nuclear ribonucleoprotein hnRNPLL.

Differentiation of memory cells involves DNA-sequence changes in B lymphocytes but is less clearly defined in T cells. RNA rearrangement is identified here as a key event in memory T cell differentiation by analysis of a mouse mutation that altered the proportions of naive and memory T cells and crippled the process of Ptprc exon silencing needed to generate CD45RO in memory T cells. A single substitution in a memory-induced RNA-binding protein, hnRNPLL, destabilized an RNA-recognition domain that bound with micromolar affinity to RNA containing the Ptprc exon-silencing sequence. Hnrpll mutation selectively diminished T cell accumulation in peripheral lymphoid tissues but not proliferation. Exon-array analysis of Hnrpll mutant naive and memory T cells revealed an extensive program of alternative mRNA splicing in memory T cells, coordinated by hnRNPLL. A remarkable overlap with alternative splicing in neural tissues may reflect a co-opted strategy for diversifying memory T cells.

Flaviviral protease inhibitors identified by fragment-based library docking into a structure generated by molecular dynamics

Fragment-based docking was used to select a conformation for virtual screening from a molecular dynamics trajectory of the West Nile virus nonstructural 3 protease. This conformation was chosen from an ensemble of 100 molecular dynamics snapshots because it optimally accommodates benzene, the most common ring in known drugs, and two positively charged fragments (methylguanidinium and 2-phenylimidazoline). The latter fragments were used as probes because of the large number of hydrogen bond acceptors in the substrate binding site of the protease. Upon high-throughput docking of a diversity set of 18,694 molecules and pose filtering, only five compounds were chosen for experimental validation, and two of them are active in the low micromolar range in an enzymatic assay and a tryptophan fluorescence quenching assay. Evidence for specific binding to the protease active site is provided by nuclear magnetic resonance spectroscopy. The two inhibitors have different scaffolds (diphenylurea and diphenyl ester) and are promising lead candidates because they have a molecular weight of about 300 Da.

NMR Analysis of the Dynamic Exchange of the NS2B Cofactor between Open and Closed Conformations of the West Nile Virus NS2B-NS3 Protease

Background The two-component NS2B-NS3 proteases of West Nile and dengue viruses are essential for viral replication and established targets for drug development. In all crystal structures of the proteases to date, the NS2B cofactor is located far from the substrate binding site (open conformation) in the absence of inhibitor and lining the substrate binding site (closed conformation) in the presence of an inhibitor. Methods In this work, nuclear magnetic resonance (NMR) spectroscopy of isotope and spin-labeled samples of the West Nile virus protease was used to investigate the occurrence of equilibria between open and closed conformations in solution. Findings In solution, the closed form of the West Nile virus protease is the predominant conformation irrespective of the presence or absence of inhibitors. Nonetheless, dissociation of the C-terminal part of the NS2B cofactor from the NS3 protease (open conformation) occurs in both the presence and the absence of inhibitors. Low-molecular-weight inhibitors can shift the conformational exchange equilibria so that over 90% of the West Nile virus protease molecules assume the closed conformation. The West Nile virus protease differs from the dengue virus protease, where the open conformation is the predominant form in the absence of inhibitors. Conclusion Partial dissociation of NS2B from NS3 has implications for the way in which the NS3 protease can be positioned with respect to the host cell membrane when NS2B is membrane associated via N- and C-terminal segments present in the polyprotein. In the case of the West Nile virus protease, discovery of low-molecular-weight inhibitors that act by breaking the association of the NS2B cofactor with the NS3 protease is impeded by the natural affinity of the cofactor to the NS3 protease. The same strategy can be more successful in the case of the dengue virus NS2B-NS3 protease.