Yu-Hou Chen

High-throughput identification of compounds targeting influenza RNA-dependent RNA polymerase activity

As influenza viruses have developed resistance towards current drugs, new inhibitors that prevent viral replication through different inhibitory mechanisms are useful. In this study, we developed a screening procedure to search for new antiinfluenza inhibitors from 1,200,000 compounds and identified previously reported as well as new antiinfluenza compounds. Several antiinfluenza compounds were inhibitory to the influenza RNA-dependent RNA polymerase (RdRP), including nucleozin and its analogs. The most potent nucleozin analog, 3061 (FA-2), inhibited the replication of the influenza A/WSN/33 (H1N1) virus in MDCK cells at submicromolar concentrations and protected the lethal H1N1 infection of mice. Influenza variants resistant to 3061 (FA-2) were isolated and shown to have the mutation on nucleoprotein (NP) that is distinct from the recently reported resistant mutation of Y289H [Kao R, et al. (2010) Nat Biotechnol 28:600]. Recombinant influenza carrying the Y52H NP is also resistant to 3061 (FA-2), and NP aggregation induced by 3061 (FA-2) was identified as the most likely cause for inhibition. In addition, we identified another antiinfluenza RdRP inhibitor 367 which targets PB1 protein but not NP. A mutant resistant to 367 has H456P mutation at the PB1 protein and both the recombinant influenza and the RdRP expressing the PB1 H456P mutation have elevated resistance to 367. Our high-throughput screening (HTS) campaign thus resulted in the identification of antiinfluenza compounds targeting RdRP activity.

E339…R416 salt bridge of nucleoprotein as a feasible target for influenza virus inhibitors

The nucleoprotein (NP) of the influenza virus exists as trimers, and its tail-loop binding pocket has been suggested as a potential target for antiinfluenza therapeutics. The possibility of NP as a drug target was validated by the recent reports that nucleozin and its analogs can inhibit viral replication by inducing aggregation of NP trimers. However, these inhibitors were identified by random screening, and the binding site and inhibition mechanism are unclear. We report a rational approach to target influenza virus with a new mechanism—disruption of NP–NP interaction. Consistent with recent work, E339A, R416A, and deletion mutant Δ402–428 were unable to support viral replication in the absence of WT NP. However, only E339A and R416A could form hetero complex with WT NP, but the complex was unable to bind the RNA polymerase, leading to inhibition of viral replication. These results demonstrate the importance of the E339…R416 salt bridge in viral survival and establish the salt bridge as a sensitive antiinfluenza target. To provide further support, we showed that peptides encompassing R416 can disrupt NP–NP interaction and inhibit viral replication. Finally we performed virtual screening to target E339…R416, and some small molecules identified were shown to disrupt the formation of NP trimers and inhibit replication of WT and nucleozin-resistant strains. This work provides a new approach to design antiinfluenza drugs.

Interception of teicoplanin oxidation intermediates yields new antimicrobial scaffolds

In the search for new efficacious antibiotics, biosynthetic engineering offers attractive opportunities to introduce minor alterations to antibiotic structures that may overcome resistance. Dbv29, a flavin-containing oxidase, catalyzes the four-electron oxidation of a vancomycin-like glycopeptide to yield A40926. Structural and biochemical examination of Dbv29 now provides insights into residues that govern flavinylation and activity, protein conformation and reaction mechanism. In particular, the serendipitous discovery of a reaction intermediate in the crystal structure led us to identify an unexpected opportunity to intercept the normal enzyme mechanism at two different points to create new teicoplanin analogs. Using this method, we synthesized families of antibiotic analogs with amidated and aminated lipid chains, some of which showed marked potency and efficacy against multidrug resistant pathogens. This method offers a new strategy for the development of chemical diversity to combat antibacterial resistance.

Intermolecular Binding between TIFA-FHA and TIFA-pT Mediates Tumor Necrosis Factor Alpha Stimulation and NF-κB Activation

ABSTRACT The forkhead-associated (FHA) domain recognizes phosphothreonine (pT) with high specificity and functional diversity. TIFA (TRAF-interacting protein with an FHA domain) is the smallest FHA-containing human protein. Its overexpression was previously suggested to provoke NF-κB activation, yet its exact roles in this signaling pathway and the underlying molecular mechanism remain unclear. Here we identify a novel threonine phosphorylation site on TIFA and show that this phosphorylated threonine (pT) binds with the FHA domain of TIFA, leading to TIFA oligomerization and TIFA-mediated NF-κB activation. Detailed analysis indicated that unphosphorylated TIFA exists as an intrinsic dimer and that the FHA-pT9 binding occurs between different dimers of TIFA. In addition, silencing of endogenous TIFA resulted in attenuation of tumor necrosis factor alpha (TNF-α)-mediated downstream signaling. We therefore propose that the TIFA FHA-pT9 binding provides a previously unidentified link between TNF-α stimulation and NF-κB activation. The intermolecular FHA-pT9 binding between dimers also represents a new mechanism for the FHA domain.

Uncovering the Mechanism of Forkhead-Associated Domain-Mediated TIFA Oligomerization That Plays a Central Role in Immune Responses.

Forkhead-associated (FHA) domain is the only signaling domain that recognizes phosphothreonine (pThr) specifically. TRAF-interacting protein with an FHA domain (TIFA) was shown to be involved in immune responses by binding with TRAF2 and TRAF6. We recently reported that TIFA is a dimer in solution and that, upon stimulation by TNF-α, TIFA is phosphorylated at Thr9, which triggers TIFA oligomerization via pThr9-FHA domain binding and activates nuclear factor κB (NF-κB). However, the structural mechanism for the functionally important TIFA oligomerization remains to be established. While FHA domain-pThr binding is known to mediate protein dimerization, its role in oligomerization has not been demonstrated at the structural level. Here we report the crystal structures of TIFA (residues 1-150, with the unstructured C-terminal tail truncated) and its complex with the N-terminal pThr9 peptide (residues 1-15), which show unique features in the FHA structure (intrinsic dimer and extra β-strand) and in its interaction with the pThr peptide (with residues preceding rather than following pThr). These structural features support previous and additional functional analyses. Furthermore, the structure of the complex suggests that the pThr9-FHA domain interaction can occur only between different sets of dimers rather than between the two protomers within a dimer, providing the structural mechanism for TIFA oligomerization. Our results uncover the mechanism of FHA domain-mediated oligomerization in a key step of immune responses and expand the paradigm of FHA domain structure and function.

Anomalous Pr ordering in the new TlBa2PrCu2O7−δ compound

Abstract Magnetic measurements on the new tetragonal Tl(1212) compound TlBa2PrCu2O7−δ( a = 3.910 A and c = 12.53 A ) reveal an anomalously high antiferromagnetic Neel temperature TN≅8 K. This transition temperature is higher than TN=2.2 K for isostructural TlBa2GdCu2O7−δ. Corroborative evidence comes from calorimetric studies, in which a magnetic entropy was obtained ΔS≅0.5 Rln 2 between 2 and 10 K, as well as a sizeable linear term in specific heat comparable to those found in heavy-fermion systems. Through structural consideration, these observations can not only be correlated to the almost identical but still puzzling features of PrBa2Cu3O7−y, but also remove some ambiguity in the interpretation of earlier results.

The effect of Ag doping on the low temperature specific heats ofNd1.85Ce0.15CuAgxO4−y

Abstract The specific heat measurements were performed in the temperature range 2K 1.85 Ce 0.15 CuAg x O 4− y where X=0, 0.1 and 0.2. The resistivity data showed that Ag doping in Nd 1.85 Ce 0.15 CuO 4− y did not make marked change in superconducting transition temperature T c . A couple of features in the specific heat data of these compounds were noticed. First Ag doping in Nd 1.85 Ce 0.15 CuO 4− y slightly decreased the specific heat of Nd ions magnetic ordering for T 1.85 Ce 0.15 CuAg x O 4− y were raised drastically by Ag doping. As the specific heats of electronic contributions and lattice contributions were estimated, the coefficients of electronic specific heat of Nd 1.85 Ce 0.15 CuO 4− y and Nd 1.85 Ce 0.15 CuAg 0.2 O 4− y were then calculated as ⋍ 10 mJ/mole K 2 and ⋍ 18mJ/mole K 2 respectively, and Debye temperature for the former was 175K and 160K for the latter.

Calorimetric study of YBa2Cu3Ox, Bi1.6Pb0.4Sr2Ca2Cu3Oy, & Nd1.85Ce0.15CuOz between 300 and 750 K

Abstract The specific heat of the superconductors YBa 2 Cu 3 O x , Bi 1.6 Pb 0.4 Sr 2 Ca 2 Cu 3 O y , & Nd 1.85 Ce 0.15 CuO z was studied as a function of temperature between 300 and 750 K by means of Differential Scanning Calorimetry technique. For Y-based system, it goes from endotherm for orthorhomic structure to exotherm for tetragonal structure in the temperature range roughly between 600 and 750 K. For Bi- and Nd-based systems, The DSC (or TG) data are linearly increased (or decreased) functions of temperature between 300 and 750 K; this suggests that the oxygen is gradually desorpted.

Phospho-priming Confers Functionally Relevant Specificities for Rad53 Kinase Autophosphorylation

The vast majority of in vitro structural and functional studies of the activation mechanism of protein kinases use the kinase domain alone. Well-demonstrated effects of regulatory domains or allosteric factors are scarce for serine/threonine kinases. Here we use a site-specifically phosphorylated SCD1-FHA1-kinase three-domain construct of the serine/threonine kinase Rad53 to show the effect of phospho-priming, an in vivo regulatory mechanism, on the autophosphorylation intermediate and specificity. Unphosphorylated Rad53 is a flexible monomer in solution but is captured in an asymmetric enzyme:substrate complex in crystal with the two FHA domains separated from each other. Phospho-priming induces formation of a stable dimer via intermolecular pT-FHA binding in solution. Importantly, autophosphorylation of unprimed and phospho-primed Rad53 produced predominantly inactive pS350-Rad53 and active pT354-Rad53, respectively. The latter mechanism was also demonstrated in vivo. Our results show that, while Rad53 can display active conformations under various conditions, simulation of in vivo regulatory conditions confers functionally relevant autophosphorylation.