|CD1||Crystal complex structures of Saccharomyces cerevisiae geranylgeranyl pyrophosphate synthase|
Rey-Ting Guo1, Tzu-Ping Ko1, Tao-Hsin Chang1, Wen-Yih Jeng1, Cammy K.-M. Chen1, Po-Huang Liang1, and Andrew H.-J. Wang1
1Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.
Geranylgeranyl pyrophosphate synthase (GGPPs) catalyzes a condensation reaction of farnesyl pyrophosphate (FPP) with isopentenyl pyrophosphate (IPP) to generate C20 geranylgeranyl pyrophosphate (GGPP), a precursor for carotenoids, chlorophylls, geranylgeranylated proteins, and archaeal ether linked lipid. In this study, we solve the crystal structures of S. cerevisiae GGPPs in complex with IPP, the substrate analog farnesyl thiopyrophosphate (FsPP) and product GGPP. IPP is bound in a positively-charged pocket with Arg39, His68, Arg85 and Tyr205. From the binding mode of the product GGPP, the pyrophosphate of GGPP binds to the IPP pyrophosphate binding site and the hydrocarbon moiety sits in the FPP hydrocarbon binding site. The binding mode for GGPP in yeast GGPPs structure is distinct from that of the product in the proposed inhibitory site of human GGPPs.
|CD2||Characterization of Enterovirus 3C Protease and Evaluation of Its Inhibitors as Potential Antiviral Agents|
Chih-Jung Kuo1,2, Jiun-Jie Shie3, Kui-Jung Yen4, Jim-Min Fang3,5, John T.-A. Hsu4, and Po-Huang Liang1,2,3
1Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan
Human enteroviruses (EV) belong to the picoravirus family which consists of over 200 medically relevant viruses. A chymotrypsin-like protease in EV which is essential for viral replication is a potential target for anti-viral therapy. In this study, we have expressed, purified, and characterized this protease and synthesized analogues of a lead compound that has been discovered as rhinovirus protease inhibitor and entered the clinical trial for common cold to evaluate their in-vitro protease inhibitory activity and the ability to reduce the virus in cell-based assays. The protease showed an optimal activity toward a fluorogenic peptide substrate containing Dabcyl-Edans quenching pair in the N- and C-termini. While cleaved by the protease, the fluorescence intensity was elevated due to the lost fluorescence resonance energy transfer. Using the FRET assay, the kcat and Km of the protease were determined to be 0.44 min-1 and 7.5¡Ó0.9£gM, respectively. According to analytic gel filtration analysis, the protease mainly formed a monomer and the dimer Kd is ranged at 0.4£gM. C-terminal His-tag used for purification was found not interfering with the activity, but N-terminal His-tag significantly reduced the activity. Peptidomimetic inhibitors with an aldehyde group at the P1¡¦ position showed better inhibition against the protease and the best inhibitor showed EC50 of 7 nM, more potent than the lead compound. The current data serves as a basis of drug development for enterovirus 71 targeting the main protease.
|CD3||Cooperative Lactonization, Regioselective de-Lactonization, and C-H¡P¡P¡PO Hydrogen Bonding Cooperativity in OligoSialic Acid Lactone|
Chien-Sheng Chen 1,2, Jim-Min Fang 1,2, Shih-Hsiung Wu 1
1 Institute of Biological Chemistry and The Genomics Research Center, Academia Sinica No. 128, Academia Road Section 2, Nan-Kang, Taipei, 115, Taiwan;
The conformation of the trisialic acid lactone was analyzed by a combination of NMR spectroscopy, molecular modeling, and molecular dynamic (MD) calculations. First, an approximate model of hydrolytic reactivity of tri-sialic acid, £\2,8-(NeuAc)3, lactone is studied with 1.0-ns in-water MD simulation and presented as the neutral hydrolysis of £_-lactone with two water-layers. The priority of stepwise de-lactonization of £\2,8-(NeuAc)3 lactone relies on water attendance near the carbonyl carbon of lactones in the 3.5 Å water-shell. Second, the two pairs of inter-residue C-H¡P¡P¡PO H-bonds and the inter-residue NOEs provided 16 important distance restraints for the molecular simulation. We conclude that the polysialic acid lactone is a right-hand helix with a rotation angle, £g, of 240o and a repeating unit, n, of 1.5 residues. Third, the unambiguous evidences indicat a 3J-correlation through the C-H¡P¡P¡PO hydrogen bond and support the hydrogen bonding cooperativity and cooperative lactonization in oligosialic acid.
|CD4||Structural basis of mercury- and zinc-conjugated complexes for inhibition of SARS-CoV 3C-like protease|
Institute of Biological Chemistry, Academia Sinica
In developing potent therapeutics against a re-emerging severe acute respiratory syndrome-associated coronavirus (SARS-CoV), we examined the co-crystal structures of five active mental-conjugated inhibitors complexed with the SARS-CoV 3C-like protease (3CLpro). The five 3CLpro structures are in complex with the mercury-conjugated inhibitor PMA (3CLpro-PMA), and the zinc-conjugated inhibitors TDT (3CLpro-TDT), EPDTC (3CLpro-EPDTC), JMF1586 (3CLpro-JMF1586) and JMF1600 (3CLpro-JMF1600). Using X-ray crystallographic analysis, we observed that PMA coordinately binds to C44, M49 and Y54 to form a Hg-centered square planar geometry at the S4 pocket. For 3CLpro-TDT and 3CLpro-EPTDC, the zinc ions of TDT and EPTDC are coordinately bound to H41 and C145 at the active sites in a zinc-centered tetrahedral geometry. Following the binding modes of 3CLpro-TDT and 3CLpro-EPTDC, we proceeded to synthesize and determine coordination structures of two zinc-based inhibitors JMF 1586 and JMF 1600 (with Ki values of 0.05 and 0.32 ƒÝM for 3CLpro, respectively). Our results reveal that the zinc ion-centered coordination pattern would serve as a platform for inhibitor optimization and the development of potential drug for SARS therapies.
|CD5||The interaction of hFGF-2 and suramine studied by NMR|
Ming-Ju Yen, Chin Yu
Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan
Fibroblast growth factors (FGFs) play crucial role in the regulation of key cellular processes such as angiogenesis, differentiation, wound healing, and tumor growth. Suramine, a polysulfonated naphthylurea, is known to be a potent inhibitor of FGF-induced angiogenesis. From the results of two-dimensional NMR spectroscopy, suramine will bind to the residues of human basic fibroblast growth factor (hFGF-2) in heparin binding pocket. By using isothermal titration calorimetry (ITC), we demonstrate that hFGF-2 binds to suramine with high affinity.