|PD1||Inhibitors and maturation mechanism of SARS main protease|
Chih-Jung Kuo(³¢Pºa) 1.2, Jiun-Jie Shie(ÁÂ«Tµ²) 2, Min-Feng Hsu(³\±Ó®p) 2, Jim-Min Fang(¤è«T¥Á) 2, Chi-Huey Wong(¯Î±Ò´f) 2, Andrew H.-J. Wang(¤ý´f¶v) 2 , Po-Huang Liang(±ç³Õ·×) 2
1 Taiwan International Graduate Program, Academia Sinica, Nan-Kang, Taipei, 115, Taiwan
Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by a
novel human coronavirus. The viral maturation requires a main protease (3CLpro)
to cleave the virus-encoded polyproteins. We report here that the 3CLpro
containing N- and/or C-terminal segments of the polyprotein sequences undergoes
autoactivation and yields the mature protease in vitro. The dimeric 3-D structure
of the C145A mutant protease shows that the active site of one protomer binds with the
C-terminal six amino acids of the protomer from another asymmetric unit, mimicking the
product-bound form and suggesting a possible mechanism for maturation. The tagged C145A
mutant protein served as a substrate for the wild-type protease and the N-terminus was
first digested (55-fold faster) at the Gln(-1)-Ser1 site followed by the C-terminal
cleavage at the Gln306-Gly307 site. Analytic ultracentrifuge of the quaternary structures
of the tagged and mature proteases reveals the remarkably tighter dimer formation for the
mature enzyme (Kd=0.35 nM) than for the mutant (C145A) containing the
N-terminal or the C-terminal 10 extra amino acids. Taken together, the study here provides
insights to the design of new structure-based inhibitors.
|PD2||Enzymatic and Nonenzymatic Synthesis of Glutathione Conjugates: Application to the Understanding of Parasite¡¦s Defense System and Alternative to the Discovery of Potent Schistosoma Japonica and Human Glutathione S-Transferase Inhibitors|
Wing See Lama, Yu-Ting Hsua, Wei-Jen Loc, Yu-Ching Chioud, Ming-Yun Changd, Shu-Chuan Jaob, and Wen-Shan Lia
aInstitute of Chemistry, bInstitute of Biological Chemistry, Academia Sinica, Taipei, cDepartment of Chemistry & Biochemistry, National Chung Cheng University, Chia-Yi, dDepartment of Chemistry, National Taiwan Normal University, Taipei, Taiwan.
In search of electrophilic compounds with improved preference for interacting with Schistosoma japonicum glutathione S-transferase (SjGST), a series of aromatic (1-8) or aliphatic (9-10) halides, epoxides (11-20), and £\,£]-unsaturated esters (21-22) or amides (23-24) have been designed, synthesized, and evaluated to understand parasite¡¦s defense system. The kcat values for 23 and 18 are about 886-fold and about 14-fold larger than that for 5 with SjGST, indicating that 23 is a very good substrate compared to other electrophiles and sequential conjugation product 55a also shows its ability as a substrate for SjGST to form 55b. Both enzymatic and nonenzymatic products were generated during SjGST-activated GSH conjugation and the rapid formation of nonenzymatic GSH conjugates may block the activity of enzyme reaction. Nonenzymatic GSH conjugates 41, 48, 53, and 54 display IC50 values of 1.95, 19.0, 152, and 0.36 £gM toward SjGST, respectively, and also exhibit moderate inhibitory activity against human GSTA2 in the same manner.
|PD3||Crystal structures of SARS 3CL protease complexed with mechanism-based irreversible inhibitors|
Min-Feng Hsua,b,c, Syaulan Yangd, Shu-Jen Chend, Hui-Lin Shrb,c, Tzu-Ping Kob, Ming-Chu Hsud*, and Andrew H.-J. Wanga,b,c*
aInstitute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan; bInstitute of Biological Chemistry, Academia Sinica; cCore Facility for Protein X-ray Crystallography, Academia Sinica, Taipei 115, Taiwan; dTaiGen Biotechnology, Taipei 114, Taiwan R.O.C.
Severe acute respiratory syndrome (SRAS) is a highly infectious disease caused by a novel human coronavirus. 3CL protease is the main protease in the replicase that regulates the polyprotein maturation and mediates virus gene replication. Inhibitors with substrate-like peptidyl-binding elements specific for SARS 3CL protease undergo a Michael reaction mediated by nucleophilic addition of the enzyme¡¦s catalytic Cys-145, resulting in covalent-bound formation and irreversible inactivation of the viral protease. The co-crystallized structures with high resolution are presented in this paper. For structural-based drug design, the first crystal structure of protease-inhibitor (TG-0203770) complex stimulates us for most effective inhibitors which produced conformational induced-fitting to improve hydrophobic interactions of protease and inhibitors. This bioinformaticsa strategy for short-term viral main protease inhibitor design is sufficient to resist numerous acute highly spread viral diseases.
|PD4||A 3D-QSAR study of Celebrex-based PDK1 Inhibitors Using CoFMA Method|
Wen-Hung Wang and Ying-Chieh Sun*
Department of Chemistry, National Taiwan Normal University, 88, TingChow Road Section 4, Taipei 116, Taiwan
A 3D-QSAR study of celebrex-based compounds of PDK1 inhibitors using the comparative molecular field analysis (CoFMA) was carried out. Structures of the compounds were obtained by using molecular mechanics calculation in SYBYL package combined with quantum chemistry calculation. CoFMA calculations for a number of grouped subsets of compounds gave the q2 values of correlation ranged from 0 to 0.8. For those of low q2 values, this should be mainly due to the narrow span of biological activity. Calculations for several subsets of 11-13 compounds gave high q2 values of 0.5-0.8. Factors affecting the calculated results, including alignment and the charge sets used in force field, are discussed. The calculated results with high q2 values suggest further chemical modifications of the compounds for gaining better activity and should be of aid in design of celebrex-based cancer drug.
|PD5||Investigating the role of apoprotein in the self resistance of Streptomyces carzinostaticus against enediyne antibiotic chromoprotein, neocarzinostatin|
Yu-Wei Chang, Parameswaran Hariharan, Der-Hang Chin*
Department of Chemistry, National Chung Hsing University, Taichung, Taiwan, ROC.
Steptomycetes are high G+C Gram-positive, antibiotic-producing, mycelial soil
bacteria. Neocarzinostatin, the first discovered potent member of the enediyne antitumor
antibiotic family, is produced by Streptomyces carzinostaticus ATCC15944 and
consists of a carrier apoprotein (encoded by the ncsA gene) and a nonpeptidic chromophore.
The apoprotein plays an important role in the protection of neocarzinostatin chromophore
and regulation of its release. Without protection from the apoprotein, the chromophore
would be potentially cytotoxic by means of a radical-based DNA cleavage mechanism.
Yet the mechanism of self resistance of the produced organisms to those among
chromoprotein members has remained a mystery. In 2003, John B. Biggins and coworkers
demonstrated a self-sacrificing paradigm for resistance against highly reactive
antibiotics in the producer organism, whereby involving a protein, CalC, which protects
the calicheamicin £^1 has been reported. Among enediynes, there exist enediyne binding
proteins that serve to stabilize the enediyne and possibly aid in self-protection.
To understand the possibility for the operation of such a self protection mechanism in
Streptomyces carzinostaticus, knock out of the gene coding for apoprotein has
been employed. We describe an efficient procedure for creating precise gene replacements
in the cosmid clones by using PCR targeting and £f-Red-mediated recombination.
The strategy for PCR-targeting for mutagenesis of Streptomyces carzinostaticus
is to replace a chromosomal sequence within a Streptomyces carzinostaticus cosmid
by a selectable marker that has been generated by PCR. The cloned Streptomyces genes are
replaced with a cassette containing a selectable antibiotic resistance and oriTRK2
for efficient transfer to Streptomyces by RP4-mediated intergeneric conjugation
or by polyethylene glycol-mediated protoplast transformation. Supercos-1 does not
replicate in Streptomyces, but the clones readily undergo double-crossover recombination,
thus creating gene replacements. The knock out of aponeocarzinostatin is expected to
shed light towards the self resistance mechanism of enediyne antibiotic chromoproteins.
|PD6||Studies of caffeine-induced inhibition in neocarzinostatin mediated DNA cleavage|
Chia-Wen Liu, Huang-Shien Lee, and Der-Hang Chin*
Department of Chemistry, National Chung Hsing University, Taichung, Taiwan, Rep. of China
Neocarzinostatin is a potent antitumor antibiotic complex comprised of a DNA damaging
agent, an enediyne chromophore, and an all £]-sheet aponeocarzinostatin. The biological
activity of the drug is stored in the protein-bound chromophore that contains a
dienediyne moiety. A thiolate attack on the chromophore cyclizes the dienediyne ring
and produces an active diradical species that abstracts hydrogen from DNA.
|PD7||The factors involved in the conversion of chlorohydrin into epoxide in neocarzinostatin chromophore|
Hsin-Yun Cheng, Chin-Jui Tseng, and Der-Hang Chin*
Department of Chemistry, National Chung Hsing University, Taichung, Taiwan, Rep. of China.
Neocarzinostatin, an antitumor antibiotic produced from the culture of Streptomyces
carzinostaticus, consists of two components: an acidic apoprotein and a noncovalently bound
labile nonprotein chromophore. Neocarzinostatin chromophore is primarily responsible for
the biological activity of neocarzinostatin, while the apoprotein plays an important role
as a carrier and stabilizer.
|PD8||Structural analysis of coxsackievirus B3 3C protease and its inhibitor complexes: implications in antiviral drug design|
Yao-Chen Teuia, b, Min-Feng Hsu b, c, Cheng-Chung Lee b, c, d and Andrew H.-J. Wanga, b, c*
aInstitute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan;
Coxsackieviruse B3 (CVB3) causes acute or chronic myocarditis, which may either heal or
lead to death, belong to the enterovirus of the Picornavidae. The genome of CVB3 consists
of a positive-sense single-stranded RNA of ~7.5 kb encoding a polyprotein of roughly
2,200 amino acids. During the life cycle of CVB3, this polyprotein is processed by the
viral proteases 2A, 3C and 3CD (the precursor of 3C protease and the RNA polymerase 3D).