PD1 Inhibitors and maturation mechanism of SARS main protease

Chih-Jung Kuo(Pa) 1.2, Jiun-Jie Shie(«T) 2, Min-Feng Hsu(\Ӯp) 2, Jim-Min Fang(T) 2, Chi-Huey Wong(αҴf) 2, Andrew H.-J. Wang(fv) 2 , Po-Huang Liang(շ) 2

1 Taiwan International Graduate Program, Academia Sinica, Nan-Kang, Taipei, 115, Taiwan
2 Institute of Biological Chemistry and The Genomics Research Center, Academia Sinica No. 128, Academia Road Section 2, 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.

Based on the structure, a series of inhibitors including symmetric diols, peptidomimetic \,]-unsaturated esters, anilides, metal-conjugated compounds, benzotriazole esters, and natural products were prepared and evaluated for inhibition of the 3CL protease. The structure-and-activity relationship was examined to derive more potent inhibitors. The most potent inhibitor has Ki of ~7 nM. Some of the potent inhibitors also showed anti-SARS-CoV activity in the cell-based assay.



PD2 Enzymatic and Nonenzymatic Synthesis of Glutathione Conjugates: Application to the Understanding of Parasites 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 parasites 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 enzymes 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[1] 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.

[1] John B. Biggins, Kenolisa C. Onwueme, Jon S. Thorson, Scinece, 2003, 301, 1537-1541



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.

Coffee is one of the most popular and widely consumed beverages through the world due to its pleasant taste and aroma. To examine coffee, most literature reports concern caffeine and its pharmacological effects. Moreover, caffeine was demonstrated to act in vitro as an antioxidant

Neocarzinostatin reveals potent antitumor activity, but it is also a mutagen. Whether caffeine can be a novel free radical scavenging molecule and whether caffeine can protect DNA from damaging in the light of neocarzinostatin are interesting questions. The present study is intended to understand the mechanism of caffeine inhibition of neocarzinostatin-mediated DNA cleavage.



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.

The most unstable site in the neocarzinostatin chromophore is the highly strained epoxide and the bicyclododecadienediyne moiety (1). It was also previously reported (2) that the epoxide moiety of the neocarzinostatin chromophore plays an important part in the cycloaromatization of the highly unsaturated bicyclo[7. 3. 0]dodecadiendiyne in the thiol-dependent, DNA-damaging reaction.

In order to properly determine the role of epoxide moiety, we synthesized different derivatives of neocarzinostatin chromophore upon opening of the epoxide. Quite interestingly we observed that chlorohydrin, one of the stable derivatives of neocarzinostatin chromophore, can convert back into the epoxide form. We studied the factors involved in the conversion of chlorohydrin into epoxide form of neocarzinostatin chromophore and found that the conversion is inefficient in hydrophobic environment but can be accelerated at high temperatures and basic pH.

(1) Koide, Y., A. Ito, K. Edo, and N. Ishida. Chem. Pharm. Bull. 34, 4425 - 4428, 1986
(2) S. H. Lee and I. H. Goldberg, Molecular pharmacology, 33, 396 V 401, 1988.



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;
bInstitute of Biological Chemistry, Academia Sinica;
cCore Facility for Protein X-ray Crystallography, Academia Sinica, Taipei 115, Taiwan;
dInstitute of Biochemistry, National Yang-Ming University, Taipei 112, 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).

CVB3 3C protease cleavages the substrates between Gln-Gly (Q-G) pairs which is similar to the substrate specificity of SARS 3C-like protease. Here we reported the first crystal structure of native CVB3 3C protease with 1.8Å resolution and its two mutants (C147A and C147S). Based on the sequence specificity of the substrates, we analyzed the inhibition activity of CVB3 3C protease with several SARS 3C-like protease inhibitors and solved structures of co-crystallized protease-inhibitor complexes. These structures show the characteristic of the 3C protease active site and provide a firm basis for antiviral drug design.