Design, synthesis and biological evaluation of inhibitors of flavivirus NS2B NS3 protease

Design, Synthesis and Biological Evaluation of Inhibitors of Flavivirus NS2B/NS3 Protease Gao Yaojun (B.Sc., Soochow University) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMISTRY NATIONAL UNINVERSITY OF SINGAPORE 2009 ACKNOWLEDGEMENTS First and foremost I offer my sincerest gratitude to my supervisor, Associate Professor Lam Yulin, for her invaluable support, encouragement, supervision and useful suggestions throughout my Ph.D. Her moral support and continuous guidance enabled me to complete my work successfully. I am also highly thankful to my co-supervisor, Dr. Cui Taian, Senior Lecturer in Singapore polytechnic for his encouragement and effort and without him this thesis, too, would not have been completed. I gratefully acknowledge the laboratory officers in CMMAC, Dept. of Chemistry, Miss Tan Geok Kheng, Mdm Han Yanhui, Mdm Wong Lai Kwan and Mdm Lai Hui Ngee for their assistance and technical support, and all others who have helped in one way or another. I deeply appreciate my group members, Fu Han, Kong Kah Hoe, He Rongjun, Gao Yongnian, Che Jun, Ching Shi Min, Fang Zhanxiong, Wong Ling Kai, William Lin Xijie and Sanjay Samanta, for all their help and encouragement during my research. Furthermore, I would like to thank the staff at SP, Miss Ang Cuixia, Dr Puah Chum Mok, Dr. Chen Gang and Dr. Liew Oi Wah, thank you for all the support given. I am as ever, especially indebted to my parents and my sister for their love and support throughout my life. Finally, I thank National University of Singapore for awarding me a research scholarship to pursue my doctorate degree. TABLE OF CONTENTS TABLE OF CONTENTS SUMMARY LIST OF TABLES LIST OF FIGURES LIST OF SCHEME LIST OF ABBREVIATIONS PUBLICATIONS i v vii viii x xi xiv Chapter 1: Introduction 1.1 Flavivirus 1.2 Flavivirus virion and viral life cycle 1.3 Flavivirus genome structure and polyprotein processing 1.4 Features of the structural and non-structural proteins 1.5 DEN/WNV NS2B/NS3 protease as drug target 13 1.6 Existing inhibitors of DEN/WNV NS2B/NS3 protease 15 1.6.1 Peptidic inhibitors 18 1.6.2 Nonpeptidic inhibitors 23 1.7 Objective of the our studies 26 Chapter 2: Biosynthesis of an Acyclic Permutant of Kalata B1 from a Recombinant Fusion Protein with Thioredoxin 2.1 Introduction 27 2.2 Results and Discussion 29 2.2.1 29 Construction of expression vectors i 2.2.2 Expression and purification of fusion protein 30 2.2.3 Enterokinase cleavage and the H2O2 effect 32 2.2.4 Isolation and purification 35 2.2.5 Characterization of recombinant ac kalata B1 36 2.2.6 Thermal stability studies 37 2.3 Conclusion 37 2.4 Experimental section 38 2.4.1 Synthetic peptides and oligonucleotides 38 2.4.2 Fusion protein expression and purification 38 2.4.3 Release of the target peptide by enterokinase cleavage 39 2.4.4 Purification of recombinant ac kalata B1 40 2.4.5 Mass spectrometric analysis of the target peptide 41 2.4.6 Thermal stability studies 41 Chapter 3: Design and disulfide bond connectivity-activity studies of a kalata B1-inspired cyclopeptide against dengue NS2B/NS3 protease 3.1 Introduction 43 3.2 Results and Discussion 48 3.2.1 Design and oxidative refolding of cyclopeptide 48 3.2.2 Determination of disulfide bond connectivity in cyclopeptide 50 Inhibition of dengue NS2B-NS3 protease 54 3.2.3 ii 3.3 Conclusion 56 3.4 Experimental Section 57 3.4.1 Chemicals and synthetic peptides 57 3.4.2 Oxidative refolding of cyclopeptide and purification of the 57 individual isomers 3.4.3 Mass spectrophotometric analysis of disulfide bond 58 connectivity in cyclopeptide 3.4.4 Inhibitory activity assay against DEN2 NS2b-NS3 protease 60 Chapter 4: Synthesis and biological evaluation of small molecule inhibitors of West Nile Virus NS2B/NS3 Protease 4.1 4.2 Introduction 63 4.1.1 Chemistry 65 4.1.2 Biological test 68 Results and Discussion 70 4.2.1 70 SAR study 4.2.2 Kinetic analysis of inhibition by enantiomer 74 4.2.3 76 Preliminary molecular docking 4.3 Conclusion 77 4.4 Experimental Section 78 iii Chapter 5: Synthesis of Pyrazolo[5,1-d][1,2,3,5]tetrazine-4(3H)- ones 5.1 Introduction 99 5.2 Results and Discussion 100 5.2.1 Solution-phase Synthesis 100 5.2.2 Solid-phase Synthesis 102 5.3 Conclusion 105 5.4 Experimental Section 105 5.4.1 Materials and methods 105 5.4.2 Experimental procedure 106 Chapter 6: References 117 Appendix 132 iv SUMMARY This thesis is divided into two parts. The first part which is the main focus of the thesis involves the design, synthesis and biological evaluation of inhibitors of Dengue and West Nile virus NS2B-NS3 protease. For the design of dengue NS2B-NS3 protease inhibitors, we were inspired by the unique structural and diverse biological activities found in cyclotides to design cyclopeptides as inhibitors of dengue virus protease. Firstly, we designed a new approach to obtain some acyclic cyclotides based on the bacterial expression of a thioredoxin-ac kalata B1 fusion protein and subsequent liberation of ac kalata B1 by enterokinase cleavage of the precursor. Secondly, using the new approach and chemical synthetic method, we prepared various kalata B1 analogues by varying its amino acid sequence and found the two fully oxidized forms of a cyclopeptide showed potent inhibition with Ki value of 1.39 ± 0.35 and 3.03 ± 0.75 μM, respectively. To our best knowledge, these were among the most potent peptide inhibitors achieved for the dengue viral protease. For the design of West Nile virus NS2B-NS3 protease inhibitors, initially, a library of more than 100 compounds was screened for WNV NS3 protease inhibition assays by high throughput screening (HTS). Through HTS, we found several “hits” that inhibited the WNV NS2B/NS3 protease. Among these “hits” compounds, a compound showed the best inhibition and was chosen for structure activity v relationship (SAR) exploration on WNV NS3 protease inhibition assays. In the studies, a potent, stable molecule with Ki value of 1.82±0.58 μM was identified to be an uncompetitive inhibitor. To our knowledge, this is the most potent compound amongst the stable small molecule inhibitors of WNV NS2B-NS3 protease reported so for. The second part of this thesis involves the methodology development of the solid-phase synthesis methodology, a of pyrazolo[5,1-d][1,2,3,5]tetrazine-4(3H)-ones. one-pot reaction from 5-aminopyrazoles In to the the pyrazolo[5,1-d][1,2,3,5] tetrazine-4(3H)-ones which provided the compounds in good yields was demonstrated. A representative set of 16 pyrazolo[5,1-d][1,2,3,5]tetrazine-4(3H)-ones was prepared. vi LIST OF TABLES Table 1.1 Characteristics and functions of flavivirus proteins Table 1.2 Summary of peptidic inhibitors of NS2B/NS3pro 22 Table 3.1 Peptides designed as potential inhibitors to DEN2 NS2B/NS3 47 protease Table 3.2 Fragments after NH4OH cleavage for assignment of disulfide bond 52 connectivity Table 3.3 Fragments after trypsin cleavage for assignment of disulfide bond 54 connectivity of isomer 1C Table 3.3 Inhibition of dengue NS2B-NS3 protease by isomers 1B and 1C 56 Table 4.1 Optimization of cyclization reaction to prepare compound 4-6 67 Table 4.2 WNV NS3 protease inhibitor analogues and their inhibition results 73 Table 4.3 The inhibition results of enantiomers from selected racemic 74 compounds Table 5.1 Nitrile analogs and their reaction times 110 vii LIST OF FIGURES Figure1.1 Image of the flavivirus virion. Figure 1.2 Schematic representation of flavivirus genome organization and polyprotein processing Figure 1.3 Nomenclature for peptide residues (P3-P3’) and their 14 Crystal structures of WNV NS2B/NS3pro and predicted 18 corresponding binding sites (S3-S3’) in the enzyme Figure 1.4 substrate and membrane interactions Figure 1.5 Non-peptidic inhibitors of DEN and WNV NS2B/NS3 proteases 25 and their inhibitory potencies Figure 2.1 Construction of expression vectors 29 Figure 2.2 Expression and enterokinase-catalyzed cleavage of recombinant 31 thioredoxin- ackalata B1 fusion protein studied by SDS-PAGE Figure 2.3 Time-dependent and hydrogen peroxide-dependent cleavage of 34 recombinant thioredoxin-ac kalata B1 fusion protein by enterokinase Figure 2.4 HPLC chromatogram and MS spectra of ac-kalata B1 35 Figure 3.1 Structure of Ciluprevir, a cyclic peptide inhibitor of HCV 44 NS3pro viii (162) Sofuku, S. M., M.; Hagitani, A. Bull. Chem. Soc. Jpn. 1970, 43, 177-181. (163) Werner, T.; Barrett, A. G. M. J. Org. Chem. 2006, 71, 4302-4304. (164) Reidlinger, C.; Dworczak, R.; Junek, H.; Graubaum, H. Monatsh. Chem. 1998, 129, 1313-1318. (165) Markwalder, J. A.; Arnone, M. R.; Benfield, P. A.; Boisclair, M.; Burton, C. R.; Chang, C.-H.; Cox, S. S.; Czerniak, P. M.; Dean, C. L.; Doleniak, D.; Grafstrom, R.; Harrison, B. A.; Kaltenbach, R. F.; Nugiel, D. A.; Rossi, K. A.; Sherk, S. R.; Sisk, L. M.; Stouten, P.; Trainor, G. L.; Worland, P.; Seitz, S. P. J. Med. Chem. 2004, 47, 5894-5911. (166) Hauske, J. R. D., P. Tetrahedron Lett. 1995, 36, 1589-1592. (167) Lee, M.-r. S., I. Angew. Chem Int. Ed. 2005, 44, 2881-2884. (168) Wang, S.-S. J. Am. Chem. Soc. 1973, 95, 1328-1333. 131 Appendix The peptides designed as potential inhibitors to DEN2 NS2B-NS3 protease. The residues in bold are the recognition sequences of the NS2B-NS3 protease: Peptide Kalata B1 Acyclic Kalata B1 Polypeptide Polypeptide Polypeptide Polypeptide Cyclopeptide Sequence Cyclo(GLPVCGETCVGGTCNTPGCTCSWPVCTRN) GLPVCGETCVGGTCNTPGCTCSWPVCTRN GLPVCGETCVGGTCNTPGCTCSWPVCTRR TPGCTCSRRSCGRRGLPVCGRTCVGCTCN GLPVCRRSCKRGCNTPGCTCSRRSCGRR LPVCGSEESRRGCNTPGCRRSWPVCTRRG Cyclo(LPVCGSEESRRGCNTPGCRRSWPVCTRRG) Table 3.1, Peptides designed as potential inhibitors to DEN2 NS2B/NS3 protease (Chapter 3) DEN2 NS2B/NS3 Protease Inhibition Assay 2500 2000 RFU 1500 1000 500 11 10 95 85 75 65 55 45 35 25 15 Time (mins) Negative control Peptide3 Acyclic kalata B1 Cyclopeptide Peptide2 Positive control Peptide4 Peptide1 Kalata B1 Screening results of the aforementioned polypeptides which are inhibitors against DEN2 NS2B-NS3 protease (each peptide was at 0.1 mg/mL (~33 μM) final concentration). Negative control is Dabcyl-KGRRSSKL-Edans. Positive control is Dabcyl-KGRRSSKL-Edans + NS2B-NS3 protease. 132 HPLC profile of cyclopeptide 1: Mass spectrum of cyclopeptide 1: In t e n . ( x , 0 , 0 ) .0 .7 0 .7 [M + H ]5+ [M + H ]4+ .0 .0 [M + H ]3+ .0 [M + H ]6+ .1 .5 .0 .2 .0 500 750 1000 1250 1500 1750 m /z Deconvolution for the above mass spectrum shows that cyclopeptide has M.W 3201 Da: 133 Mass spectrum of isomer 1A (Figure 3-5a in Chapter 3): [M + H ]5+ [M + H ]4+ [M + H ]3+ [M + H ]6+ Mass spectrum of isomer 1B ((Figure 3-5a in Chapter 3): [M + H ]4+ [M + H ] 5+ [M + H ]3+ [M + H ]6+ Mass spectrum of isomer 1C (Figure 3-5a in Chapter 3): [M +5H] 5+ [M +4H] 4+ [M +6H] 6+ [M +3H] 3+ 134 Mass spectrum of peak of HPLC of isomer 1B as shown in Figure 3-5b in Chapter 3: [M +4H] 4+ [M +5H] 5+ [M +3H] 3+ Mass spectrum of peak of HPLC of isomer 1B as shown in Figure 3-5b in Chapter 3: [M+4H]4+ [M+5H]5+ [M+3H]3+ Mass spectrum of peak of HPLC of isomer 1B as shown in Figure 3-5b in Chapter 3: [M + H ] + [M + H ] + [M + H ] + [M + H ] + 135 Mass spectrum of peak of HPLC of isomer 1B as shown in Figure 3-5b in Chapter 3: [M+5H] 5+ [M+4H]4+ [M+3H] 3+ Mass spectrum of fragments obtained after NH4OH cleavage (Table 3.2) : Itz-CTRRGLPVCGSEE GPTNCGRRS [M + 4H ] 4+ [M + 3H ] 3+ Itz-CRRSWPV [M+3H]3+ [M+2H]2+ 136 One nick [M + H ] + [M + H ] + [M + H ] + [M + H ] + HPLC profile of isomer 1C after partial reduction via strategy (as shown in Scheme 3.2) 40.710 mV(x100) Detector A:220nm 4.0 3.0 38.680 2.0 1.0 0.0 10 20 30 40 Elution time = 38.7 min, fully reduced peptide; Elution time = 40.7 min, partial reduced peptide 137 HPLC profile and mass spectrum of: (a) NEM alkylated cyclopeptide mV(x100) Detector A:220nm 10.0 7.5 40.677 5.0 2.5 0.0 10 20 30 40 Elution time = 40.677 Mass spectrum [M +5H] 5+ [M +4H] 4+ [M +6H] 6+ 138 (b) fully alkylated cyclopeptide mV(x10) Detector A:220nm 6.0 5.0 43.785 4.0 3.0 2.0 1.0 0.0 10 20 30 40 Elution time = 43.785 Mass spectrum [M +5H] 5+ [M +4H] 4+ 139 Mass spectrum of fragments obtained after trypsin cleavage (Table 3.3): GLPVC(CONH2)GSEESR [M + H ] + GLPVC(CONH2)CGSEESR(R) [M + H ]3+ [M + H ]2+ RGC(NEM)NTPGC(NEM)RRSWPVC(CONH2)TR(R) [M + H ] + [M + H ] + 140 SWPVC(CONH2)TR [M +2H] 2+ [M +3H] 3+ [M +H] + Amino acid sequence of thioredoxin fused with a DEN2 NS2B-NS3. Residues to 109 which specifies thioredoxin are shown in italics. Residues 116 to 121 specifying the N-terminal His6 tag are underlined. The enterokinase recognition sequence (residues 153 to 157) is underlined and the arrow indicates the site of cleavage to yield NS2B/NS3 with no unwanted residues. Residues 158 to 240 indicated in bold typeface comprise the 83-amino-acid hydrophilic core sequence derived from the DEN2 NS2B sequence (GenBank accession no. NP_739586). The 181 amino acids of the NS3 sequence (residues 241 to 421) (PDB entry 1BEF) are highlighted in gray: 141 190 180 170 160 150 7.2 6.8 140 6.4 130 2.4217 1.0000 5.4073 2.3666 4.0301 1.9672 O HO 6.0 Cl 120 N Cl 5.6 5.2 O HO N O 110 S O 4.8 4.4 S 100 90 4.0 80 3.6 3.2 70 2.8 60 40.0028 39.8352 39.7551 39.6676 39.5000 39.3324 39.1648 38.9972 37.2555 7.6 61.5734 8.0 120.9946 8.4 149.2696 137.8430 136.3928 136.2034 134.8406 132.7127 130.6650 129.8415 128.9889 128.5006 128.3913 128.2237 127.5824 8.8 155.7627 9.2 159.5011 9.6 164.6751 13 187.7396 Integral 2.5038 2.5000 2.4962 4.5298 4.5235 4.4970 4.4680 4.4415 6.1599 7.7661 7.7484 7.5379 7.5215 7.5064 7.4194 7.3967 7.3828 7.3299 7.3135 7.2958 7.2769 7.2630 7.2479 H NMR of 4-6k in Chapter NN S Cl (ppm) 2.4 50 2.0 1.6 40 1.2 30 0.8 20 0.4 10 0.0 C NMR of 4-6k in Chapter NN S Cl (ppm) 142 190 180 170 160 150 7.2 6.8 140 6.4 130 6.0 Cl 120 N 5.6 5.2 O HO 110 S O 4.8 N 4.4 S O 100 90 4.0 80 3.6 3.2 70 2.8 60 2.4 50 2.0 1.6 40 1.2 30 12.8574 HO 22.2727 7.6 2.5000 2.4962 3.2325 3.2186 3.2073 3.1921 3.1795 3.1669 3.1531 3.1404 6.0994 7.7661 7.7497 7.4900 7.4736 7.3135 7.2971 0.9644 0.9493 0.9354 Cl 2.9570 S 1.7095 1.6957 1.6805 1.6667 1.6515 1.6377 N N 1.9735 2.4714 O 39.9955 39.8279 39.6676 39.5000 39.3324 39.1648 38.9972 35.2952 8.0 1.0000 7.8526 Cl 61.3621 8.4 118.5679 8.8 137.1872 137.1434 136.7936 136.4147 136.3928 136.3710 132.2318 130.6067 129.7103 127.9832 9.2 155.5732 9.6 159.9092 13 186.5518 Integral H NMR of 4-6o in Chapter (ppm) 0.8 20 0.4 10 0.0 C NMR of 4-6o in Chapter N N S Cl (ppm) 143 210 200 190 180 170 160 150 6.4 6.0 140 130 5.6 5.2 120 4.8 Cl 110 4.4 4.0 HO 100 3.6 O N N 90 3.2 S 80 2.8 70 2.4 60 2.0 50 1.6 40 3.1305 S 2.1044 N 1.2 0.8 30 20 13.2946 6.8 N 2.0001 O 40.0028 39.9227 39.8352 39.7551 39.6676 39.5000 39.3324 39.1648 38.9972 33.1017 30.8717 21.0848 7.2 2.3907 HO 61.4933 7.6 1.0000 1.9508 3.8649 1.9483 Integral Cl 120.0910 8.0 137.4349 136.5677 135.4673 132.5232 130.6358 129.7978 128.2383 128.1290 8.4 155.4857 8.8 160.1132 9.2 165.1342 9.6 187.2878 13 210.5637 1.6679 1.6528 1.6389 1.6238 1.6087 1.4120 1.3969 1.3830 1.3679 1.3527 1.3389 0.8825 0.8674 0.8535 2.5000 3.2615 3.2464 3.2350 3.2211 3.2060 3.1909 3.1770 3.1657 3.1505 6.1347 7.7648 7.7484 7.5227 7.5101 7.4963 7.3248 7.3084 H NMR of 4-6p in Chapter N S O Cl (ppm) 0.4 0.0 C NMR of 4-6p in Chapter N S O Cl (ppm) 10 144 IR Spectrum of Resin 5-11 O O N N H2N CH3 CN X-ray Structure of 5-1l 145 210 200 190 180 170 Ph 160 13 150 140 130 120 110 N N 100 90 Ph N 80 70 60 50 -1 40 30 13.0014 3.0000 4.9549 N 40.0534 39.7730 39.5000 39.2196 38.9392 38.6662 10 88.0697 11 110.8419 12 129.9761 129.2455 126.6038 13 139.2665 137.0379 14 148.1362 157.4119 Integral 2.6184 2.5012 3.3392 7.6583 7.6282 H NMR Spectrum of 5-1a O N N CH3 N CN (ppm) -2 -3 20 -4 C NMR Spectrum of 5-1a O N N CH3 N CN (ppm) 10 146 [...]... for polyprotein processing and RNA replication The N-terminal third of the protein is the catalytic domain of the NS2B- NS3 serine protease complex55-57 In addition to cleaving the NS2A /NS2B, NS2B/ NS3, NS3/ NS4A, and NS4B/NS5 junctions, the protease generates the C-termini of mature capsid protein13,27 and NS4A14, and can cleave at internal sites within NS2A and NS3 (Figue 1.2 and Table 1.1) Since the... structures of WNV NS2B/ NS3pro and predicted substrate and membrane interactions NS2B (red), NS3pro (blue) and catalytic triad (magenta in B) A and B, polypeptide backbone and side chains of NS2B residues identified as important for proteolytic activity are in yellow and correspond to A, site 1, NS2B5 9-62 and B, site 2, NS2B7 5-87 Potential target sites for blocking cofactor association with NS3pro are... representation of a cyclotide (kalata B1) structure 46 Figure 3.3 Possible isomers of cyclopeptide 1 48 Figure 3.4 HPLC profiles 49 Figure 3.5 Inhibition of WNV by protease inhibitors 56 Figure 4.1 Structures and IC50 values of WNV NS2B- NS3pro inhibitors 65 confirmed in the HTS Figure 4.2 Uncompetitive mechanism of inhibition of WNV NS2B- NS3pro 76 by the (-) enantiomer of compound 4-6o Figure 5.1 Library of synthesized... responsible for cleavage [vir C/anchC] cleavage site: RR↓S, NS2B- NS3 Protease PrM: TGG↓V, Signalase M: ARR↓A, Furin E 53-54 kD AYS↓A, Signalase NS1 46 kD VGA↓D, Signalase NS2A 24 kD VTA↓G, Unknown NS2B 14 kD RR↓S, NS2B- NS3 Protease NS3 70 kD RR↓S, NS2B- NS3 Protease NS4A 16 kD RR↓G, NS2B- NS3 Protease NS4B 28 kD VAA↓N, Signalase NS5 103 kD RR↓G, NS2B- NS3 Protease Function/enzymatic activity Capsid protein,... between NS3 and its cofactor NS2B To date most attention has been focused on the development of inhibitors that compete for the substrate-binding cleft The preference of the substrate binding cleft of flaviviral proteases for ligands 15 with consecutive basic residues at P1 and P2 is not usual for mammalian proteases and therefore might be exploited to provide inhibitors with specificity for NS2B/ NS3pro... charged nature of the interactions of such basic residues makes the design of nonpeptidic inhibitors extremely challenging The recently reported crystal structures of the NS2B/ NS3 protease, together with the results of mutagenesis studies and solution structure-activity relationships of substrates /inhibitors, provides a basis for rational drug design and for structural optimization of inhibitors that... acts as a cofactor for the NS2B- NS3 serine protease5 1 The cofactor activity lies in a central peptide that intercalates within the fold of the serine protease domain52, similar to the hepatitis C 10 virus (HCV) NS4A cofactor Mutation of conserved residues in NS2B can have dramatic effects on autoproteolytic cleavage at the NS2B/ NS3 junction and transcleavage activities53,54 The NS3 protein The NS3 is a... this enzyme family, but reinforce an unusually flexible mode of substrate binding in the S1 pocket Single chain proteases have been recently created by genetically fusing the NS2B cofactor region with the NS3 protease domain60,61 The structures of the WNV and DENV-2 NS2B- 3 proteases reveal that the cofactor region of NS2B contributes a β-strand to forming the chymotrypsin-like fold, similar to what 11... (P3-P3’) and their corresponding binding sites (S3-S3’) in the enzyme Further biochemical analysis localised viral protease activity to the N-terminal 184 amino acids of NS3 and showed the protease activity to be dependent upon association with a hydrophilic domain within NS2B5 6,70-72 An advance in the understanding of the active protease was provided by the crystal structures of the NS2B/ NS3 proteases... to Leu79 and Phe85 cofactor residues and to interfere with attachment of this region87 As this flexible region of NS2B forms part of the substrate binding cleft, blocking of its association is thereby likely to prevent substrate binding and cleavage Further optimization and development of inhibitors targeted to these sites could potentially lead to the generation of a novel antiviral drug candidate . focus of the thesis involves the design, synthesis and biological evaluation of inhibitors of Dengue and West Nile virus NS2B- NS3 protease. For the design of dengue NS2B- NS3 protease inhibitors, . Design, Synthesis and Biological Evaluation of Inhibitors of Flavivirus NS2B/ NS3 Protease Gao Yaojun (B.Sc., Soochow University) A THESIS SUBMITTED FOR THE DEGREE OF. Yulin Lam. Synthesis and biological evaluation of small molecule inhibitors of West Nile Virus NS2B/ NS3 Protease. ( In preparation). 2. Yaojun Gao, Taian Cui, Yulin Lam. Synthesis and disulfide