Optimization of Assay Conditions fo r Dengue Virus Protease: Effect of Various Polyols and Nonionic Detergents

Abstract

The aim of this work was to perform a systematic study of the effect of nonionic detergents on the activity of the dengue virus NS2B-NS3 protease. To ensure a high activity of the protease, the assay procedures for the dengue virus and other flaviviral proteases published to date are performed in the presence of up to 35% glycerol, which does not represent the cellular physicochemical environment. In addition, the high viscosity of glycerol-containing solutions leads to various experimental problems in miniaturized assays. Using an internally quenched peptide substrate, the authors show that glycerol is not essential for enzymatic activity if certain nonionic detergents are added to the assay buffer. In addition, nonionic detergents may help to avoid false-positive screening results caused by “promiscuous” inhibitors. Other polyalcohols can substitute glycerol and have less effect on the viscosity of the assay buffer. The assay was used to screen a compound library and allowed the identification of small-molecular nonpeptidic inhibitors of dengue NS3 protease. Finally, the authors discuss the mode of action of nonionic detergents and the influence that they may have on the conformational properties of the NS2B-NS3 protease. (Journal of Biomolecular Screening 2009:1102-1108)

Keywords

dengue virus protease high-throughput screening promiscuous inhibitors nonionic detergents

References

Mukhopadhyay S. , Kuhn RJ , Rossmann MG: A structural perspective of the flavivirus life cycle. Nat Rev Microbiol 2005;3:13-22.

Bera AK , Kuhn RJ , Smith JL: Functional characterization of cis and trans activity of the flavivirus NS2B-NS3 protease. J Biol Chem 2007;282: 12883-12892.

Falgout B. , Pethel M. , Zhang YM , Lai CJ: Both nonstructural proteins NS2B and NS3 are required for the proteolytic processing of dengue virus nonstructural proteins. J Virol 1991;65:2467-2475.

Preugschat F. , Yao CW , Strauss JH: In vitro processing of dengue virus type 2 nonstructural proteins NS2A, NS2B, and NS3. J Virol 1990;64: 4364-4374.

Falgout B. , Miller RH , Lai CJ: Deletion analysis of dengue virus type 4 nonstructural protein NS2B: identification of a domain required for NS2B-NS3 protease activity. J Virol 1993;67:2034-2042.

Aleshin AE , Shiryaev SA , Strongin AY , Liddington RC: Structural evidence for regulation and specificity of flaviviral proteases and evolution of the Flaviviridae fold. Protein Sci 2007;16:795-806.

Niyomrattanakit P. , Winoyanuwattikun P. , Chanprapaph S. , Angsuthanasombat C. , Panyim S. , Katzenmeier G: Identification of residues in the dengue virus type 2 NS2B cofactor that are critical for NS3 protease activation. J Virol 2004;78:13708-13716.

Leung D. , Schroder K. , White H. , Fang NX , Stoermer MJ , Abbenante G. , et al: Activity of recombinant dengue 2 virus NS3 protease in the presence of a truncated NS2B co-factor, small peptide substrates, and inhibitors. J Biol Chem 2001;276:45762-45771.

Niyomrattanakit P. , Yahorava S. , Mutule I. , Mutulis F. , Petrovska R. , Prusis P. , et al: Probing the substrate specificity of the dengue virus type 2 NS3 serine protease by using internally quenched fluorescent peptides. Biochem J 2006;397:203-211.

10.

LiJ. , Lim SP , Beer D. , Patel V. , Wen D. , Tumanut C. , et al: Functional profiling of recombinant NS3 proteases from all four serotypes of dengue virus using tetrapeptide and octapeptide substrate libraries . J Biol Chem 2005; 280:28766-28774.

11.

Gekko K. , Timasheff SN : Mechanism of protein stabilization by glycerol: preferential hydration in glycerol-water mixtures. Biochemistry 1981 ;20: 4667-4676.

12.

Merrifield RB : Solid-phase peptide syntheses. Endeavour 1965;24:3-7.

13.

Chang CD , Meienhofer J. : Solid-phase peptide synthesis using mild base cleavage of N alpha-fluorenylmethyloxycarbonylamino acids, exemplified by a synthesis of dihydrosomatostatin. Int J Pept Protein Res 1978; 11:246-249.

14.

Nall TA , Chappell KJ , Stoermer MJ , Fang NX , Tyndall JD , Young PR , et al: Enzymatic characterization and homology model of a catalytically active recombinant West Nile virus NS3 protease. J Biol Chem 2004;279: 48535-48542.

15.

Zhang JH , Chung TD , Oldenburg KR : A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 1999;4:67-73.

16.

Gouvea IE , Izidoro MA , Judice WA , Cezari MH , Caliendo G. , Santagada V. , et al: Substrate specificity of recombinant dengue 2 virus NS2B-NS3 protease: influence of natural and unnatural basic amino acids on hydrolysis of synthetic fluorescent substrates. Arch Biochem Biophys 2007;457: 187-196.

17.

McGovern SL , Caselli E. , Grigorieff N. , Shoichet BK : A common mechanism underlying promiscuous inhibitors from virtual and high-throughput screening. J Med Chem 2002;45:1712-1722.

18.

Chappell KJ , Stoermer MJ , Fairlie DP , Young PR : Generation and characterization of proteolytically active and highly stable truncated and full-length recombinant West Nile virus NS3. Protein Expr Purif 2007;53: 87-96.

19.

Chappell KJ , Nall TA , Stoermer MJ , Fang NX , Tyndall JD , Fairlie DP , et al: Site-directed mutagenesis and kinetic studies of the West Nile Virus NS3 protease identify key enzyme-substrate interactions. J Biol Chem 2005;280:2896-2903.

20.

Ganesh VK , Muller N. , Judge K. , Luan CH , Padmanabhan R. , Murthy KH : Identification and characterization of nonsubstrate based inhibitors of the essential dengue and West Nile virus proteases. Bioorg Med Chem 2005;13:257-264.

21.

Knox JE , Ma NL , Yin Z. , Patel SJ , Wang WL , Chan WL , et al: Peptide inhibitors of West Nile NS3 protease: SAR study of tetrapeptide aldehyde inhibitors. J Med Chem 2006;49:6585-6590.

22.

Lin CW , Huang HD , Shiu SY , Chen WJ , Tsai MH , Huang SH , et al: Functional determinants of NS2B for activation of Japanese encephalitis virus NS3 protease. Virus Res 2007;127:88-94.

23.

Lohr K. , Knox JE , Phong WY , Ma NL , Yin Z. , Sampath A. , et al: Yellow fever virus NS3 protease: peptide-inhibition studies . J Gen Virol 2007;88: 2223-2227.

24.

Mueller NH , Yon C. , Ganesh VK , Padmanabhan R. : Characterization of the West Nile virus protease substrate specificity and inhibitors. Int J Biochem Cell Biol 2007 ;39:606-614.

25.

Radichev I. , Shiryaev SA , Aleshin AE , Ratnikov BI , Smith JW , Liddington RC , et al: Structure-based mutagenesis identifies important novel determinants of the NS2B cofactor of the West Nile virus two-component NS2B-NS3 proteinase. J Gen Virol 2008;89:636-641.

26.

Shiryaev SA , Ratnikov BI , Chekanov AV , Sikora S. , Rozanov DV , Godzik A. , et al: Cleavage targets and the D-arginine-based inhibitors of the West Nile virus NS3 processing proteinase. Biochem J 2006;393: 503-511.

27.

Shiryaev SA , Ratnikov BI , Aleshin AE , Kozlov IA , Nelson NA , Lebl M. , et al: Switching the substrate specificity of the two-component NS2B-NS3 flavivirus proteinase by structure-based mutagenesis . J Virol 2007;81: 4501-4509.

28.

Johnston PA , Phillips J. , Shun TY , Shinde S. , Lazo JS , Huryn DM , et al: HTS identifies novel and specific uncompetitive inhibitors of the two-component NS2B-NS3 proteinase of West Nile virus. Assay Drug Dev Technol 2007;5:737-750.

29.

Stoermer MJ , Chappell KJ , Liebscher S. , Jensen CM , Gan CH , Gupta PK , et al: Potent cationic inhibitors of West Nile virus NS2B/NS3 protease with serum stability, cell permeability and antiviral activity. J Med Chem 2008;51:5714-5721.

30.

Yin Z. , Patel SJ , Wang WL , Chan WL , Ranga Rao KR , Wang G. , et al: Peptide inhibitors of dengue virus NS3 protease: Part 2. SAR study of tetrapeptide aldehyde inhibitors. Bioorg Med Chem Lett 2006;16:40-43.

31.

Hait SK , Moulik SP : Determination of critical micelle concentration (CMC) of nonionic surfactants by donor-acceptor interaction with iodine and correlation of CMC with hydrophile-lipophile balance and other parameters of the surfactants . J Surfactants Detergents 2001;4:303-309.

32.

le Maire M. , Champeil P. , Moller JV : Interaction of membrane proteins and lipids with solubilizing detergents. Biochim Biophys Acta 2000;1508: 86-111.

33.

Pennell KD , Adinolfi AM , Abriola LM , Diallo MS : Solubilization of dodecane, tetrachloroethylene, and 1,2-dichlorobenzene in micellar solutions of ethoxylated nonionic surfactants. Environ Sci Technol 1997;31: 1382-1389.