Tetrapeptides as Potent Protease Inhibitors of Hepatitis C Virus Full-Length NS3 (Protease-Helicase/NTPase) Anja Johansson, a Anton Poliakov, b EvaA ˚ kerblom, a Gunnar Lindeberg, a Susanne Winiwarter, a Bertil Samuelsson, c U. Helena Danielson b and Anders Hallberg a, * a Department of Medicinal Chemistry, Uppsala University, BMC, Box 574, SE-751 23 Uppsala, Sweden b Department of Biochemistry, Uppsala University, BMC, Box 576, SE-751 23 Uppsala, Sweden c Medivir AB, Lunastigen 7, SE-141 44 Huddinge, Sweden Received 14 May 2002; accepted 18 July 2002 Abstract—AlibraryoftetrapeptideswasevaluatedforHepatitisCVirusNS3proteaseinhibitoractivityinaninvitroassaysystem comprising the native bifunctional full-length NS3 (protease-helicase/NTPase) protein. Tetrapeptides with K i values in the high nanomolar range were identified, for example Suc-Chg-Glu-2-Nal-Cys (K i =0.27  0.03 mM) and Suc-Dif-Glu-Glu-Cys (K i =0.40  0.10 mM).Furthermore,itwasshownthattheinhibitorypotenciesarenotaffectedsignificantlybyassayionicstrength. As suggested by molecular modelling, potential binding interactions of the tetrapeptide inhibitors with the helicase domain might explainthedataandstructure–activityrelationshipsthusobtained.Hence,wepostulatethatthefull-lengthNS3assayisarelevant system for inhibitor identification, offering new opportunities for inhibitor design. # 2002ElsevierScienceLtd.Allrightsreserved. Introduction Hepatitis C Virus (HCV) infections constitute a global health problem. About 3% of the world’s population suffersfromchronichepatitisC,whichcouldultimately developintolivercirrhosis,hepatocellularcarcinomaor liver failure. The currently available therapies are inter- feron-a (IFN-a)anditscombinationwithribavirinand, more recently, pegylated IFN-a. Unfortunately, the successofthesetherapiesismodestandvariesdepending on the specific HCV genotype. 1 4 Consequently, new andmoreeffectivetherapiesaremuchneeded. The RNA genome of HCV is translated into the poly- proteinNH 2 -C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A- NS5B-COOH, which is proteolytically cleaved into 10 mature viral proteins. The non-structural protein 3 (NS3) is a multifunctional enzyme possessing serine protease activity in the N-terminal third of the protein and RNA helicase/NTPase activity in the C-terminal portion. The NS3 protease is responsible for auto cata- lytic cis cleavage at the NS3/NS4A junction and for trans cleavages at the NS4A/NS4B, NS4B/NS5A and NS5A/NS5Bjunctions. 5 7 TheNS3proteasehasbeenthe subject of extensive studies and represents the primary target in the search for effective antiviral treatments againstHCV. 2,8 10 The NS3 protease is inhibited by both classical serine protease inhibitors with an electrophilic serine-trap functionalityintheP1position 11 21 and,uniquely,byits N-terminal cleavage products and derivatives thereof, containing a C-terminal COOH group. 10,12,19,22 26 Fur- thermore,itwasdemonstratedwithaseriesofproduct- basedhexapeptideinhibitorsthattheinhibitorypotencies were affected significantly by the ionic strength of the assay medium. 24 Thus, considerably lower activities were reported at physiological NaCl concentrations (  150 mM) than in the absence of salt additives, employing an assay using the protease domain of NS3, thatisnotthenativeformoftheenzyme. 24 Recently,wereportedthatthestructure–activityrelation- ships(SARs)ofasmallseriesofHCVNS3proteaseinhi- bitorsisdependentontheassayused. 27 Mostimportantly, we found that shortened product-based inhibitors retained much better inhibitory potencies in the native bifunctionalfull-lengthNS3(protease-helicase/NTPase) 0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII:S0968-0896(02)00310-3 Bioorganic & Medicinal Chemistry (2002) 3915–3922 *Corresponding author. Tel.: +46-18-471-4284; fax: +46-18-471- 4474; e-mail: anders.hallberg@bmc.uu.se