Discovery of a novel HCV helicase inhibitor by a de novo drug design approach Sahar Kandil a , Sonia Biondaro a , Dimitrios Vlachakis a , Anna-Claire Cummins a , Antonio Coluccia a , Colin Berry b , Pieter Leyssen c , Johan Neyts c , Andrea Brancale a, * a The Welsh School of Pharmacy, Cardiff University, Cardiff, United Kingdom b Cardiff School of Biosciences, Cardiff University, Cardiff, United Kingdom c Rega Institute for Medical Research, KULeuven, Leuven, Belgium article info Article history: Received 9 March 2009 Revised 14 April 2009 Accepted 17 April 2009 Available online 22 April 2009 Keywords: HCV Helicase inhibitor De novo Molecular modelling abstract Herein we report a successful application of a computer-aided design approach to identify a novel HCV helicase inhibitor. A de novo drug design methodology was used to generate an initial set of structures that could potentially bind to a putative binding site. Further structure refinement was carried out through docking a series of focused virtual libraries. The most promising compound was synthesised and it exhibited a submicromolar inhibition of the HCV helicase. Ó 2009 Elsevier Ltd. All rights reserved. HCV infection is the second most common chronic viral infec- tion in the world with global prevalence. An estimated 180 million people are chronically infected with this hepatitis C virus (HCV) and thus at increased risk of developing serious life threatening li- ver diseases including cirrhosis that may progress to hepatocellular carcinoma. 1,2 There is no prophylactic or therapeutic vaccine available against HCV and the development of such a vaccine is not expected to oc- cur soon. Current standard therapy, that is, the combination of pegylated interferon with ribavirin is only effective in about 60% of the patients and is associated with important side-effects. 3 Sev- eral drugs that target various stages of the replication cycle of HCV are currently in preclinical or clinical development; these include NS3 protease inhibitors, nucleoside and non-nucleoside polymer- ase inhibitors and cyclophylin binding compounds. 4,5 So far the NS3 helicase has not been extensively explored as a target for inhibition of HCV replication. 6–9 We devised a rational approach for the design of selective inhib- itors of the HCV NS3 helicase. Several crystal structures of the HCV NS3 have been reported, that is, as individual domains (protease and helicase) as well as the full length protein. The structure re- ported by Kim 10 was co-crystallised with a strand of DNA bound to the helicase domain (PDB 1A1V). It was decided to use this structure as the starting point for the de novo design of novel po- tential inhibitors that could compete for the nucleic acid binding site. The first step was to define a potential binding site for a small molecule; we focused therefore on the area around Arg393. This residue is conserved in all HCV isolates and is also known to play a key role in activity of the enzyme since mutation R393A has a detrimental effect on the unwinding activity of the enzyme. 11 Tar- geting this specific amino acid represented a considerable chal- lenge: after removing the nucleic acid from the structure it became apparent that the area around Arg393 was completely ex- posed to the solvent, making it more difficult for a small molecule to bind tightly to it without being replaced by water or the nucleic acid itself. However, on further inspection it was observed that the sulfur atom of Cys431 (a residue not involved directly in the nu- cleic acid binding and that is situated 10 Å away from Arg393) had interacted to form an adduct with a molecule of mercap- toethanol present in the crystallisation water. This observation may suggest that Cys431 is accessible to a small molecule and that it is sufficiently reactive to establish a covalent bond with an appropriate compound. These observations led us to formulate the hypothesis that a molecule that would (i) interact with Arg393 and (ii) at the same time form a covalent bond with Cys431 through a chemically reactive group may have the poten- tial to inhibit helicase activity. Such a covalent binder would be very difficult to displace by water or the incoming nucleic acid. In the initial stages of inhibitor design, the de novo software package LigBuilder was used. 12 Programs of this type normally re- quire the user to define an initial ‘seed’ in a binding site, after which the computer builds a series of molecules by adding to the growing structures the most suitable fragments taken from a given library. 13 The major drawback of this approach is that very often the proposed structures are highly complex and not synthetically 0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2009.04.074 * Corresponding author. Tel.: +44 29 20874485. E-mail address: brancalea@cf.ac.uk (A. Brancale). Bioorganic & Medicinal Chemistry Letters 19 (2009) 2935–2937 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl