Antiviral Research 82 (2009) 51–58 Contents lists available at ScienceDirect Antiviral Research journal homepage: www.elsevier.com/locate/antiviral Design of oseltamivir analogs inhibiting neuraminidase of avian influenza virus H5N1 Thanyada Rungrotmongkol a,1 , Vladimir Frecer a,2 , Wanchai De-Eknamkul b , Supot Hannongbua c , Stanislav Miertus a,∗ a International Centre for Science and High Technology, UNIDO, AREA Science Park, Padriciano 99, Trieste I-34012, Italy b Department of Chemistry, Faculty of Science, Chulalongkorn University, Phayathai Road, Pathumwan, Bangkok 10330, Thailand c Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Phayathai Road, Pathumwan, Bangkok 10330, Thailand article info Article history: Received 29 August 2008 Received in revised form 4 December 2008 Accepted 26 January 2009 Keywords: Avian influenza virus subtype H5N1 Neuraminidase inhibitors Oseltamivir analogs Computer-assisted combinatorial library design In silico screening abstract Neuraminidase is an important target for design of antiviral agents in the prophylaxis and treatment of avian influenza virus infections. We have shown the applicability of computer-assisted combinatorial techniques in the design, focusing and in silico screening of a virtual library of analogs of oseltamivir (Tamiflu) with the goal to find potent inhibitors of influenza A neuraminidase N1 that fill the cavity found adjacent to the active site. Crystal structure of oseltamivir–N1 complex was used in the structure-based focusing and virtual screening of the designed library. A target-specific Piecewise Linear Potential type 1 scoring function fitted for a training set of 14 carbocyclic inhibitors and validated for three other inhibitors was used to select virtual hits with predicted inhibitory activities in the subnanomolar range. The results of this computational study are useful as a rational guide for synthetic and medicinal chemists who are developing new drugs against the avian influenza virus H5N1. © 2009 Elsevier B.V. All rights reserved. 1. Introduction In the recent years, the emergence and worldwide spread of the avian influenza A virus subtype H5N1 has raised concerns of pos- sible easy human-to-human transmission, which calls for the need to develop more potent antiviral drugs to be used for the prophy- laxis and treatment of influenza infections. Neuraminidase (NA), a membrane glycoprotein of the influenza virus, which is required for the release of budding virions from the host cell, is one of the potential drug targets of antiviral agents. Several potent and specific inhibitors of the NA have been developed through structure-based rational design, however, only two of them, oseltamivir (Tamiflu) and zanamivir (Relenza), have been approved for human use (Fig. 1) (Kim et al., 1997; Gubareva et al., 2000). A third NA inhibitor, namely peramivir (BCX-1812), failed to show statistically significant viral inhibition due to the relatively low blood levels obtained after oral administration (Bantia et al., 2006). High mutation rate and emerg- ∗ Corresponding author. Fax: +39 040 922 8115. E-mail address: stanislav.miertus@ics.trieste.it (S. Miertus). 1 Permanent address: Department of Chemistry, Faculty of Science, Chulalongkorn University, Phayathai Road, Patumwan, Bangkok 10330, Thailand. 2 Permanent address: Cancer Research Institute, Slovak Academy of Sciences, Vlarska 7, Bratislava SK-83391, Slovakia. ing drug resistance to the commercially available drugs, especially to oseltamivir, have been widely reported (Gubareva et al., 2001; Moscona, 2005). Therefore, finding novel potent inhibitors of NA less affected by cross-resistance as well as identification of new drug targets is a vital goal. Based on the recent X-ray crystallographic studies of influenza A virus NAs of the group-1, it was shown that their structures con- tain a larger cavity adjacent to the active site, formed by residues 147–152 (150-loop) (Russell et al., 2006). This cavity was not found in the group-2 NAs. The 150-loop switches from open to closed con- formation upon the ligand binding, however, the group-1 NAs can bind ligands in both conformational states of the loop. The closed conformation of the loop is similar to that observed in the group- 2 NAs (Russell et al., 2006). Residues of the flexible 150-loop are located in the vicinity and interact mainly with the amino and acetamide groups of the oseltamivir moiety (Fig. 2). The active sites of both NA groups retain a conserved structure of three arginine residues (Arg118, Arg292 and Arg371) that bind the carboxylate group, Arg152 that interacts with the acetamido group of the sialic acid substrate and Glu276 that forms hydrogen bonds with the substrate hydroxyl groups. Shikimic acid is an important metabolic intermediate in the shikimate pathway found in microorganisms and plants, commonly known as a precursor for the biosynthesis of aromatic amino acids and aromatic secondary metabolites. Recently, this phytochemical 0166-3542/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.antiviral.2009.01.008