Structure-guided fragment-based in silico drug design of dengue protease inhibitors Tim Knehans • Andreas Schu ¨ ller • Danny N. Doan • Kassoum Nacro • Jeffrey Hill • Peter Gu ¨ ntert • M. S. Madhusudhan • Tanja Weil • Subhash G. Vasudevan Received: 23 November 2010 / Accepted: 7 February 2011 / Published online: 23 February 2011 Ó Springer Science+Business Media B.V. 2011 Abstract An in silico fragment-based drug design approach was devised and applied towards the identifica- tion of small molecule inhibitors of the dengue virus (DENV) NS2B-NS3 protease. Currently, no DENV prote- ase co-crystal structure with bound inhibitor and fully formed substrate binding site is available. Therefore a homology model of DENV NS2B-NS3 protease was gen- erated employing a multiple template spatial restraints method and used for structure-based design. A library of molecular fragments was derived from the ZINC screening database with help of the retrosynthetic combinatorial analysis procedure (RECAP). 150,000 molecular fragments were docked to the DENV protease homology model and the docking poses were rescored using a target-specific scoring function. High scoring fragments were assembled to small molecule candidates by an implicit linking cas- cade. The cascade included substructure searching and structural filters focusing on interactions with the S1 and S2 pockets of the protease. The chemical space adjacent to the promising candidates was further explored by neigh- borhood searching. A total of 23 compounds were tested experimentally and two compounds were discovered to inhibit dengue protease (IC 50 = 7.7 lM and 37.9 lM, respectively) and the related West Nile virus protease (IC 50 = 6.3 lM and 39.0 lM, respectively). This study demonstrates the successful application of a structure-gui- ded fragment-based in silico drug design approach for dengue protease inhibitors providing straightforward hit generation using a combination of homology modeling, fragment docking, chemical similarity and structural filters. Keywords Dengue virus  NS2B-NS3 protease  West Nile virus  Protease inhibitor  Fragment-based drug design (FBDD)  Homology modeling Introduction The incidence of dengue has grown dramatically around the world in recent decades. Some 2.5 billion people—two fifths of the world’s population—are now at risk from dengue [1]. The WHO currently estimates that 50 million dengue virus (DENV) infections occur worldwide every Tim Knehans and Andreas Schu ¨ller have contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s10822-011-9418-0) contains supplementary material, which is available to authorized users. T. Knehans  P. Gu ¨ntert Institute of Biophysical Chemistry, Center for Biomolecular Magnetic Resonance and Frankfurt Institute for Advanced Studies, J.W. Goethe-University, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany T. Knehans  A. Schu ¨ller  D. N. Doan  S. G. Vasudevan (&) Program in Emerging Infectious Diseases, Duke-NUS Graduate Medical School, 8 College Road, Singapore 169857, Singapore e-mail: subhash.vasudevan@duke-nus.edu.sg K. Nacro  J. Hill Experimental Therapeutic Centre, Agency for Science, Technology and Research (A*STAR), 31 Biopolis Way, Nanos Level 3, Singapore 138669, Singapore M. S. Madhusudhan Bioinformatics Institute, Agency for Science, Technology and Research (A*STAR), 30 Biopolis Street, #07-01 Matrix, Singapore 138671, Singapore T. Weil Institute of Organic Chemistry III/Macromolecular Chemistry, University of Ulm, Albert-Einstein-Allee 11, 89069 Ulm, Germany 123 J Comput Aided Mol Des (2011) 25:263–274 DOI 10.1007/s10822-011-9418-0