Hybrid Structure-Based Virtual Screening Protocol for the Identification of Novel BACE1 Inhibitors R. S. K Vijayan, M. Prabu, Nahren Manuel Mascarenhas, and Nanda Ghoshal* Structural Biology and Bioinformatics Division, Indian Institute of Chemical Biology (A unit of CSIR), Kolkata 700032, India Received October 17, 2008 BACE1, also called -secretase or memapsin 2, is an extensively studied aspartic protease, involved in etiopathogenesis and progression of Alzheimer’s disease (AD). We report herein a modified structure-based virtual screening protocol that augments the lead identification process against BACE1 during virtual screening endeavors. A hybrid structure-based virtual screening protocol that incorporates elements from both ligand- based and structure-based techniques was used for the identification of prospective small molecule inhibitors. Virtual screening, using an active-site-derived pharmacophore, followed by ROCS (rapid overlay of chemical structures)-based GOLD (genetic optimization in ligand docking) docking was used to identify a library of focused candidates. The efficacy of the ROCS-based GOLD docking method together with our customized weighted consensus scoring function was evaluated against conventional docking methods for its ability to discern true positives from a screening library. An in-depth structural analysis of the binding mode of the top-ranking molecules reveals that emulation of the curial interaction patterns deemed necessary for BACE1 inhibition. The results obtained from our validation study ensure the superiority of our docking methodology over conventional docking methods in yielding higher enrichment rates. INTRODUCTION Alzheimer’s disease (AD) is a common age-related neu- rodegenerative disorder, first described by Alois Alzheimer 1 in 1906, that continues to increase worldwide. A major histopathological manifestation of Alzheimer’s disease (AD) involves the accumulation of extra cellular -amyloid (A) neuritic plaques. 2 One of the two isoforms of proteases termed BACE1, involved in the cleavage of  amyloid precursor protein (APP), plays a central role in the etiology of Alzheimer’s disease. 3,4 Reducing the production of A or increasing its clearance offers an axiomatic strategy for the treatment of Alzheimer’s disease. 5 Hence inhibition of the enzyme BACE1 offers a tractable target for drug development that would aid in the management and delay the progression of AD. Though considerable attention has been paid toward the development of aspartic protease inhibitors, the development of inhibitors targeting proteases of human origin is challenging because BACE1 is a cellular protein and presumably has some normal physiological functions. Further the existence of other homologous aspartlyl proteases like BACE2 and a ubiquitous protein termed cathepsin D obviously reminds one of the need to consider selectivity aspects as a part of the investigation. Therefore, identifying selective nonpeptidic BACE1 inhibitors with CNS penetration and good pharmacokinetic properties would be demanding. 6 Working toward this end, we carried out a hybrid structure-based virtual screening program that could provide greater strides for the identification of novel non- peptidic BACE1 inhibitors. A drug discovery project conducted by Merck research laboratories and NeoGenesis pharmaceuticals identified a series of aminopentyl oxacetamides as potent and selective inhibitors. 7 The published crystal structure of BACE1 in complex with an aminopentyl oxacetamide (PDB ID 1TQF) served as a starting point to pursue our structure-based drug design program. This cocrystallized complex was chosen because it reveals a binding mode unconventional to aspartyl proteases and more importantly it characterizes the nontran- sition state of the enzyme complex. To begin with, an active- site-directed pharmacophore model was generated on the basis of the geometric disposition of the protein-inhibitor complex using the program LigandScout. 8 Hits identified by pharmacophores generated through direct approach are more reliable because they impose the necessary constraints required for interaction and selectivity. In the next phase, scaffold hopping was performed against multiconformational databases using pharmacophore-derived queries. Molecular docking, a widely used approach in virtual screening was used for post processing hits that lack complementarities with the target protein outside the phar- macophoric definition. 9 Though the concept of protein-ligand docking brought about a paradigm shift in early stage drug discovery, it is apparent that there are certain lacunas that impede the identification of high quality leads during a virtual screen. 10 High quality lead identification is an important prerequisite in drug discovery research; hence optimization of docking applications represents the next step in attaining better enrichment rates during virtual screening. While other efforts center on addressing issues like receptor flexibility and development of improved scoring functions for protein- * To whom correspondence should be addressed. Phone: +91-33-2473- 3491 ext. 254/236. Fax: +91-33-2473-0284/5197. E-mail: nghoshal@iicb.res.in. Address: Structural Biology and Bioinformatics Division, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Jadavpur, Kolkata 700032, India. J. Chem. Inf. Model. 2009, 49, 647–657 647 10.1021/ci800386v CCC: $40.75  2009 American Chemical Society Published on Web 02/13/2009