Published: March 31, 2011 r2011 American Chemical Society 3081 dx.doi.org/10.1021/jm101568y | J. Med. Chem. 2011, 54, 3081–3085 BRIEF ARTICLE pubs.acs.org/jmc On a Possible Neutral Charge State for the Catalytic Dyad in β-Secretase When Bound to Hydroxyethylene Transition State Analogue Inhibitors † Fredy Sussman,* ,‡ Jos  e M. Otero, § M. Carmen Villaverde, ‡ Marian Castro, || Jos  e L. Domínguez, ‡ Lucía Gonz  alez-Louro, ‡,§ Ram on J. Est  evez, § and J. Carlos Est  evez* ,§ ‡ Molecular Modeling Research Group, Departamento de Química Org  anica, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain § Carbohydrate Research Group, Departamento de Química Org  anica, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain ) Departamento de Farmacología, Instituto de Farmacia Industrial, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain b S Supporting Information ABSTRACT: β-Secretase is one of the aspartic proteases involved in the formation of amyloid plaques in Alzheimer’s disease patients. Our previous results using a combination of surface plasmon resonance experiments with molecular modeling calculations suggested that the Asp dyad in β-secretase bound to hydroxylethylene containing inhibitors adopts a neutral charged state. In this work, we show that the Asp dyad diprotonated state reproduced the binding ranking of a set of these inhibitors better than alternative protonation states. ’ INTRODUCTION Alzheimer’s disease (AD) is a widespread, neurodegenerative, dementia-inducing disorder, characterized by the formation of amyloid plaques in the brain. 1 The genesis of this construct is catalyzed by a tandem of two proteases identified as β- and γ- secretases. 1,2 It is known that the former enzyme (also referred to as BACE-1 and Memapsin 2) participates in the rate-limiting step of the hydrolytic process that leads to the APP fragments, 13 a fact that has converted β-secretase into a major target for drugs against Alzheimer’s disease. 2,4,5 There are several issues related to the computer-aided design of novel ligands that could enhance the success of in silico high throughput screening protocols but that have not been fully addressed yet. One of the most vital ones is the protonation state of the many buried acidic residues found in this protein, including the active site Asp dyad, which lends to this enzyme an optimal catalytic activity at low pH. 3 There have been many attempts to predict the charged state of the active site Asp dyad by computational methods. 69 The results of those calculations support the hypothesis that the most favored Asp dyad charge state is the one that has only one of the Asp residues protonated. Nevertheless, quantum mechanical (QM) based calculations have left open the possibility that the Asp dyad might be neutral at the acidic optimal pH of the protein. 8 Recently, we have determined the effect of the pH on the affinities of a set of inhibitors with a variety of chemical motifs to the ectodomain BACE-1 region by a surface plasmon resonance (SPR) biosensor based assay. 10 To understand the molecular interactions that underlie the diverse optimum pH for the binding of the various inhibitors as observed experimentally, we calculated the titration curves for a set of BACE-1 ligand complexes. 10 One of our most striking results of that work relates to the protonation state of peptidic inhibitors with hydroxylethy- lene (HE) based isosteres. For these inhibitors, our calculations predicted that the Asp dyad will be diprotonated at low pH, as opposed to all previous studies that pointed to a monoproto- nated Asp dyad state. 10 To further investigate this issue, in this work we have assembled a set of transition state analogue peptidic inhibitors with HE isosteres (see Table 1), some of which have been already synthe- sized and tested by other authors, while others have been produced and assayed by our group. Using molecular mechanics based protocols we show that the diprotonated state reproduced the binding ranking of our set of peptidic inhibitors better than the ones based on either of the monoprotoned Asp dyad bearing enzymes proposed by previous studies, 69 a result that provides further support to our enzyme’s charge assignment when bound to this type of transition state analogue inhibitors. Finally, we discuss the proposed Asp dyad protonation state in relation to the possible existence of low barrier hydrogen bonds (LBHBs) in these systems. ’ RESULTS AND DISCUSSION Chemistry. The δ-amino acid isosters of inhibitors 3 and 4 were prepared as shown in Scheme 1, through a methodology that allows the introduction of a variety of side chain groups at P1/P1 0 using as a key intermediate the oxiranelactone 9, easily Received: October 18, 2010