a-Amylase Modulation: Discovery of Inhibitors Using a Multi-Pharmacophore Approach for Virtual Screening Jamil Al-Asri, [a] Gyçngyi GyØmµnt, [b] Erika Fazekas, [b] Gµbor Lehoczki, [b] Matthias F. Melzig, [a] Gerhard Wolber,* [a] and JØrØmie Mortier* [a] Better control of postprandial hyperglycemia can be achieved by delaying the absorption of glucose resulting from carbohy- drate digestion. Because a-amylase initiates the hydrolysis of polysaccharides, the design of a-amylase inhibitors can lead to the development of new treatments for metabolic disorders such as type II diabetes and obesity. In this study, a rational computer-aided approach was developed to identify novel a- amylase inhibitors. Three-dimensional pharmacophores were developed based on the binding mode analysis of six different families of compounds that bind to this enzyme. In a stepwise virtual screening workflow, seven molecules were selected from a library of 1.4 million. Five out of seven biologically tested compounds showed a-amylase inhibition, and the two most potent compounds inhibited a-amylase with IC 50 values of 17 and 27 mm. The scaffold benzylideneacetohydrazide was shared by four of the discovered inhibitors, emerging as a novel drug-like non-carbohydrate fragment and constituting a promising lead scaffold for a-amylase inhibition. Metabolic diseases such as obesity and type II diabetes are characterized by high levels of blood glucose. [1] Therefore, en- zymes that regulate rate-controlling steps in the gluconeogen- ic or glycogenolytic pathways are key biological targets for therapeutic interventions. [2] Among drugable enzymes targeted to regulate such clinical manifestations is a-amylase. [3] This di- gestive enzyme controls the rate-limiting step of starch metab- olism by hydrolyzing the a-1,4-glycosidic bond in starchy foods and producing oligomaltose chains. [4] The latter are sub- jected to hydrolysis by other enzymes to produce glucose that can be absorbed into the bloodstream. [5] In recent years, a- amylase has been in the spotlight to identify novel anti-obesity and anti-diabetes drugs. [6] This 56 kDa enzyme has a simple structure formed by a single polypeptide chain. Its largest domain includes the cat- alytic pocket with a cavity spanning from subsites À4 to + 3. [7] The cleavage site is positioned between subsites À1 and + 1, where the catalytic triad is located (Asp197, Glu233, and Asp300). This large binding site can be blocked by carbohy- drate-based inhibitors such as members of trestatin families (acarbose, acarviostatins). Flavonoids are also known to bind to a-amylase. For example, natural flavonoids (anthocyanins) extracted from sour cherry were identified recently as potent human salivary a-amylase (HSA) inhibitors. [8] If the specificity of these natural products is usually low and their inhibitory po- tency remains weaker than sugar-based compounds, [9] a careful analysis of their binding mode can be very valuable for the ra- tional design of novel a-amylase inhibitors. Acarbose is the ref- erence compound in a-amylase inhibition and it is used as anti-diabetic agent; however, it shows undesirable gastrointes- tinal disturbances as side effects due to its inhibition of a-glu- cosidase. [10] Acarbose has a mechanism of inhibition that is very different in each binding pocket: acarbose reacts with a- amylase and undergoes a chemical rearrangement beforehand to block the catalytic center of a-amylase, [7] whereas acarbose is accommodated in the binding site of a-glucosidase with no preliminary rearrangement. [11] Therefore, the design of small non-carbohydrate HSA inhibitors that are able to block the function of this enzyme selectively holds great potential for anti-diabetic therapies. Recently, we published a computer-based approach for the identification of chemical fragments binding to HSA. [12] This preliminary work was the first successful discovery of novel drug-like a-amylase inhibitors with a rational structure-based strategy targeting subunits À1 and + 1 of the catalytic pocket. In the continuation of this study, a computer-aided method was developed to compile key ligand–enzyme interactions for a-amylase inhibition and to use this information to identify novel drug-like compounds able to bind a-amylase specifically. Analyzing ligand conformations to identify required 3D chemi- cal feature arrangements for optimal binding is state of the art in structure-based drug design and virtual screening strat- egies. [13] Recently, human pancreatic a-amylase (HPA) has been crystallized with the ligand myricetin (IC 50 = 30.2 mm), [14] the first non-carbohydrate ligand co-crystallized with HPA (PDB ID: 4GQR). [15] In this crystal structure, key residues (including cata- lytic residue Asp300) in the active site show different orienta- tions from that observed with all other macromolecules bound to carbohydrate-based derivatives. As HSA and HPA share a high sequence identity (97 %), this important new structural information can lead to the identification of different chemical scaffolds in a novel virtual screening strategy. While one main pharmacophore was applied in a first screening campaign, [12] [a] J. Al-Asri, M. F. Melzig, G. Wolber, J. Mortier Institute of Pharmacy, Department of Pharmaceutical & Medicinal Chemis- try, Freie Universität Berlin, Kçnigin-Luise Str. 2-4, 14195 Berlin (Germany) E-mail : gerhard.wolber@fu-berlin.de jeremie.mortier@fu-berlin.de [b] G. GyØmµnt, E. Fazekas, G. Lehoczki Department of Inorganic & Analytical Chemistry, University of Debrecen, Egyetem ter 1, PO Box 21, 4032 Debrecen (Hungary) Supporting information and the ORCID identification number(s) for the author(s) of this article can be found under http://dx.doi.org/10.1002/ cmdc.201600427. ChemMedChem 2016, 11,1–7  2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1 These are not the final page numbers! ÞÞ These are not the final page numbers! ÞÞ Communications DOI: 10.1002/cmdc.201600427 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10 11 11 12 12 13 13 14 14 15 15 16 16 17 17 18 18 19 19 20 20 21 21 22 22 23 23 24 24 25 25 26 26 27 27 28 28 29 29 30 30 31 31 32 32 33 33 34 34 35 35 36 36 37 37 38 38 39 39 40 40 41 41 42 42 43 43 44 44 45 45 46 46 47 47 48 48 49 49 50 50 51 51 52 52 53 53 54 54 55 55 56 56 57 57