Original article Identification by in silico and in vitro screenings of small organic molecules acting as reversible inhibitors of kallikreins Xiao Tan a , Claudia Bertonati b , Lixian Qin a , Laetitia Furio c , Chahrazade El Amri a, * , Alain Hovnanian c, d, *** , Michèle Reboud-Ravaux a , Bruno O. Villoutreix b, ** a Enzymologie Moléculaire et Fonctionnelle, UR4, Université Pierre et Marie Curie e Sorbonne Universités (UPMC), case courrier 256, 7, quai St Bernard, 75252 Paris Cedex 05, France b Molécules Thérapeutiques in silico (MTi), Université Paris-Diderot, Inserm UMR-S, 973 Lamarck Building, 35 rue Hélène Brion, 75205 Paris Cedex 13, France c INSERM U781, Université Paris Descartes e Sorbonne Paris Cité, Institut Imagine, Necker Hospital, 149 rue de Sèvres, France d Department of Genetics, Necker Hospital, 149 rue de Sèvres, 75743 Paris Cedex 15, France article info Article history: Received 2 October 2013 Accepted 12 October 2013 Available online 22 October 2013 Keywords: Netherton syndrome Skin diseases Kallikreins Small organic molecules Virtual screening In vitro screening Reversible inhibitors abstract Netherton syndrome is caused by loss-of-function mutations in SPINK5 encoding the Kazal-type inhibitor LEKTI-1 leading to dysregulation of proteolytic cascades involving several kallikreins. We used both structure-based and ligand-based virtual screening computations to identify commercially available non- covalent inhibitors of human kallikrein 5 (hK5), a serine protease (trypsin-like) that plays a central role in the initiation of the molecular cascades leading to the Netherton syndrome phenotype. The efficacy and mechanism of inhibition of the identified new families of organic compounds were analyzed not only for hK5 but also on other proteases implicated in the cascades (hK7, hK14 and matriptase). These inhibitors are nontoxic on healthy human keratinocytes and are structurally different from traditional serine protease inhibitors validating their potential utility as initial hits to control proteolytic disorders observed in dermatological pathologies such as Netherton syndrome. Ó 2013 Elsevier Masson SAS. All rights reserved. 1. Introduction The skin stratum corneum functions as the body’s main barrier against dehydration, physical and chemical damage, and biological pathogens [1]. The integrity of stratum corneum is maintained by cell adhesion via desmosomes and corneodesmosmes. Shedding of the outermost layer of corneocytes (desquamation) is a serine protease dependent process that is essential to control the thickness of the stratum corneum [1,2]. This process is tightly regulated by a balance between proteolytic enzymes and macromolecular in- hibitors. Among them, human tissue kallikreins (hK)5, 7 and 14 and their natural macromolecular lympho-epithelial Kazal-type-related inhibitor LEKTI play a major role in proteolytic cascades governing skin homeostasis. They contribute to the regulation of desquama- tion, innate immunity and cutaneous inflammation processes. Netherton syndrome (NS) is an autosomal recessive skin disor- der associated with mutations in the SPINK5 gene which encodes the LEKTI [3,4]. The majority of SPINK5 mutations results in com- plete absence of LEKTI and sometimes dysfunctional protein with a reduced capacity to inhibit hKs expressed in the skin. Experimental data provide evidence that hK5 plays an important role in initiating the molecular cascades leading to the disease phenotype. Recently, the role of matriptase, an activator of hKLK5, was also depicted in NS [5]. Its inactivation in LEKTI-deficient mice eliminated aberrant protease activity, prevented detachment of the stratum corneum and improved the barrier function of the epidermis. Currently only symptomatic treatments of NS are used to prevent and treat dehydration, cutaneous and systemic infections and allergic man- ifestations [6]. Although these treatments may improve patients condition, they are not satisfactory and innovative therapies are needed. Endogeneous serine proteases controlled by LEKTI constitute plausible drug targets, especially the trypsin-like hK5 Abbreviations: AMC, 3-amino-7-methyl-coumarin; DMSO, dimethylsulfoxide; hK, human kallikrein; LBVLS, ligand-based virtual ligand screening; SBVLS, struc- ture-based virtual ligand screening; LEKTI, lympho-epithelial Kazal-type inhibitor. * Corresponding author. ** Corresponding author. *** Corresponding author. INSERM U781, Université Paris Descartes e Sorbonne Paris Cité, Institut Imagine, Necker Hospital, 149 rue de Sèvres, France. E-mail addresses: chahrazade.el_amri@upmc.fr (C. El Amri), alain.hovnanian@ inserm.fr (A. Hovnanian), bruno.villoutreix@gmail.com (B.O. Villoutreix). Contents lists available at ScienceDirect European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech 0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.ejmech.2013.10.040 European Journal of Medicinal Chemistry 70 (2013) 661e668