In Silico Discovery of a Compound with Nanomolar Affinity to Antithrombin Causing Partial Activation and Increased Heparin Affinity J. Navarro-Ferna ́ ndez, †,∥ H. Pe ́ rez-Sa ́ nchez, ‡,§,∥ I. Martínez-Martínez, † I. Meliciani, ‡ J. A. Guerrero, † V. Vicente, † J. Corral,* ,† and W. Wenzel* ,‡ † Servicio de Hematología y Oncología Mé dica, H. U. Morales Meseguer, Centro Regional de Hemodonació n, University of Murcia, Spain ‡ Institute of Nanotechnologie, Karlsruhe Institute of Technology, Karlsruhe, Germany § Department of Computer Engineering, School of Computer Science, University of Murcia, Spain * S Supporting Information ABSTRACT: The medical and socioeconomic relevance of thromboembolic disorders promotes an ongoing effort to develop new anticoagulants. Heparin is widely used as activator of antithrombin but incurs side effects. We screened a large database in silico to find alternative molecules and predicted D-myo-inositol 3,4,5,6-tetrakisphosphate (TMI) to strongly interact with antithrombin. Isothermal titration calorimetry confirmed a TMI affinity of 45 nM, higher than the heparin affinity (273 nM). Functional studies, fluorescence analysis, and citrullination experiments revealed that TMI induced a partial activation of antithrombin that facilitated the interaction with heparin and low affinity heparins. TMI improved antithrombin inhibitory function of plasma from homozygous patients with antithrombin deficiency with a heparin binding defect and also in a model with endothelial cells. Our in silico screen identified a new, non-polysaccharide scaffold able to interact with the heparin binding domain of antithrombin. The functional consequences of this interaction were experimentally characterized and suggest potential anticoagulant therapeutic applications. ■ INTRODUCTION Ever since the discovery of the anticoagulant properties of hirudin from the leech saliva, the increasing relevance of thromboembolic diseases has encouraged a continuous search for new compounds with anticoagulant activity, which has led to the development of the new commercially available anticoagulants. 1 One of the targets for prophylaxis and treatment of thromboembolic diseases is the plasma antico- agulant antithrombin. Antithrombin is a member of the serpin superfamily of protease inhibitors. As in other serpins, antithrombin inhibits its proteases by an unusual branched pathway suicide substrate mechanism in which the reactive center loop of the inhibitor is cleaved by the protease as a normal substrate but is trapped as an acyl intermediate covalent complex. 2,3 However, antithrombin circulates in blood in a metastable conformation in which the reactive center loop is partially inserted and is only activated by heparin and heparan sulfate glycosaminoglycans on the injured subendothelium. 4−6 Accordingly sulfated polysaccharide heparin chains with different size, from unfractionated to the essential pentasac- charide, have been used successfully in anticoagulant therapy and thromboprophilaxis. 7 Since the discovery of the anticoagulant activity of heparins isolated from canine liver in 1916, 8 several new molecules able to bind antithrombin have been identified. The strategies used in this search have been based mainly on the synthesis or chemical modification of existing drugs or in the application of natural compounds with properties similar to those currently used. 9 Examples of such compounds are lignins and flavonoids, 9,10 highly sulfated small organic ligands that seem to have similar properties to heparins. An alternative approach is to screen a large database in silico and use affinity-ranking to identify some at least weakly binding molecules for further refinement. Aided by ever-increasing computational power, 11,12 virtual screening is an appealing and cost-effective approach to tap into the wealth of available structural information. 13 However, despite several success stories, limitations in current in silico screening approaches restrict their accuracy and general applicability. 14,15 Here we have pursued an in silico discovery strategy in order to find molecules with non-polysaccharide scaffolds in the ZINC database 16 with strong interactions with the heparin binding domain of antithrombin. The ligand with highest score, D-myo-inositol 3,4,5,6-tetrakisphosphate (TMI), was exper- imentally validated confirming that this compound binds to Received: March 20, 2012 Published: June 28, 2012 Article pubs.acs.org/jmc © 2012 American Chemical Society 6403 dx.doi.org/10.1021/jm300621j | J. Med. Chem. 2012, 55, 6403−6412