Application of encoded library technology (ELT) to a protein–protein interaction target: Discovery of a potent class of integrin lymphocyte function-associated antigen 1 (LFA-1) antagonists Christopher S. Kollmann a , Xiaopeng Bai a , Ching-Hsuan Tsai a , Hongfang Yang a , Kenneth E. Lind a , Steven R. Skinner a , Zhengrong Zhu a , David I. Israel a , John W. Cuozzo a,  , Barry A. Morgan a , Koichi Yuki b,à , Can Xie b,§ , Timothy A. Springer b , Motomu Shimaoka b,– , Ghotas Evindar a,⇑ a GlaxoSmithKline, Platform Technology & Science, MDR Boston, 830 Winter Street, Waltham, MA 02451, USA b Immune Disease Institute, Children’s Hospital Boston, Harvard Medical School, Program in Cellular and Molecular Medicine, Department of Biological Chemistry and Molecular Pharmacology, 3 Blackfan Circle, Rm. 3100, Boston, MA 02115, USA article info Article history: Received 25 November 2013 Revised 15 January 2014 Accepted 24 January 2014 Available online 7 February 2014 Keywords: Encoded Library Technology DNA-encoded libraries Affinity-based selections Lymphocyte Function-associated Antigen 1 Intercellular Adhesion Molecule 1 Protein-Protein Interactions abstract The inhibition of protein–protein interactions remains a challenge for traditional small molecule drug discovery. Here we describe the use of DNA-encoded library technology for the discovery of small molecules that are potent inhibitors of the interaction between lymphocyte function-associated antigen 1 and its ligand intercellular adhesion molecule 1. A DNA-encoded library with a potential complexity of 4.1 billion compounds was exposed to the I-domain of the target protein and the bound ligands were affinity selected, yielding an enriched small-molecule hit family. Compounds representing this family were synthesized without their DNA encoding moiety and found to inhibit the lymphocyte function- associated antigen 1/intercellular adhesion molecule-1 interaction with submicromolar potency in both ELISA and cell adhesion assays. Re-synthesized compounds conjugated to DNA or a fluorophore were demonstrated to bind to cells expressing the target protein. Ó 2014 Elsevier Ltd. All rights reserved. 1. Introduction Targeting small molecule drugs to the interfaces between pro- teins has great therapeutic potential, but remains a challenge for traditional high-throughput screening drug discovery efforts. Three quarters of marketed pharmaceuticals target either enzymes or G-protein coupled receptors target classes known for containing classical ligand pockets that are amenable to traditional drug discovery efforts. 1 Most protein–protein interactions (PPI) occur over large, flat surface areas that have proved more difficult to target than classical ligand binding pockets. 2,3 Integrin LFA-1 (lymphocyte function-associated antigen-1) is a major leukocyte cell adhesion molecule that binds to its major ligand ICAM-1 (intercellular adhesion molecule-1) on endothelial cells and dendritic cells. 4 LFA-1 plays a pivotal role in regulating leukocyte trafficking to sites of inflammation as well as in inducing immune responses, thereby representing an established therapeutic target for autoimmune and inflammatory diseases. 5 A therapeutic antibody to LFA-1, efalizumab (Raptiva™) has been demonstrated to be effective for the treatment of patients with psoriasis, a T-cell mediated autoimmune disease in the skin. 6 In addition, LFA-1 inhibitors are currently being investigated in clinical trials for the treatment of uveitis 7 and other ocular inflammation. 8 The ligand binding domain of LFA-1 termed inserted (I) domain adopts a Rossmann fold that contains a metal ion-dependent adhe- sion site (MIDAS) located on the top, whereas its C- and N-terminal connections are located on the distal bottom face. 9 The ability of the I domain to bind ligand is regulated by conformational changes, as the affinity for its ligand is dramatically enhanced by a ‘piston-like’ downward axial displacement of its C-terminal helix. http://dx.doi.org/10.1016/j.bmc.2014.01.050 0968-0896/Ó 2014 Elsevier Ltd. All rights reserved. Abbreviations: ELT, encoded library technology; LFA-1, lymphocyte function- associated antigen 1; ICAM-1, intercellular adhesion molecule 1; PPI, protein– protein interactions. ⇑ Corresponding author. Tel.: +1 781 795 4423; fax: +1 781 795 4496. E-mail address: ghotas.x.evindar@gsk.com (G. Evindar).   Present address: X-Chem Inc, 100 Beaver Street Suite 101, Waltham, MA 02453, USA. à Present address: Peking University, State Key Laboratory of Biomembrane and Membrane Biotechnology, Laboratory of Receptor Biology, 5 Yiheyuan Road, Beijing 100871, China. § Present address: Department of Anesthesiology, Perioperative and Pain Medicine, Children’s Hospital Boston, and Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115, USA. – Present address: Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu-City, Mie 514-8507, Japan. Bioorganic & Medicinal Chemistry 22 (2014) 2353–2365 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc