Published: August 29, 2011 r2011 American Chemical Society 1232 dx.doi.org/10.1021/cb200180m | ACS Chem. Biol. 2011, 6, 1232–1243 ARTICLES pubs.acs.org/acschemicalbiology Targeting Multiple Conformations Leads to Small Molecule Inhibitors of the uPAR 3 uPA ProteinÀProtein Interaction That Block Cancer Cell Invasion May Khanna, † Fang Wang, † Inha Jo, † W. Eric Knabe, † Sarah M. Wilson, ||, 3 Liwei Li, †, § Khuchtumur Bum-Erdene, §, z Jing Li, † George W. Sledge, Jr., ^,# Rajesh Khanna, ‡, ||, 3 and Samy O. Meroueh †, §, ||, ^, z, * † Department of Biochemistry and Molecular Biology, ‡ Department of Pharmacology and Toxicology, § Center for Computational Biology and Bioinformatics, ) Stark Neuroscience Research Institute, ^ Indiana University Cancer Center, # Department of Medicine, and 3 Medical Neuroscience Program, Indiana University School of Medicine, Indianapolis, Indiana 46202, United States z Department of Chemistry and Chemical Biology, Indiana University Purdue University Indianapolis (IUPUI), Indianapolis, Indiana 46202, United States b S Supporting Information ABSTRACT: Interaction of the urokinase receptor (uPAR) with its binding partners such as the urokinase-type plasminogen activator (uPA) at the cell surface triggers a series of proteolytic and signaling events that promote invasion and metastasis. Here, we report the discovery of a small molecule (IPR-456) and its derivatives that inhibit the tight uPAR 3 uPA proteinÀprotein interaction. IPR-456 was discovered by virtual screening against multiple conformations of uPAR sampled from explicit-solvent molecular dynamics simulations. Biochemical characterization reveal that the compound binds to uPAR with submicromolar affinity (K d = 310 nM) and inhibits the tight proteinÀprotein interaction with an IC 50 of 10 μM. Free energy calculations based on explicit-solvent molecular dynamics simulations suggested the importance of a carboxylate moiety on IPR-456, which was confirmed by the activity of several derivatives including IPR-803. Immunofluorescence imaging showed that IPR-456 inhibited uPA binding to uPAR of breast MDA- MB-231 tumor cells with an IC 50 of 8 μM. The compounds blocked MDA-MB-231 cell invasion, but IPR-456 showed little effect on MDA-MB-231 migration and no effect on adhesion, suggesting that uPAR mediates these processes through its other binding partners. T he interaction of the glycosylphosphatidylinositol (GPI)- anchored cell-surface urokinase receptor (uPAR) with its serine protease ligand urokinase-type plasminogen activator (uPA) has been implicated in nearly every step of tumor formation and progression, including tumorigenesis, 1 cell proliferation, 2À4 cell migration, 5,6 adhesion, 3,7 angiogenesis, 8,9 and invasion. 3,4,10,11 The uPAR 3 uPA complex formation enhances pericellular pro- teolysis through activation of plasminogen, 7 culminating in the active degradation of extracellular matrix (ECM) components. Whereas uPAR has no inherent signaling capability, a large body of evidence has shown that the uPAR 3 uPA complex promotes signaling by actively associating to cell surface receptors such as integrins, 12 receptor tyrosine kinases (RTKs), 13,14 and G-protein coupled receptors (GPCRs). 15 Furthermore, uPAR binding to vitronectin components indicates interactions that extend beyond the cell surface to engage the microenvironment and further promotes metastasis. To date, while peptides and antibodies have been reported to inhibit the uPAR 3 uPA complex, 16 there is not a single small organic molecule that inhibits the tight subnanomolar uPAR 3 uPA interaction. Moreover, most efforts have concentrated on inhibiting the enzymatic activity of uPA, neglecting the signaling capabilities of the receptor. The uPAR 3 uPA complex (Figure 1a) poses the same challenges that have plagued prior efforts to inhibit proteinÀprotein interactions with small molecules, 17 due to the tightness of the proteinÀprotein interaction, 18,19 the large Received: June 5, 2011 Accepted: August 29, 2011