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1.  Probing Binding and Cellular Activity of Pyrrolidinone and Piperidinone Small Molecules Targeting the Urokinase Receptor 
ChemMedChem  2013;8(12):1963-1977.
The urokinase receptor (uPAR) is a cell-surface protein that is part of an intricate web of transient and tight protein interactions that promote cancer cell invasion and metastasis. Here we evaluate the binding and biological activity of a new class of pyrrolidinone (3) and piperidinone (4) compounds, along with derivatives of previously-identified pyrazole (1) and propylamine (2) compounds. Competition assays revealed that the compounds displaced a fluorescently-labeled peptide (AE147-FAM) with inhibition constant Ki ranging from 6 to 63 μM. Structure-based computational pharmacophore analysis followed by extensive explicit-solvent molecular dynamics simulations and free energy calculations suggested pyrazole-based 1a and piperidinone-based 4 adopt different binding modes, despite their similar two-dimensional structures. In cells, compounds 1b and 1f showed significant inhibition of breast MDA-MB-231 and pancreatic ductal adenocarcinoma (PDAC) cell proliferation, but 4b exhibited no cytotoxicity even at concentrations of 100 μM. 1f impaired MDA-MB-231 invasion, adhesion, and migration in a concentration-dependent manner, while 4b inhibited only invasion. 1f inhibited gelatinase (MMP-9) activity in a concentration-dependent manner, while 4b showed no effect suggesting different mechanisms for inhibition of cell invasion. Signaling studies further highlighted these differences, showing that pyrazole compounds completely inhibited ERK phosphorylation and impaired HIF1α and NF-κB signaling, while pyrrolidinone and piperidinone (3 and 4b) had no effect. Annexin V staining suggested that the effect of pyrazole-based 1f on proliferation was due to cell killing through an apoptotic mechanism.
PMCID: PMC4058332  PMID: 24115356
2.  Small-Molecule Inhibition of the uPAR·uPA Interaction: Synthesis, Biochemical, Cellular, in vivo Pharmacokinetics and Efficacy Studies in Breast Cancer Metastasis 
Bioorganic & medicinal chemistry  2013;21(7):2145-2155.
The uPAR·uPA protein-protein interaction (PPI) is involved in signaling and proteolytic events that promote tumor invasion and metastasis. A previous study had identified 4 (IPR-803) from computational screening of a commercial chemical library and shown that the compound inhibited uPAR·uPA PPI in competition biochemical assays and invasion cellular studies. Here, we synthesize 4 to evaluate in vivo pharmacokinetic (PK) and efficacy studies in a murine breast cancer metastasis model. First, we show, using fluorescence polarization and saturation transfer difference (STD) NMR, that 4 binds directly to uPAR with sub-micromolar affinity of 0.2 μM. We show that 4 blocks invasion of breast MDA-MB-231, and inhibits matrix metalloproteinase (MMP) breakdown of the extracellular matrix (ECM). Derivatives of 4 also inhibited MMP activity and blocked invasion in a concentration-dependent manner. 4 also impaired MDA-MB-231 cell adhesion and migration. Extensive in vivo PK studies in NOD-SCID mice revealed a half-life of nearly 5 hours and peak concentration of 5 μM. Similar levels of the inhibitor were detected in tumor tissue up to 10 hours. Female NSG mice inoculated with highly malignant TMD-MDA-MB-231 in their mammary fat pads showed that 4 impaired metastasis to the lungs with only four of the treated mice showing severe or marked metastasis compared to ten for the untreated mice. Compound 4 is a promising template for the development of compounds with enhanced PK parameters and greater efficacy.
PMCID: PMC3625246  PMID: 23411397
3.  Virtual Screening Targeting the Urokinase Receptor, Biochemical and Cell-Based Studies, Synthesis, Pharmacokinetic Characterization, and Effect on Breast Tumor Metastasis 
Journal of Medicinal Chemistry  2011;54(20):7193-7205.
Virtual screening targeting the urokinase receptor (uPAR) led to (3R)-4-cyclohexyl-3-(hexahydrobenzo[d][1,3]dioxol-5-yl)-N-((hexahydrobenzo[d][1,3]dioxol-5-yl)methyl)butan-1-aminium 1 (IPR-1) and 4-(4-((3,5-dimethylcyclohexyl)carbamoyl)-2-(4-isopropylcyclohexyl)pyrazolidin-3-yl)piperidin-1-ium 3 (IPR-69). Synthesis of an analog of 1, namely 2 (IPR-9), and 3 led to breast MDA-MB-231 invasion, migration and adhesion assays with IC50 near 30 μM. Both compounds blocked angiogenesis with IC50 of 3 μM. Compounds 2 and 3 inhibited cell growth with IC50 of 6 and 18 μM and induced apoptosis. Biochemical assays revealed lead-like properties for 3, but not 2. Compound 3 administered orally reached peak concentration of nearly 40 μM with a half-life of about 2 hours. In NOD-SCID mice inoculated with breast TMD-231 cells in their mammary fat pads, compound 3 showed a 20% reduction in tumor volumes and less extensive metastasis was observed for the treated mice. The suitable pharmacokinetic properties of 3 and the encouraging preliminary results in metastasis make it an ideal starting point for next generation compounds.
PMCID: PMC3280887  PMID: 21851064

Results 1-3 (3)