Design, synthesis, biochemical studies, cellular characterization, and structure-based computational studies of small molecules targeting the urokinase receptor

Abstract

The urokinase receptor (uPAR) serves as a docking site to the serine protease urokinase-type plasminogen activator (uPA) to promote extracellular matrix (ECM) degradation and tumor invasion and metastasis. Previously, we had reported a small molecule inhibitor of the uPAR·uPA interaction that emerged from structure-based virtual screening. Here, we measure the affinity of a large number of derivatives from commercial sources. Synthesis of additional compounds was carried out to probe the role of various groups on the parent compound. Extensive structure-based computational studies suggested a binding mode for these compounds that led to a structure-activity relationship study. Cellular studies in non-small cell lung cancer (NSCLC) cell lines that include A549, H460 and H1299 showed that compounds blocked invasion, migration and adhesion. The effects on invasion of active compounds were consistent with their inhibition of uPA and MMP proteolytic activity. These compounds showed weak cytotoxicity consistent with the confined role of uPAR to metastasis.

Figure 1

(A) Screening of 127 compounds that emerged from a similarity search of the ZINC chemical library based on the structure of 1. Compounds were tested at a single concentration of 50 µM; (B) A concentration-dependent study of hit compounds obtained from commercial databases. Structure and inhibition constants are provided in Table 1. (C) A concentration-dependent study using our ELISA for compounds that were synthesized in this work. (D) Binding affinity (K_D) of compounds using fluorescence polarization measured at a fixed concentration of compound and increasing concentration of uPAR.

Figure 2

Structure-based analysis of uPAR/2 interactions. (A) Stereoview of the top three binding modes of compound 2 docked onto the uPA binding pocket on uPAR. The compound and the residues it interacts with were shown in sticks (carbon atoms in compound and protein were colored in yellow and green respectively; nitrogens in blue and oxygens in red). The uPAR backbone was shown as grey ribbons. (B) The MD structures were aligned onto the first snapshot in the trajectory. The RMSD was calculated based on the heavy atoms of the compound. It was reported as the average of five independent trajectories in length of 10 ns. (C) The 2D schematic illustration of compound 2 interacting with residues on the uPAR pocket. Compound explores four sub-pockets (depicted with colored curves and named as S1, S2, S3 and S4 respectively) on the uPA binding pocket.

Figure 3

Invasion in the absence (control) or with 3k for lung cancer cells with siRNA knockdown of uPAR. (A) H1299, H460 and A549 cells were transfected with siRNAs for uPAR or control siRNA (control). After 48 h, cells were lysed and analyzed by immunoblotting. (B) Representative experimental cells from control and in the presence of uPAR siRNA with or without 3k were photographed (C) for the Boyden chamber assays (×200) to illustrate the effect of 2 on invasion as quantified in C.

Figure 4

2 blocks invasion, migration and adhesion of H1299 cancer cells. (A,B) Invasion of H1299, H460 and A549 cells at increasing concentration of IPR-803, as indicated. Ten fields of each membrane were counted for cell numbers (×200). These data represent the average (SD of three independent experiments). (C,D) Migration of H1299 cells at increasing concentration of 2, as indicated. Ten fields on each membrane were counted for cell numbers (×200). These data represent the average (SD of three independent experiments). (E) The adhesion of H1299 cells to ECM components fibronectin (FN) or vitronectin (VN) in the absence or presence of 2 are shown as indicated. The numbers of attached cells were quantified by MTT assay. (F) IC₅₀ for inhibition of A549, H460, and H1299 cell proliferation by 2.

Figure 5

Proteolytic activity and signaling. (A) Fluorescence assays of cell surface-bound uPA activity in A549 cells. Cells were pre-incubated for 30 min with a range of concentrations of UK122, chloromethyl ketone or 2 on ice. Cells were washed, fluorogenic uPA-specific substrate was added and fluorescence measurements were recorded immediately. Initial rates of change in fluorescence after subtraction of background fluorescence (cells only) are presented as a percentage of control (no test compound added). Values represent means ± SD (n = 3). (B, C) Effect on ECM degradation. Gelatin zymography analysis for H1299 cells with increasing concentration of 2 (D) Cell signaling study. H460 and A549 cells were treated with 1, 10 and 50 µM compound(s) for 30 min, then immunoblotted with phospho-p44/42 MAPK and p44/42 MAPK, phospho-FAK (Tyr397), FAK; phospho-Src family (Tyr416), Src and actin respectively.

Scheme 1

Scheme 2

Reagents and conditions: a) K₂CO₃, MEK:DMF (3:1), benzyl bromide b) Cu(OAc)₂, Cu, KOAc amyl alcohol, 150–160°C.

Scheme 3

Reagents and conditions: a) Ag₂O, CHCl₃, isopropyl iodide, r.t. b) 2M NaOH (aq), MeOH, 70–80°C.

Scheme 4

Reagents and conditions: a) Ag₂O, CHCl₃, iodomethane, r.t.

Scheme 5

Synthesis of IPR-456 derivatives Reagents and Conditions: a) AlCl₃, DCM, r.t. b) Br₂, NaOAc, AcOH, reflux c) Cu(OAc)₂, Cu, KOAc amyl alcohol, 150–160°C

Scheme 6

Synthesis of IPR-456 derivatives