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

Wang, Fang; Knabe, William E.; Li, Liwei; Jo, Inha; Mani, Timmy; Roehm, Hartmut; Oh, Kyungsoo; Li, Jing; Khanna, May; Meroueh, Samy O.

doi:10.1016/j.bmc.2012.06.002

Cited by 34 publications

(49 citation statements)

References 57 publications

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“…However, recent experimental evidence indicates that some members of the MMP family behave as tumor-suppressor enzymes and may therefore be regarded as antitargets in cancer therapy (46). Moreover, small-molecule inhibitors of uPA binding to uPAR have been recently described (47,48).…”

Section: Discussionmentioning

confidence: 99%

Discovery of New Small Molecules Targeting the Vitronectin-Binding Site of the Urokinase Receptor That Block Cancer Cell Invasion

Rea

Lavecchia

Giovanni

et al. 2013

Molecular Cancer Therapeutics

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Besides focusing urokinase (uPA) proteolytic activity on the cell membrane, the uPA receptor (uPAR) is able to bind vitronectin, via a direct binding site. Furthermore, uPAR interacts with other cell surface receptors, such as integrins, receptor tyrosine kinases, and chemotaxis receptors, triggering cell-signaling pathways that promote tumor progression. The ability of uPAR to coordinate binding and degradation of extracellular matrix (ECM) and cell signaling makes it an attractive therapeutic target in cancer. We used structure-based virtual screening (SB-VS) to search for small molecules targeting the uPAR-binding site for vitronectin. Forty-one compounds were identified and tested on uPAR-negative HEK-293 epithelial cells transfected with uPAR (uPAR-293 cells), using the parental cell line transfected with the empty vector (V-293 cells) as a control. Compounds 6 and 37 selectively inhibited uPAR-293 cell adhesion to vitronectin and the resulting changes in cell morphology and signal transduction, without exerting any effect on V-293 cells. Compounds 6 and 37 inhibited uPAR-293 cell binding to vitronectin with IC 50 values of 3.6 and 1.2 mmol/L, respectively. Compounds 6 and 37 targeted S88 and R91, key residues for uPAR binding to vitronectin but also for uPAR interaction with the fMLF family of chemotaxis receptors (fMLF-Rs). As a consequence, compounds 6 and 37 impaired uPAR-293 cell migration toward fetal calf serum (FCS), uPA, and fMLF, likely by inhibiting the interaction between uPAR and FPR1, the high affinity fMLF-R. Both compounds blocked in vitro ECM invasion of several cancer cell types, thus representing new promising leads for pharmaceuticals in cancer.

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Section: Discussionmentioning

confidence: 99%

Discovery of New Small Molecules Targeting the Vitronectin-Binding Site of the Urokinase Receptor That Block Cancer Cell Invasion

Rea

Lavecchia

Giovanni

et al. 2013

Molecular Cancer Therapeutics

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“…This effect was further enhanced by addition of the exogenous uPA due to the release of fully activated Lb and was partially abolished by the addition of PAI-1. Because it was confirmed that the NSCLC cells produce uPA, 24 this may partially contribute to the inhibitory effect of KPLK on cell proliferation. In contrast, the normal cell line (HSGC) with the same treatment was unaffected, suggesting that both KPLK and Lb were not toxic to normal cells.…”

Section: Discussionmentioning

confidence: 97%

Preparation and antitumor effect of a toxin-linked conjugate targeting vascular endothelial growth factor receptor and urokinase plasminogen activator

Xiang

Huang

et al. 2014

Exp Biol Med (Maywood)

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The aberrant signaling activation of vascular endothelial growth factor receptor (VEGFR) and urokinase plasminogen activator (uPA) is a common characteristic of many tumors, including lung cancer. Accordingly, VEGFR and uPA have emerged as attractive targets for tumor. KDR (Flk-1/VEGFR-2), a member of the VEGFR family, has been recognized as an important target for antiangiogenesis in tumor. In this study, a recombinant immunotoxin was produced to specifically target KDR-expressing tumor vascular endothelial cells and uPA-expressing tumor cells and mediate antitumor angiogenesis and antitumor effect. Based on its potent inhibitory effect on protein synthesis, Luffin-beta (Lβ) ribosome-inactivating protein was selected as part of a recombinant fusion protein, a single-chain variable fragment against KDR (KDRscFv)-uPA cleavage site (uPAcs)-Lβ-KDEL (named as KPLK). The KDRscFv-uPAcs-Lβ-KDEL (KPLK) contained a single-chain variable fragment (scFv) against KDR, uPAcs, Lβ, and the retention signal for endoplasmic reticulum proteins KDEL (Lys-Asp-Glu-Leu). The KPLK-expressing vector was expressed in Escherichia coli, and the KPLK protein was isolated with nickel affinity chromatography and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis test demonstrated KPLK was effectively expressed. Result of in vitro cell viability assay on non-small cell lung cancer (NSCLC) H460 cell line (uPA-positive cell) revealed that KPLK significantly inhibited cell proliferation, induced apoptosis, and accumulated cells in S and G2/M phases, but the normal cell line (human submandibular gland cell) was unaffected. These effects were enhanced when uPA was added to digest KPLK to release Lβ. For in vivo assay of KPLK, subcutaneous xenograft tumor model of nude mice were established with H460 cells. Growth of solid tumors was significantly inhibited in animals treated with KPLK up to 21 days, tumor weights were decreased, and the expression of angiogenesis marker CD31 was downregulated; meanwhile, the apoptosis-related protein casspase-3 was upregulated. These results suggested that the recombinant KPLK may have therapeutic applications on tumors, especially uPA-overexpressing ones.

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“…6,7) Therefore, inhibition of the expression and activity of ECM degradation enzymes is important in arresting cancer cell invasion and metastasis. [8][9][10] Since transcription factor nuclear factor kappa B (NF-κB) is a major mediator of inflammation, as well as a regulator of cancer cell development and progression. Many reports have been demonstrated that NF-κB signaling has a critical role in cancer cells metastasis.…”

mentioning

confidence: 99%

Anti-invasion Effect of Crebanine and O-Methylbulbocapnine from Stephania venosa via Down-Regulated Matrix Metalloproteinases and Urokinase Plasminogen Activator

et al. 2013

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The alkaloids isolated from Stephania venosa (S. venosa) have been shown to inhibit the proliferation and to induce the apoptosis of cancer cells. However, the anti-metastatic effect of the alkaloids on cancer cell invasion is unknown. In this study, we investigated the anti-invasive properties of four alkaloids from S. venosa, crebanine (CN), O-methylbulbocapnine (OMBC), tetrahydropalmatine (THP), and N-methyltetrahydropalmatine (NMTHP), in HT1080 human fibrosacroma cells. Treatment of the cells with 15 µg/mL of CN and OMBC reduced the chemo-invasion of HT1080 cells to 45 and 50%, respectively, whereas THP and NMTHP had a negative effect. On the other hand, CN and OMBC had no effect on cell migration. Matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA) are the extracellular matrix (ECM) degradation enzymes that play an important role in cancer cell metastasis. Results from zymography and western blot analysis showed that CN and OMBC comparatively reduced MMP-2, MMP-9, MT1-MMP and uPA expression in a dose-dependent manner. However, CN and OMBC had no effect on the activity of collagenase, MMP-2 and MMP-9. We also found that CN and OMBC reduced the nuclear translocation and DNA binding activity of nuclear factor kappa B (NF-κB), which is the expressed mediator of ECM degradation enzymes. These findings demonstrated that CN and OMBC mediated HT1080 cell invasion by the reduction of MMP-2, MMP-9, uPA and MT1-MMP expression, possibly by targeting of NF-κB signaling pathway in the HT1080 cells.

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

Cited by 34 publications

References 57 publications

Discovery of New Small Molecules Targeting the Vitronectin-Binding Site of the Urokinase Receptor That Block Cancer Cell Invasion

Discovery of New Small Molecules Targeting the Vitronectin-Binding Site of the Urokinase Receptor That Block Cancer Cell Invasion

Preparation and antitumor effect of a toxin-linked conjugate targeting vascular endothelial growth factor receptor and urokinase plasminogen activator

Anti-invasion Effect of Crebanine and <i>O</i>-Methylbulbocapnine from <i>Stephania venosa</i> <i>via</i> Down-Regulated Matrix Metalloproteinases and Urokinase Plasminogen Activator

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