Probing Binding and Cellular Activity of Pyrrolidinone and Piperidinone Small Molecules Targeting the Urokinase Receptor

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 and piperidinone compounds, along with derivatives of previously‐identified pyrazole and propylamine compounds. Competition assays revealed that the compounds displace a fluorescently labeled peptide (AE147‐FAM) with inhibition constant (K_i) values ranging from 6 to 63 μM . Structure‐based computational pharmacophore analysis followed by extensive explicit‐solvent molecular dynamics (MD) simulations and free energy calculations suggested the pyrazole‐based and piperidinone‐based compounds adopt different binding modes, despite their similar two‐dimensional structures. In cells, pyrazole‐based compounds showed significant inhibition of breast adenocarcinoma (MDA‐MB‐231) and pancreatic ductal adenocarcinoma (PDAC) cell proliferation, but piperidinone‐containing compounds exhibited no cytotoxicity even at concentrations of 100 μM . One pyrazole‐based compound impaired MDA‐MB‐231 invasion, adhesion, and migration in a concentration‐dependent manner, while the piperidinone inhibited only invasion. The pyrazole derivative inhibited matrix metalloprotease‐9 (gelatinase) activity in a concentration‐dependent manner, while the piperidinone 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 pyrrolidinones and piperidinones had no effect. Annexin V staining suggested that the effect of the pyrazole‐based compound on proliferation was due to cell killing through an apoptotic mechanism. The compounds identified represent valuable leads in the design of further derivatives with higher affinities and potential probes to unravel the protein–protein interactions of uPAR.     

The Urokinase Receptor: A Multifunctional Receptor in Cancer Cell Biology. Therapeutic Implications

Proteolysis is a key event in several biological processes; proteolysis must be tightly controlled because its improper activation leads to dramatic consequences. Deregulation of proteolytic activity characterizes many pathological conditions, including cancer. The plasminogen activation (PA) system plays a key role in cancer; it includes the serine-protease urokinase-type plasminogen activator (uPA). uPA binds to a specific cellular receptor (uPAR), which concentrates proteolytic activity at the cell surface, thus supporting cell migration. However, a large body of evidence clearly showed uPAR involvement in the biology of cancer cell independently of the proteolytic activity of its ligand. In this review we will first describe this multifunctional molecule and then we will discuss how uPAR can sustain most of cancer hallmarks, which represent the biological capabilities acquired during the multistep cancer development. Finally, we will illustrate the main data available in the literature on uPAR as a cancer biomarker and a molecular target in anti-cancer therapy.     

Ab initio molecular orbital calculations on specific interactions between urokinase-type plasminogen activator and its receptor

Cancer invasions and metastases are controlled by various proteases. In particular, the binding of urokinase-type plasminogen activator (uPA) to the uPA receptor (uPAR) existing on the surface of cancer cell is considered to be a trigger for cancer invasions. In the present study, we determined the structure of uPA and uPAR complex in water and investigated the specific interactions between uPA and uPAR by ab initio molecular orbital (MO) calculations based on fragment MO method. The result indicates that the 20–26 amino acid residues of uPA are important for the binding between uPA and uPAR, and that the electrostatic interactions between the charged amino acid residues existing in both uPA and uPAR have large contribution to the binding. The influence of crystal water molecules existing between uPA and uPAR was also investigated to be significant on the specific interactions between uPA and uPAR. These results are expected to be informative for developing new medicines blocking the binding of uPA and uPAR.     

Fibrinolytic System and Cancer: Diagnostic and Therapeutic Applications

Fibrinolysis is a crucial physiological process that helps to maintain a hemostatic balance by counteracting excessive thrombosis. The components of the fibrinolytic system are well established and are associated with a wide array of physiological and pathophysiological processes. The aberrant expression of several components, especially urokinase-type plasminogen activator (uPA), its cognate receptor uPAR, and plasminogen activator inhibitor-1 (PAI-1), has shown a direct correlation with increased tumor growth, invasiveness, and metastasis. As a result, targeting the fibrinolytic system has been of great interest in the field of cancer biology. Even though there is a plethora of encouraging preclinical evidence on the potential therapeutic benefits of targeting the key oncogenic components of the fibrinolytic system, none of them made it from “bench to bedside” due to a limited number of clinical trials on them. This review summarizes our existing understanding of the various diagnostic and therapeutic strategies targeting the fibrinolytic system during cancer.     

尿激酶型纤溶酶原激活剂(uPA)系统在子宫内膜癌中的研究进展

尿激酶型纤溶酶原激活系统是纤溶系统的重要组成部分，它通过水解细胞外间质，参与组织改造和细胞迁移，在肿瘤细胞的侵袭和转移中发挥作用；讨论了uPA系统的结构、作用机理及与子宫内膜癌的关系，旨在为子宫内膜癌的诊断和治疗提供新方法。     

The Multifaceted Role of Plasminogen in Cancer

Fibrinolytic factors like plasminogen, tissue-type plasminogen activator (tPA), and urokinase plasminogen activator (uPA) dissolve clots. Though mere extracellular-matrix-degrading enzymes, fibrinolytic factors interfere with many processes during primary cancer growth and metastasis. Their many receptors give them access to cellular functions that tumor cells have widely exploited to promote tumor cell survival, growth, and metastatic abilities. They give cancer cells tools to ensure their own survival by interfering with the signaling pathways involved in senescence, anoikis, and autophagy. They can also directly promote primary tumor growth and metastasis, and endow tumor cells with mechanisms to evade myelosuppression, thus acquiring drug resistance. In this review, recent studies on the role fibrinolytic factors play in metastasis and controlling cell-death-associated processes are presented, along with studies that describe how cancer cells have exploited plasminogen receptors to escape myelosuppression.     

乌司他丁和泰索帝对人乳腺癌细胞MDA-MB-231中uPA、uPAR、ERK表达的影响

目的探讨乌司他丁(Ulinastatin,UTI)和泰索帝(Taxotere,TXT)对体外培养的人乳腺癌细胞MDA-MB-231中u-PA、uPAR、ERK表达的影响。方法将MDA-MB-231(ER-)细胞分为4组:UTI组(UTI 800 U/ml)、TXT组(TXT 3.7μg/ml)、UTI+TXT组(UTI 800 U/ml+TXT 3.7μg/ml)、对照组(等量生理盐水)。给药后24 h,分别采用荧光定量RT-PCR检测各组细胞中uPA、uPAR、ERK基因mRNA的水平,Western blot法检测各组细胞中uPA、uPAR、p-ERK1/2蛋白的表达水平。结果 UTI组和UTI+TXT组MDA-MB-231(ER-)细胞中uPA和uPAR基因mRNA的水平均明显低于对照组(P<0.05),而TXT组中二者的表达水平均明显高于对照组(P<0.01),各组间ERK基因mRNA的水平差异无统计学意义(P>0.05);UTI组和UTI+TXT组中uPA、uPAR和p-ERK1/2蛋白的表达水平均明显低于对照组(P<0.01),而TXT组中3种蛋白的表达水平均明显高于对照组(P<0.05)。结论UTI可抑制MDA-MB-231细胞中uPA、uPAR、p-ERK的表达,而TXT可上调三者的表达。     

Inhibition of Human Urokinase-Type Plasminogen Activator (uPA) Enzyme Activity and Receptor Binding by DNA Aptamers as Potential Therapeutics through Binding to the Different Forms of uPA

Urokinase-type plasminogen activator is widely discussed as a marker for cancer prognosis and diagnosis and as a target for cancer therapies. Together with its receptor, uPA plays an important role in tumorigenesis, tumor progression and metastasis. In the present study, systematic evolution of ligands by exponential enrichment (SELEX) was used to select single-stranded DNA aptamers targeting different forms of human uPA. Selected aptamers allowed the distinction between HMW-uPA and LMW-uPA, and therefore, presumably, have different binding regions. Here, uPAapt-02-FR showed highly affine binding with a K_D of 0.7 nM for HMW-uPA and 21 nM for LMW-uPA and was also able to bind to pro-uPA with a K_D of 14 nM. Furthermore, no cross-reactivity to mouse uPA or tissue-type plasminogen activator (tPA) was measured, demonstrating high specificity. Suppression of the catalytic activity of uPA and inhibition of uPAR-binding could be demonstrated through binding with different aptamers and several of their truncated variants. Since RNA aptamers are already known to inhibit uPA-uPAR binding and other pathological functions of the uPA system, these aptamers represent a novel, promising tool not only for detection of uPA but also for interfering with the pathological functions of the uPA system by additionally inhibiting uPA activity.