ABSTRACT
The serine proteinase urokinase-type plasminogen activator (uPA) is widely recognized as a potential target for anticancer therapy. Its association with cell surfaces through the uPA receptor (uPAR) is central to its function and plays an important role in cancer invasion and metastasis. In the current study, we used systematic evolution of ligands by exponential enrichment (SELEX) to select serum-stable 2'-fluoro-pyrimidine-modified RNA aptamers specifically targeting human uPA and blocking the interaction to its receptor at low nanomolar concentrations. In agreement with the inhibitory function of the aptamers, binding was found to be dependent on the presence of the growth factor domain of uPA, which mediates uPAR binding. One of the most potent uPA aptamers, upanap-12, was analyzed in more detail and could be reduced significantly in size without severe loss of its inhibitory activity. Finally, we show that the uPA-scavenging effect of the aptamers can reduce uPAR-dependent endocytosis of the uPA-PAI-1 complex and cell-surface associated plasminogen activation in cell culture experiments. uPA-scavenging 2'-fluoro-pyrimidine-modified RNA aptamers represent a novel promising principle for interfering with the pathological functions of the uPA system.
Subject(s)
Aptamers, Nucleotide/pharmacology , Receptors, Urokinase Plasminogen Activator/metabolism , Urokinase-Type Plasminogen Activator/antagonists & inhibitors , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Aptamers, Nucleotide/chemical synthesis , Base Sequence , Drug Screening Assays, Antitumor , Drug Stability , Endocytosis/drug effects , Endocytosis/physiology , Humans , Molecular Sequence Data , Multiprotein Complexes/antagonists & inhibitors , Multiprotein Complexes/metabolism , Plasminogen Activator Inhibitor 1/metabolism , Protein Binding/drug effects , Protein Structure, Tertiary , Serum/metabolism , Substrate Specificity , Urokinase-Type Plasminogen Activator/chemistry , Urokinase-Type Plasminogen Activator/genetics , Urokinase-Type Plasminogen Activator/metabolismABSTRACT
PAI-1 is a Mr ~50,000 glycoprotein, which is the primary physiological inhibitor of the two plasminogen activators uPA and tPA. PAI-1 belongs to the serpin protein family. Studies of PAI-1 have contributed significantly to the elucidation of the protease inhibitory mechanism of serpins, which is based on a metastable native state becoming stabilised by insertion of the RCL into the central beta-sheet A and formation of covalent complexes with target proteases. In PAI-1, this insertion can occur in the absence of the protease, resulting in generation of a so-called latent, inactive form of the protein. PAI-1, in its active state, also binds to the extracellular protein vitronectin. When in complex with its target proteases, it binds with high affinity to endocytosis receptors of the low density receptor family.
