Synthesis, Solution Structure, and Biological Evaluation of Urokinase Type Plasminogen Activator (uPA)-Derived Receptor Binding Domain Mimetics

Abstract

Tumor cell migration and metastasis in cancer are facilitated by interaction of the serine protease urokinase type plasminogen activator (uPA) with its receptor uPAR (CD 87). Overexpression of uPA and uPAR in cancer tissues is associated with a high incidence of disease recurrence and early death. In agreement with these findings, disruption of the protein-protein interaction between uPAR present on tumor cells and its ligand uPA evolved as an attractive intervention strategy to impair tumor growth and metastasis. For this, the uPAR antagonist cyclo[19,31][D-Cys(19)]-uPA(19)(-)(31) was optimized to efficiently interrupt binding of uPA to cellular uPAR. First, the disulfide bridge of this lead compound was shifted and then the modified peptide was shortened from the amino and carboxy terminus to generate cyclo[21,29][Cys(21,29)]-uPA(21)(-)(30). Next, cyclo[21,29][D-Cys(21)Cys(29)]-uPA(21)(-)(30) was yielded by changing the chirality of Cys(21) to D-Cys(21). For analysis of uPAR binding activity, we employed competitive flow cytofluorometric receptor binding assays, using FITC-uPA as the ligand and U937 promyeloid leukemia cells as the cellular source of uPAR. As demonstrated for cyclo[21,29][D-Cys(21)Cys(29)]-uPA(21)(-)(30), the achieved peptide modifications maintained receptor binding activity (IC(50) = 0.04 microM), which is close in order to that of the parent protein ligand, uPA (IC(50) = 0.01 microM). A detailed NMR analysis with restrained and free molecular dynamics calculations in explicit H(2)O exhibits a well-defined structure with characteristic features such as an omega-loop with two betaI-turns about Lys(3), Tyr(4), Ser(6), and Asn(7). Hydrophobic clustering of the side chains of Tyr(4), Phe(5), Ile(8), and Trp(10) is observed. Side chain mobility is analyzed with time-dependent distance restraints. The NMR structure of cyclo[21,29][D-Cys(21)Cys(29)]-uPA(21)(-)(30) is very similar to the previously reported structure of the amino terminal fragment of uPA. Systematic point mutations led to cyclo[21,29][D-Cys(21)Nle(23)Cys(29)]-uPA(21)(-)(30), which still binds to uPAR but is resistant to proteolytic cleavage, e.g., by the tumor-associated serine proteases uPA and plasmin, and is stable in blood serum or plasma. In conclusion, small cyclic peptides were created, which mimic the structure and activity of the binding epitope of uPA to uPAR and which may serve as novel therapeutic agents in cancer metastasis.

Conformational Regulation of Urokinase Receptor Function

“…Our model raises the major concern that some small binding antagonists may in fact possess undesirable agonist effects on uPAR-mediated adhesion and migration on vitronectin-rich matrices. This ambiguity of action is illustrated by the inductive effects we observe for AE234, which actually was considered a potential lead compound for pharmaceutical intervention of uPA binding (42,46). Similar concerns are, however, less relevant for compounds belonging to the AE105-derived class of inhibitors.…”

“…One peptide antagonist (AE234) is designed to mimic the ␤-hairpin region of GFD, which is responsible for the tight receptor binding properties of uPA (41,42). This compound is a small 10-mer cyclic peptide, and it inhibited the uPA-uPAR interaction with an IC 50 value of 150 nM ( Table 1 and Fig.…”

“…It is also clear from these experiments that GFD is just as efficient as pro-uPA in stimulating uPAR-dependent protrusions. (42) and AE235 is a scrambled control. b The sequences are shown in the single-letter code, where capital letters represent L-amino acids and lower case letters represent D-amino acids.…”

A Flexible Multidomain Structure Drives the Function of the Urokinase-type Plasminogen Activator Receptor (uPAR)

“…The involvement of uPAR in cancer invasion and metastasis has prompted several academic institutions and pharmaceutical companies to embark on drug development programs aimed at targeting and controlling uPAR function (31,32,53,54). The majority of the small molecules developed so far target the central uPAR cavity to antagonize uPA binding.…”

“…Recombinant uPAR DI (residues 1-92) and DIIDIII (residues 88 -283) were likewise expressed in S2 cells and affinity-purified using anti-uPAR mAbs R21 and R2, respectively (19). Receptor-binding fragments of uPA (GFD(1-48) and ATF(1-143)) and the synthetic peptide antagonists AE105 (DXFsrYLWS) and AE234 (cNKYF-SNICW) were prepared as described (19,31,32), where lowercase letters correspond to D-enantiomers, X is cyclo-hexyl-Lalanine, and the two cysteine residues in AE234 are oxidized to form a cyclic peptide. D 2 O (99.9% atom % D) was purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA), and tris(2-carboxyethyl)phosphine was obtained from Sigma-Aldrich.…”