α2-Antiplasmin (α2AP) is a glycoprotein in blood plasma that rapidly and specifically inhibits the enzyme, plasmin, which digests blood clots, whether presenting early as intravascular platelet-fibrin deposits or as partially or completely occlusive thrombi. Similarly, plasmin and α2AP activities are important to the development and survival of fibrin as occurs in inflammation, wound healing and virtually all forms of cancer and its metastases. Human α2-antiplasmin (α2AP), also known as α2-plasmin inhibitor, is the main inhibitor of plasmin. Plasmin plays a critical role in fibrin proteolysis and tissue remodeling. The inventors discovered antiplasmin-cleaving enzyme (APCE) in human plasma and showed that it is a soluble isoform or derivative of fibroblast activation protein-alpha (FAP). Like APCE, FAP is also a prolyl-specific enzyme that exhibits both endopeptidase and dipeptidyl peptidase activities.
FAP significantly over-expresses in >90% of epithelial-derived cancers [1-3]. FAP is produced transiently by activated stromal fibroblasts during embryogenesis [4] and wound healing [3], but other than an occasional normal fibroblast or pancreatic islet α-cell, it is not expressed by normal adult tissues or benign tumors [2, 3, 5]. FAP is prominent on the membranes of proliferating fibroblasts in diseases where fibrous tissue growth is a conspicuous feature, such as primary pulmonary fibrosis [6]; chronic hepatitis [7]; certain bone-associated malignancies [8, 9]; and the arthritides [10]. Selected parenchymatous cancer cells may also occasionally express FAP [11].
While a biologic substrate for the proteinase activity of FAP has not been definitively established, results indicate that FAP helps digest extracellular matrix (ECM) components as tissue is remodeled to accommodate cancer expansion [2, 16, 17]. Paradoxically, activated fibroblasts not only digest ECM, but also synthesize ECM components of the stromal scaffolding that support cell division and motility during neoplastic growth [18]. FAP has been considered a potential target in the diagnosis and therapy of cancer, with inhibition of FAP proteinase activity posed as possibly therapeutically useful [19, 20]. Santos et al. [21] have shown that genetic deletion or pharmacologic inhibition of FAP by glutamyl-proline boronic acid (Glu-boroPro) decreased stromal growth in mouse models of lung and colon cancer. However, Glu-boroPro has an exceptionally short plasma half-life before cyclizing and losing inhibitory activity [22]. Moreover, it also inhibits dipeptidyl peptidase IV (DPPIV), which is important in plasma glucose regulation and immune function [23]. Hence, despite inhibiting FAP and suppressing tumor growth, Glu-boroPro is not likely to be therapeutically useful in cancer [24].
The measurement of cellular FAP activity and inhibition is confounded by another prolyl endopeptidase: namely, Prolyl Oligopeptidase (POP) which is elevated in many cancers [25]. POP is a prolyl-specific serine proteinases, which cleaves peptides of less than about 30 amino acids in length. The enzyme is present in most tissues, but is noted to be more abundant in selected organs, e.g., brain and kidney. Recently POP has been indicated as making secondary cleavages of thymosin-β4 to yield the derivative peptide, acetyl-serine-asparagine-lysine-proline, which appears to be a potent stimulator of angiogenesis [26]. Both FAP and POP activities are commonly measured using non-specific substrates such as Z-Gly-Pro-AMC or succinyl-Gly-Pro-AMC, neither of which distinguishes between the two activities [27]. Consequently, total prolyl-specific endopeptidase activity, which is often attributed to FAP alone, may also include POP activity. This complicates interpretations about the effects of inhibiting either enzyme on cancer growth. Pre-clinical studies have suggested other promising applications of POP inhibition for managing memory, learning disorders and depression, but development of relatively benign, highly effective POP inhibitors for in vivo testing have been elusive. The results, newly described herein, indicate that POP is expressed in significant amounts by a variety of cancer cells grown in culture.
Certain compounds which specifically inhibit either one or both of FAP and POP and therefore can be used to treat various conditions which involve these proteins are desirable. Thus, the presently disclosed and claimed inventive concept(s) is directed to, but not limited to, substrates and inhibitors of FAP and/or POP, and to methods of using FAP and/or POP inhibitors for treating conditions such as but not limited to, cancers, neural disorders, and angiogenesis, and to screening methods for identifying such inhibitors, that overcome the disadvantages and defects of the prior art.