IC/PBS is a chronic disease of the bladder characterized by thinning and ulceration of the bladder epithelial layer causing severe pain, urinary frequency and urgency. Seminal work at the University of Maryland, Baltimore, showed that a specific factor was responsible for many of the characteristic pathological changes that occur in IC/PBS. This factor, called APF, was shown to have antiproliferative activity toward bladder epithelial cells at sub-nanomolar concentrations. APF caused an increase in paracellular permeability, the down regulation of several protein involved in tight junctions formation and reduced the levels of heparin-binding epidermal growth factor-like growth factor (HB-EGF). In addition, APF was also a potent antiproliferative agent against bladder tumor cells at equally low concentrations and has subsequently been shown to inhibit proliferation of other tumor cell lines. The peptide portion of APF has 100% sequence identity to a stretch of amino acids in the 6th transmebrane domain of Frizzled 8, a Wnt signaling receptor. Whereas the sugar portion, Gal(beta)1-3GalNAc(alpha)-O-Thr is the well-known Thomsen Friedenreich disaccharide, a tumor associated carbohydrate antigen used in vaccine design and in the immunotherapy of cancer. In 2006, synthesis began on a series of analogues of the asialo derivative of APF (as-APF, equipotent to the natural sialylated compound) to define the structure-activity profile of the natural glycopeptide. This was started in the lab of Dr.Chris Michejda who tragically passed away very suddenly in January of 2007. In 2008 the project was turned over to this section and to date, approximately 60 analogues have been prepared and tested. Analogues of both the peptide and sugar portions of the molecule were modified and specific clues have emerged as to portions of the molecule that are essential for activity. We have determined that most all of the molecule is necessary for full antiproliferative activity. The sugar is essential but the Thomsen Friedenreich disaccharide can be replaced with LacNAc (Gal(beta)1-4GlcNAc) alpha linked to the threonine. The peptide sequence may be truncated at the C-terminal end (removal of alanine) to an 8-mer without detriment to activity;further truncation abolishes function. Other important features for activity are 1) maintenance of charge at the termini;2) A specific arrangement of methyl groups on the N-terminal amino acid sidechains and the ability to assume some secondary structural element in the C-terminal tail. The AXXXA motif is one that is frequently found within a helical motif and involved in binding to other protein helices (protein-protein interactions). Disruption of proper arrangement of these amino acids is detrimental to activity. A very important discovery was one that showed that specific derivatives with D-amino acid substitutions are inhibitors of APF antiproliferative activity in bladder epithelial cells, and these can now be developed as therapeutic leads for IC/PBS patients. Since we have already published on the peptide portion of the molecule and are now completing a study for a series of 8-mer glycopeptides, we are concentrating on the elements of the sugar portion of the molecule that are necessary for APF to function. We know several things about the sugar requirements already, but are now modifying individual atoms and stereochemistries around the disaccharide to map the binding interactions that are involved with the sugar portion of APF. A significant advance in the research was made by our collaborators when in 2006 they published on the discovery of a cellular receptor for APF in bladder epithelium. Cytoskeletal-associated protein 4 (CKAP4) was identified and characterized as this receptor, and knock down of its function desensitized cells to APF activity. CKAP4 links the cytoskeleton to the endoplasmic reticulum, it binds surfactant protein A and tissue plasminogen activator, but little is known of its actual function or the consequences of its inhibition. We are very interested in the details of the putative interactions of CKAP4 with APF, and a small grant was awarded to us to clone and purify the extracellular domain (474 residues) which has recently been accomplished in the Protein Expression Lab of he Advanced Technology Program here at NCI Frederick. A major effort in our APF work is dedicated to the structural characterization of the preferred conformation of APF and its analogues in solution. Being a small glycopeptide, it is relatively unstructured in water solution, but we have found certain NMR observables that suggest a preferred fold in the C-terminal domain, the portion of the peptide that we have surmised needs to be structured for APF to function. We are exploring the solution biophysics on several fronts: 1) Compare the conformations of active and inactive compounds in both the 9-mer and 8-mer series;2) Investigate the possible aggregative properties of APF since it has a hydrophilic end (sugar) and a very hydrophobic peptide sequence;3) Study the binding of analogues to CKAP4 and observe trends with actives and inactive compounds and 4) Develop new force fields for glycopeptide with our collaborators at the School of Pharmacy at the U. of Maryland and perform simulations that may offer other clues to APF behavior in solution. All of these studies will be geared toward defining a pharmacophore for APFs anticancer activity to design novel mimetics that may be used to selectively shut down proliferation in tumor tissue. APF constitutes an interesting an confounding study in biosynthesis and medicinal chemistry. It is a mystery as to how a small glycosylated peptide could be produced and secreted by the bladder epithelium to act like APF. If the peptide is derived from Frizzled 8, how did the transmembrane sequence become glycosylated?? Why is the molecule so sensitive to minor changes in structure and/or hydrophobicity?? We are going to tackle the glycobiology of APF by initially attempting to identify the glycosyl transferases that are involved in its biosynthesis. We would like to use novel chemical biology approaches to dynamically tag the precursors of APF in bladder cells and follow its biosynthesis and ultimate secretion. Unraveling the mysteries of the production and function of APF in the bladder will have dramatic implications that will translate to the anticancer drug discovery arena. Overall, every aspect of this project will advance our basic understanding of novel small molecule biosynthesis function and relevance to a variety of disease states including a host of different cancers.
