Structure−Activity Studies on Antiproliferative Factor (APF) Glycooctapeptide Derivatives

Abstract

Antiproliferative factor (APF), a sialylated glycopeptide secreted by explanted bladder epithelial cells from interstitial cystitis/painful bladder syndrome (IC/PBS) patients, and its unsialylated analogue (as-APF) significantly decrease proliferation of bladder epithelial cells and/or certain carcinoma cell lines in vitro. We recently reported a structure-activity relationship profile for the peptide portion of as-APF and revealed that truncation of the C-terminal alanine did not significantly affect antiproliferative activity. To better understand the structural basis for the maintenance of activity of this truncated eight amino acid as-APF (as-APF8), we synthesized several amino acid-substituted derivatives and studied their ability to inhibit bladder epithelial cell proliferation in vitro as well as their solution conformations by CD and NMR spectroscopy. While single amino acid changes to as-APF8 often strongly reduced activity, full potency was retained when the trivaline tail was replaced with three alanines. The Ala(6-8) derivative 9 is the simplest, fully potent APF analogue synthesized to date.

Conformational Determinants of the Activity of Antiproliferative Factor Glycopeptide

“…This observation is in line with the large 3 J HNHα coupling constants (between 7.0 Hz and 8.9 Hz) experimentally observed for the trivaline region. 4, 9, 10 Thus, while the alanines also sample the folded α-helical conformations the trivaline tail does not. The presence of the commonly found α-helical AXXXA (X=V in as-APF) motif 38 in APF had initially encouraged the hypothesis of a well formed α-helical tail region in APF in the original SAR proposal.…”

“…4, 9, 10 The chosen derivatives including as-APF (compound 1 ; Scheme 1) represent a selection of as-APF derivatives with differing biological activities. The details of the synthesis of the glycopeptides have been described in detail in earlier studies.…”

“…The details of the synthesis of the glycopeptides have been described in detail in earlier studies. 4, 9, 10 MD simulations were performed using an equilibration and production strategy as tested earlier for O-linked glycoprotein systems. 11 In brief, the simulations were performed with the CHARMM program.…”

Chemical Synthesis of a Heparan Sulfate Glycopeptide: Syndecan‐1

“…The free carboxylic acid terminal was subsequently protected as benzyl ester and the N-terminal Fmoc was removed to generate the nonapeptide 33 with a free amino terminal. Coupling of octasaccharide carboxylic acid 31 with peptide 33 without any base additive [18] successfully produced glycopeptide 34 in 75% yield (Scheme 4b). It was important to omit the base during this step to avoid the β-elimination of the glycan chain from the peptide backbone.…”

“…It was shown that sulfates did not affect peptide coupling reactions. [18] Indeed, reaction of 36 with 33 produced glycopeptide 37 in 60% yield. Subsequent hydrogenation and methyl ester hydrolysis afforded the final glycopeptide 1 (Scheme 5).…”