Nearly 95% of patients who fulfill modified NIDDK criteria for interstitial cystitis/painful bladder syndrome (IC/PBS) secrete an antiproliferative factor (APF) in their urine that inhibits the proliferation of and elicits changes in bladder epithelial cells at subnanomolar concentrations (in vitro). APF has been identified as a nine amino-acid sialoglycopeptide (Neu5Acα2-3Galβ1-3GalNAcα-O-TVPAAVVVA) that has a peculiar structure-activity relationship (SAR). The sialic acid moiety is not required for activity, but removal of the glycan completely abolishes antiproliferative activity. Comparison of CD spectra of closely related (un)glycosylated peptides suggests that glycosylation is essential for structure (and perhaps function) of the glycopeptide. Furthermore, α-O-glycosylation appears to be essential for activity. Accordingly, we investigated the structural effects of glycosylation upon asialo-APF (Galβ1-3GalNAcα-O-TVPAAVVVA). Using Hamiltonian replica exchange (HREX) molecular dynamics (MD) simulations, we were able to predict a SAR for the active conformation of asialo-APF. Three probable conformational sampling regions (and two major conformations) were identified for asialo-APF, although no single structural model correlated with the observed activity profile. In this work, we seek to validate the structural models resulting from the HREX MD simulations by measuring 13C one-bond scalar coupling constants and RDCs for asialo-APF using J-modulated constant-time INEPT CT-HSQC (CTi-CT-HSQC) experiments. The structural models were refined using 15N RDCs alone, which has met with some success in differentiating the orientation of the peptide backbone in the two major conformational models. However, information about the orientation of the glycan is accessible only via incorporation of the measured 13C RDCs into the structural models. The combination of NMR input (3J values, NOEs and RDCs), and HREX MD simulations can provide powerful insight into the solution conformation of asialo-APF. The method detailed herein may further elucidate the complex interactions between glycan and peptide, while also serving as a guide to lead optimization of more potent APF analogues with potential utility as antiproliferative agents.

Unlocking the secrets of asialo-APF: Combining NMR spectroscopy and molecular dynamics to refine the complex structure-activity relationship of a (seemingly) simple antiproliferative glycopeptide

254th National Meeting (2017)

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