N-Methylation of Amino Acids in Gelatinase Biosynthesis-Activating Pheromone Identifies Key Site for Stability Enhancement with Retention of the Enterococcus faecalis fsr Quorum Sensing Circuit Response

Abstract

The growing prevalence of multiantibiotic-resistant bacteria necessitates looking at potential alternative approaches for attenuating infections by bacteria while reducing the rate of antibiotic resistance development. Enterococcus faecalis is responsible for a large percentage of clinical enterococci infections, and its pathogenicity has been demonstrated to be influenced by quorum sensing (QS). In this study, we report the systematic study of the relationship between backbone hydrogens and the ability to activate the FsrC receptor. We demonstrate that N-methylation was particularly well-tolerated at one site (Phe7) and granted stability against protease digestion, increasing the peptide half-life relative to the native signal by more than 6-fold. The inclusion of the N-Me-Phe7 modification may be useful for improving the pharmacological properties of E. faecalis QS inhibitors as part of the development of future therapeutic candidates.

Keywords: gelatinase biosynthesis-activating pheromone; metabolic stability; peptidomimetics; quorum sensing.

Figure 1.

Comparison of GBAP in PBS at 37 °C without (A and B) and with (C and D) treatment with 50 nM chymotrypsin. Full analytical HPLC spectra (A and C) are compared with zoomed views (B and D) of the same respective spectra. Solid blue lines indicate the GBAP traces prior to incubation. Dashed red lines indicate the GBAP traces after 8 hours of incubation. In both cases, the half-life for GBAP is approximately 4 hours, however the difference in products indicates that chymotrypsin can successfully digest GBAP.

Figure 2:

Degradation trends in PBS at 37 °C without (A) and with (B) treatment with 50 nM chymotrypsin. GBAP is shown in blue with circles, linear GBAP is shown in green with triangles, and GBAP-N-MeF7 is shown in orange with squares.