Assay for drugs affecting lysine decarboxylase activity associated with gingivitis

Objectives: Lysine decarboxylase (Ldc-E) from Eikenella corrodens (EC) converts lysine to cadaverine in dental biofilms from gingivally healthy adults after a week of restricted oral hygiene. Most lysine comes from gingival crevicular fluid (GCF) and its depletion to cadaverine impairs the dental epithelial attachment necessary to maintain a barrier to bacterial products (PMID:26110450). Lysine also activates Ldc-E (PMID:11312612). An in silico study, detected a site within the N-terminal region that bound lysine but more strongly to many molecules containing quaternary or protonated tertiary amines (AADR March 2016, Abstract 867). The second strongest binding agent was nilotinib. Aims were to develop an assay for identifying drugs that alter Ldc-E activity, determine whether nilotinib activates or inhibits, and compare the results with tranexamic acid (TA), a known Ldc-E inhibitor.
Methods: Ldc-E was obtained from cell surface extracts of early stationary phase EC. Ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC–MS/MS) was set to detect cadaverine and deuterated cadaverine. For calibration, a cadaverine standard solution of 64 mM was serially diluted to give seven calibrator wells, 0 – 64 mM, and a fixed amount of deuterated cadaverine was added to all control and test samples (internal standard). The ratio of each cadaverine peak area resulting from the enzyme reaction to the internal standard peak area was converted to mM cadaverine.
Results: Assay results show that nilotinib (13 μg/mL) increased cadaverine production in Ldc-E preparations and that TA (8 μg/mL) reduced cadaverine production (Figure 1).
Conclusions: uHPLC-MS/MS can identify drugs that alter Ldc-E activity. Nilotinib is likely an allosteric activator at the N-terminal site detected in silico.