Fig. 4.
L-dopa decarboxylation by E. faecalis is inhibited by AFMT but not the host-targeted drug carbidopa. (A) Carbidopa and AFMT. (B) Carbidopa preferentially inhibits human AADC over TyrDC. AADC or TyrDC were incubated with inhibitor, and reaction rates were measured with LC-MS/MS. “% Activity” represents the rate relative to a no inhibitor (vehicle) control. Results are mean ± SEM (n = 3 replicates). (C) Activity of carbidopa and AFMT in cultures of E. faecalis grown for 16 hours anaerobically with 0.5 mM L-dopa. Error bars represent the mean ± SEM for three biological replicates. (D) Activity of carbidopa in a human fecal microbiota from a Parkinson’s patient. The sample was cultured anaerobically with carbidopa and 1 mM d3-phenyl-L-dopa for 72 hours. Error bars represent the mean ± SEM for three biological replicates. (E) AFMT preferentially inhibits TyrDC over AADC in vitro. AADC or TyrDC were incubated with inhibitor, and reaction rates were measured with LC-MS/MS. “% Activity” represents the rate relative to a no inhibitor (vehicle) control. Error bars represent the mean ± SEM for three biological replicates. (F) Detection of an AFMT-PLP covalent adduct after incubation of TyrDC or AADC with AFMT for 1 hour. The data shown is the extracted ion chromatogram of the mass of the predicted covalent adduct. (G) Action of AFMT in human fecal microbiotas from Parkinson’s patients incubated anaerobically with AFMT and 1 mM d3-phenyl-L-dopa for 72 hours. Error bars represent the mean ± SEM for three biological replicates. (H) Pharmacokinetic analysis in gnotobiotic mice colonized with E. faecalis and given L-dopa + carbidopa + AFMT demonstrates higher serum L-dopa relative to vehicle controls. Error bars represent the mean ± SEM. (I) The maximum serum concentration (Cmax) of L-dopa is significantly higher with AFMT relative to vehicle controls. In (H) and (I), *P < 0.05, Mann-Whitney U test; n = 4 to 5 mice per group.