Cell-Effective Transition-State Analogue of Phenylethanolamine -Methyltransferase.

Phenylethanolamine -methyltransferase (PNMT) catalyzes the -adenosyl-l-methionine (SAM)-dependent methylation of norepinephrine to form epinephrine. Epinephrine is implicated in the regulation of blood pressure, respiration, Alzheimer's disease, and post-traumatic stress disorder (PTSD). Transition-state (TS) analogues bind their target enzymes orders of magnitude more tightly than their substrates. A synthetic strategy for first-generation TS analogues of human PNMT (hPNMT) permitted structural analysis of hPNMT and revealed potential for second-generation inhibitors [Mahmoodi, N.; 2020, 142, 14222-14233]. A second-generation TS analogue inhibitor of PNMT was designed, synthesized, and characterized to yield a value of 1.2 nM. PNMT isothermal titration calorimetry (ITC) measurements of inhibitor indicated a negative cooperative binding mechanism driven by large favorable entropic contributions and smaller enthalpic contributions. Cell-based assays with HEK293T cells expressing PNMT revealed a cell permeable, intracellular PNMT inhibitor with an IC value of 81 nM. Structural analysis demonstrated inhibitor filling catalytic site regions to recapitulate both norepinephrine and SAM interactions. Conformation of the second-generation inhibitor in the catalytic site of PNMT improves contacts relative to those from the first-generation inhibitors. Inhibitor demonstrates up to 51,000-fold specificity for PNMT relative to DNA and protein methyltransferases. Inhibitor also exhibits a 12,000-fold specificity for PNMT over the α-adrenoceptor.