Structure-Based Drug Design of Bisubstrate Inhibitors of Phenylethanolamine N-Methyltransferase Possessing Low Nanomolar Affinity at Both Substrate Binding Domains¹

Abstract

The enzyme phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.28) catalyzes the final step in the biosynthesis of epinephrine and is a potential drug target, primarily for the control of hypertension. Unfortunately, many potent PNMT inhibitors also possess significant affinity for the a 2adrenoceptor, which complicates the interpretation of their pharmacology. A bisubstrate analogue approach offers the potential for development of highly selective inhibitors of PNMT. This paper documents the design, synthesis, and evaluation of such analogues, several of which were found to possess human PNMT (hPNMT) inhibitory potency <5 nM versus AdoMet. Site-directed mutagenesis studies were consistent with bisubstrate binding. Two of these compounds (19 and 29) were co-crystallized with hPNMT and the resulting structures revealed both compounds bound as predicted, simultaneously occupying both substrate binding domains. This bisubstrate inhibitor approach has resulted in one of the most potent (20) and selective (vs the a 2-adrenoceptor) inhibitors of hPNMT yet reported.

Bisubstrate Strategies to Target Methyltransferases

“…Moreover, all the data described in this review may provide a new starting point in the search for more effective and selective bisubstrate compounds. [57] (73) and Grunewald and co-workers [58] (74). [59]…”

“…At the same time, Grunewald and co-workers also reported the synthesis of bisubstrate PNMT inhibitors with low nanomolar affinity. [58] Here, the amino acid chain was removed to retain the sulfur atom of the SAM structure. A propylamino linker was first Figure 19.…”

“…Figure 20. Transition state of norepinephrine methylation yielding epinephrine and bisubstrate inhibitors of hPNMT developed by Schramm and co-workers[57] (73) and Grunewald and co-workers[58] (74).…”

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

“…Bisubstrate inhibitors confer a thermodynamic advantage by incorporating two binding moieties within one molecule . This approach has provided improved specificity for a PNMT inhibitor relative to the α 2 -adrenoceptor. , A similar approach has been employed to develop bisubstrate inhibitors of nicotinamide N -methyltransferase (NNMT) and protein N -terminal methyltransferase 1 (NTMT1), with high selectivity for their enzymes. , …”

“…18 This approach has provided improved specificity for a PNMT inhibitor relative to the α 2 -adrenoceptor. 19,20 A similar approach has been employed to develop bisubstrate inhibitors of nicotinamide N-methyltransferase (NNMT) and protein N-terminal methyltransferase 1 (NTMT1), with high selectivity for their enzymes. 21,22 Transition-state analogues (TSA) mimic the geometry and electrostatic properties of an enzymatic transition state in chemically stable structures and have the potential to bind their cognate active sites orders of magnitude more tightly than substrate analogues.…”