Esterase-Sensitive Prodrugs of a Potent Bisubstrate Inhibitor of Nicotinamide N-Methyltransferase (NNMT) Display Cellular Activity

Abstract

A recently discovered bisubstrate inhibitor of Nicotinamide N-methyltransferase (NNMT) was found to be highly potent in biochemical assays with a single digit nanomolar IC50 value but lacking in cellular activity. We, here, report a prodrug strategy designed to translate the observed potent biochemical inhibitory activity of this inhibitor into strong cellular activity. This prodrug strategy relies on the temporary protection of the amine and carboxylic acid moieties of the highly polar amino acid side chain present in the bisubstrate inhibitor. The modification of the carboxylic acid into a range of esters in the absence or presence of a trimethyl-lock (TML) amine protecting group yielded a range of candidate prodrugs. Based on the stability in an aqueous buffer, and the confirmed esterase-dependent conversion to the parent compound, the isopropyl ester was selected as the preferred acid prodrug. The isopropyl ester and isopropyl ester-TML prodrugs exhibit improved cell permeability, which also translates to significantly enhanced cellular activity as established using assays designed to measure the enzymatic activity of NNMT in live cells.

Structure–Activity Relationship Studies on Cell-Potent Nicotinamide N-Methyltransferase Bisubstrate Inhibitors

“…While several NNMT inhibitors have been reported, only 6-methoxynicotinamide ( JBSNF-88) and 5-amino-1-methylquinoline ( 5-MQ ) are commonly used in cellular and animal studies (Figure ). , Both compounds target the nicotinamide substrate-binding site with comparable biochemical and cellular activity. , However, concerns remain regarding their modest potency (IC 50 values ranging from 1 to 10 μM), selectivity, and low metabolic stability, limiting their utility in elucidating the biological functions of NNMT. , To address these limitations, bisubstrate inhibitors that target both nicotinamide and cofactor binding have been developed to confer high selectivity and nM inhibitory activities on purified recombinant NNMT. , Nevertheless, their cellular activity has been hampered by poor uptake, exemplified by prodrugs of two top bisubstrate inhibitors NS1 and LL320 . , For instance, the methyl ester of NS1 reduced MNA levels in U2OS cells by only 30% at the concentration of 31.6 μM; the ethyl ester of LL320 displayed poor cell permeability even at 100 μM. , Similarly, an alkene-linked bisubstrate GYZ-319 showed poor cell permeability even with a less polar cyano group, and its prodrug only showed a modest inhibitory effect on cell viability at 100 μM . Thus, there is a need for more effective NNMT inhibitors with enhanced cellular activity.…”

Biomarkers of Bladder Cancer: Cell-Free DNA, Epigenetic Modifications and Non-Coding RNAs

“…A known predictor of poor prognosis, Aurora Kinase A affects NNMT expression [ 136 ]. Research on NNMT is currently focused on inhibitors, as therapeutic agents of many NNMT-related pathways, such as cancer or diabetes [ 137 , 138 , 139 ].…”

Overexpression of NNMT in Glioma Aggravates Tumor Cell Progression: An Emerging Therapeutic Target

“…It has been pointed out in the literature that the design of predrug strategies for NNMT inhibitors that can deliver polar NNMT inhibitors into the cells allows NNMT inhibitors to exert stronger efficacy. This provides valuable new insights for the design and optimization of NNMT inhibitors at a later stage [ 51 ]. Therefore, NNMT is a key factor influencing the diagnosis, treatment, and prognosis.…”