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.

中文翻译：

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烟酰胺 N-甲基转移酶 (NNMT) 的强效双底物抑制剂的酯酶敏感性前药显示细胞活性

最近发现的一种烟酰胺N-甲基转移酶 (NNMT) 的双底物抑制剂在生化分析中非常有效，IC _{50为单位数纳摩尔}价值但缺乏细胞活性。我们在这里报告了一种前药策略，旨在将观察到的这种抑制剂的有效生化抑制活性转化为强细胞活性。这种前药策略依赖于双底物抑制剂中存在的高极性氨基酸侧链的胺和羧酸部分的临时保护。在不存在或存在三甲基锁 (TML) 胺保护基团的情况下将羧酸修饰成一系列酯，从而产生了一系列候选前药。基于在水性缓冲液中的稳定性和证实的酯酶依赖性转化为母体化合物，异丙酯被选为优选的酸前药。异丙酯和异丙酯-TML前药表现出改善的细胞渗透性，