Nicotinamide N-methyltransferase (NNMT) is a methyltransferase that is highly expressed in the human liver and adipose tissue. It is a phase II metabolic enzyme that uses the methyl donor S-adenosylmethionine (SAM) to catalyze the methylation of nicotinamide (NAM) and structurally related compounds, resulting in the formation of S-adenosyl-l-homocysteine (SAH) and N1-methyl-nicotinamide (MNAM) [1], [2]. In 1951, Giulio Cantoni partially purified the enzyme from the rat liver and characterized its methyltransferase activity for the first time [3]. In 1952, SAM was identified as a methyl donor in NAM methylation. Furthermore, SAM participates in most cellular methylation reactions, where it provides methyl groups [4]. In the 1990 s, Weinshilboum et al. successfully cloned the NNMT gene in vitro and expressed it in human liver tissue [5]. For a long time, NNMT was believed to be involved in niacin metabolism and xenobiotic detoxification of endogenous metabolites. However, further research revealed that NNMT is extensively involved in various physiological metabolic and regulatory processes. NNMT expression plays a crucial role in NAM metabolism [6]. NAM is a precursor of NAD+, which is one of the most abundant molecules in the human body [7]. NAD+ serves as a coenzyme and provides electrons to mitochondrial complex I in the electron transport chain, thus participating in numerous redox reactions in metabolic pathways, including glycolysis, the citric acid cycle, oxidative phosphorylation, and other basic bioenergetic pathways [8]. Under stress conditions, NAD+ plays multiple roles in cellular division, DNA repair, chromatin remodeling, and epigenetic signaling [9]. Specifically, NAM, either by itself or by altering NAD+ levels, regulates mitochondrial function and activity, thereby affecting cellular vitality and metabolism [10]. In contrast, NNMT influences methyl donor SAM consumption and overall methylation. Studies have shown that NNMT plays complex roles and exhibits different effects in different types of cancer. In certain tumors, high NNMT expression promotes cell proliferation and invasion [11], [12], [13], [14]. For example, in invasive cancer renal cell carcinoma (RCC), NNMT is highly expressed in the clear cell renal cell carcinoma (ccRCC) type [15]. However, in other tumors, low NNMT expression may be an adaptive mechanism for maintaining specific tumor cell phenotypes. For example, in hepatocellular carcinoma (HCC), NNMT expression is suppressed [16]. Regardless of the level of expression, NNMT influences cell metabolism and epigenetic reprogramming and plays complex roles in cancer progression. Moreover, NNMT is highly expressed in human and rodent adipose tissues. In 2014, Kahn et al. reported increased NNMT expression in adipose and liver cells of obese and diabetic mice [17]. Research has found that in conditions such as cardiovascular diseases [18], neurodegenerative disorders [19], [20], [21], and metabolic disorders [22], [23], NNMT expression also demonstrates an increasing trend.

Owing to the regulatory role of NNMT in cellular epigenetics and metabolism and its extensive involvement in several human diseases, it can be considered a novel potential pharmacological target for the treatment of various metabolic disorders, cancer, and other pathologies. In recent years, the development of effective and selective inhibitors targeting NNMT has accelerated as studies have noted its significance.

The crystal structure published in 2011 provided the first evidence of the interaction between NNMT and its substrate NAM and cofactor analog SAH. this finding facilitated the development of rationally designed small-molecule inhibitors for NNMT [24]. As a product of SAM, SAH serves as a natural feedback inhibitor of NNMT, with an IC50 of 26.3 ± 4.4 μM. Structurally similar to SAH, Sinefungin, a natural product isolated from Streptomyces griseus, exhibits inhibitory activity and has an IC50 of 3.9 ± 0.3 μM. Initially considered a potential antifungal agent, Sinefungin later became a research tool for the inhibition of pan-methyltransferase and was categorized as a moderate inhibitor. Similarly, as a metabolite of NAM, 1-Methylnicotinamide (MNAM) serves as a feedback inhibitor of NNMT and has an IC50 of 24.6 μM [2]. MNAM, a prototype NNMT inhibitor involved in the binding site of NAM, has been conventionally used in pharmacological evaluations of NNMT function. Further studies on structurally similar compounds revealed that other N-methylation products formed by various substrate heterocycles exhibited similar levels of inhibitory activity. An example is 1-MQ, with an IC50 of 12.1 ± 3.1 μM [25]. In 2021, Sanofi performed high-throughput screening (HTS) of compound AK-4 (IC50 = 0.07 μM) and identified a series of competitive tricyclic NAM inhibitors, represented by AK-2 (IC50 = 1.6 μM) and AK-3 (IC50 = 0.18 μM) [26].

Among the numerous inhibitors that have been developed, those developed using a bisubstrate inhibition strategy are shown to achieve high inhibitory activity. Guided by the Bi-Bi kinetic mechanism of NNMT, bisubstrate inhibitors have been developed that can occupy both the substrate and cofactor-binding sites to better mimic the transition state [27]. The structure activity relationship has shown that many functional groups present in SAM and NAM are essential for binding; even slight changes in the chemical structure of the bisubstrate compounds could have a substantial impact on their activity. In 2017, Martin et al. established a library of bisubstrate-like compounds that inhibit NNMT [28]. The most representative bisubstrate inhibitor is VH45 (IC50 = 29.2 ± 4.0 μM). Based on the crystal structure of NNMT (PDB ID: 3ROD), the distance between the pyridine nitrogen atom of NAM and the sulfur atom of SAH is between 3.5 and 4.2 Å. In 2019, Nathaniel et al. optimized the structure of bisubstrate inhibitors and conducted an in-depth SAR study of the amino acid and niacinamide units. Based on the π-π stacking interaction between the tyrosine (Tyr) residue Y204 and niacinamide substrate, they designed and discovered a compound with a naphthyl group, GYZ-78 (IC50 = 1.4 μM) [29]. In 2019, Shair et al. developed high-affinity alkyne-based bisubstrate inhibitors of NNMT using a combination of structure-based design and molecular docking [30]. A distinguishing feature of these inhibitors was the rigid alkyne linker designed to mimic the linear 180° transition-state geometry during the methylation reaction. The most potent inhibitor NS1 was designed to direct the alkyne toward the NAM-binding pocket via the C6 chiral center, resulting in a Ki value of 500 pM. Huang et al. designed and synthesized a tightly bound bisubstrate inhibitor, LL320, using a novel propargyl linker and achieved a Ki value as low as 1.6 nM [31]. In 2022, Martin et al. introduced various aromatic and truncated naphthalene moieties to further modify and optimize the bisubstrate inhibitor, ultimately discovering the novel NNMT inhibitor GYZ-17u (IC50 = 3.7 ± 0.2 nM) [32]. In 2023, Huang et al. designed and synthesized a series of analogs, among which compound II559 (Ki = 1.2 nM) exhibited robust cellular inhibitory activity, with a cellular IC50 value of approximately 150 nM [33]. (Fig. 1).

We analyzed the spatial environment of the adenine moiety at position 7 of the NNMT dual-substrate inhibitor through modeling. Subsequently, we synthesized compounds with activities superior to LL320 and further enhanced their inhibitory effects.