Isocitrate dehydrogenase (IDH) catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate, following the reaction: isocitrate + NADP+ → α-ketoglutarate + CO2 + NADPH + H+. There are three types of IDH in eukaryotic cells (Al-Khallaf, 2017, Alzial et al., 2022). NAD+-dependent IDH (IDH3), which is well known for its central role in energy production within the Krebs cycle, is localized in the mitochondrial matrix (Ramachandran and Colman, 1980). In contrast, IDH1 and IDH2 are NADP+-dependent IDHs located in the cytoplasm (Jennings et al., 1994) and mitochondria (Haselbeck and Mcalisterhenn, 1993), respectively. These enzymes play a crucial role in cellular defense against oxidative stress by serving as sources of NADPH (Jo et al., 2001, Kim and Park, 2003, Lee et al., 2002). IDH3, which exists as a heterotetramer (α2βγ), is tightly regulated by allosteric effectors, presenting a key regulatory point for mitochondrial energy production (Chen and Ding, 2023, Sun et al., 2020). Conversely, the regulation of IDH1 and IDH2, which are involved in various cellular processes, is less well understood compared to IDH3. Both IDH1 and IDH2 are homodimers and share similar overall structures, consisting of three domains: the active site comprises large and small domains, while dimerization occurs through small and clasp domains (Ceccarelli et al., 2002, Cho et al., 2018, Xu et al., 2004).

IDH1 is primarily located in the cytosol and peroxisomes, where it plays a crucial role in generating NADPH, which is essential for reductive biosynthesis in the cytosol. NADP+-linked IDH1 serves as the major source of NADPH (Bruinenberg et al., 1983), and the NADPH produced by IDH1 acts as a cofactor necessary for fatty acid synthesis (Nam et al., 2012, van Roermund et al., 1998). Consequently, IDH1 presents a potential target for fat reduction, and inhibitors of IDH1 are currently under development as therapeutic agents for obesity (Carosi et al., 2024, Koh et al., 2004). The functions of IDH1 have been implicated in numerous metabolic diseases and tumors (Ivanov et al., 2024). Mutations in IDH1 are frequently found in melanoma patients (Linos and Tafe, 2018), and inhibition of IDH1 has proven effective in the treatment of melanoma (Zarei et al., 2022).

Oxalomalate serves as a potent inhibitor of IDH1 (Ruffo et al., 1974, Yang and Park, 2003). The structural similarity between oxalomalate and isocitrate allows oxalomalate to occupy the active site, thereby obstructing substrate access and diminishing the enzyme's activity. Oxaloacetate can also bind at the active site. IDH from pig liver cytoplasm catalyzes the reduction of oxaloacetate, resulting in the production of D-malate (Illingworth and Tipton, 1970). Oxaloacetate is recognized as a competitive inhibitor of IDH1, as it binds to the active site and directly competes with isocitrate. In the presence of oxaloacetate, a bacterial NADP+-IDH showed a reduced activity (Alvarado and Flores, 2003). Together, oxaloacetate and glyoxylate inhibit IDH, similar to oxalomalate (Ingebretsen, 1976). Furthermore, oxaloacetate facilitates the generation of the potent inhibitor oxalomalate in the presence of glyoxylate (Johanson and Reeves, 1977, Nimmo, 1986). Inhibitors of IDH1 can significantly influence the intricate regulation of cellular metabolism by modulating the enzyme's activity in response to the cell's metabolic state.

Previously, we determined crystal structures of mouse IDH1 (Cho et al., 2018). The protein was heterologously expressed for crystallization, and NADP+ derived from Escherichia coli was identified within the protein. In the crystal structure, additional electron density was observed at the active site, likely resulting from the binding of a metabolite from the cells that is compatible with the site. To explore potential interaction modes of small molecules that can bind to the active site of IDH1, we determined crystal structures of IDH1 complexed with isocitrate and oxaloacetate. Unlike isocitrate, which was located at the active site, oxaloacetate binds not only at the active site but also at an allosteric site. The IDH1 activity assay demonstrated that the inhibition caused by oxaloacetate is due to its interaction at the allosteric site. The allosteric interaction of oxaloacetate is exclusive to the NADP+-bound conformation of IDH1, and this interaction restricts the conversion of IDH1 to its NADP+-free conformation. This allosteric site may serve as a target for the development of novel inhibitors for IDH1.