Numerous small molecule inhibitors of DGAT1 have been developed for treating metabolic diseases characterized by TG overaccumulation23. In clinical trials, some inhibitors were found to cause dose-related gastrointestinal side effects, limiting their utility for treating chronic metabolic disorders24. However, studies using these inhibitors in animal models also suggest they might provide therapeutics to treat cancers with altered TG metabolism, including glioblastoma and prostate cancer7,25.

The molecular mechanisms of these compounds for inhibiting DGAT1 and TG synthesis remain uncertain. Moreover, our understanding of small-molecule inhibition of MBOAT enzymes, in general, is limited. Inhibitors have been developed for other MBOAT enzymes, such as PORCN26 and HHAT27, that acylate the signaling proteins Wnt and Shh, respectively, or MBOAT528 and MBOAT721 that acylate phospholipids. Some are in clinical trials26,29. But how much inhibitors achieve specificity for the different MBOAT enzymes and whether there are common themes for enzyme inhibition are not understood.

To address these questions, we investigated how potent and selective DGAT1 inhibitors block TG synthesis. Using cryo-EM, we resolved the complex structures of human DGAT1 with inhibitors T863 or DGAT1IN1, each at ~3.2 Å resolution. The structures, together with structure-based biochemical analyses, show how these compounds inhibit DGAT1 and provide insights into how they achieve MBOAT selectivity. The shared chemistry between the compounds and their binding modes further suggest chemical features to consider in developing MBOAT inhibitors.