Fig. 4: Structural basis of inhibition selectivity of T863 and DGAT1IN1 for DGAT1 versus ACAT1.

a Structural superposition of monomeric DGAT1 (blue) and ACAT1 (orange, PDB: 6VUM)¹⁶. The structure of human DGAT1 and ACAT1 monomers are depicted as cylinders. TM transmembrane helix. The bound T863 in DGAT1 is also shown (green). b Detailed view of the T863 binding pocket in DGAT1 (left) and the corresponding region in ACAT1 (middle). Note the loop region labeled red in ACAT1 adopts a different conformation than that in DGAT1 (right). c T863 inefficiently inhibits ACAT1 but does so with higher potency for the Asn487Ala mutant. d ACAT1 inhibitor ART101 exhibits potent inhibition on ACAT1. e Sequence alignment of the red region in (b) among DGAT1 and ACAT1 proteins. The squiggles and solid lines on the top represent transmembrane helices and loop regions, respectively. The solid triangle denotes the Asn487 in ACAT1 protruding into the corresponding pocket in DGAT1 at T863 binding pocket. f DGAT1IN1 exhibits inhibition on the wild-type ACAT1 and increased inhibition on the Asn487Ala mutant. Experiments in c and f were repeated three times independently (n = 3) with similar results. The data shown are one representative result. Data points are shown as mean ± s.d., calculated from three technical replicates. FFA free fatty acid, NS non-specific band. T863 and DGAT1IN1 were used at 10 μM, and ART101 was used at 1 μM in inhibition studies. Statistical analysis by one-way ANOVA (c, d, f). The raw TLC plates are shown in Supplementary Fig. 8a–c. g Proposed DGAT1 inhibition mechanism by small-molecule inhibitors. The catalytic His⁴¹⁵ and Asn³⁷⁸ residues localize in the ER membrane. Type I inhibitors (e.g., T863 and DGAT1IN1) compete with the fatty acyl CoA substrate for binding at the cytoplasmic side of the enzyme; the type II inhibitors enter the catalytic center from the lateral opening. Amide bond-containing inhibitors may act by mechanism-based inhibition, locking the catalytic His and Asn residues (right panel).