Mechanism of action for small-molecule inhibitors of triacylglycerol synthesis

Triacylglycerols (TGs) serve as the primary energy storage form in many organisms, storing reduced carbon acyl chains esterified to a glycerol backbone. The accumulation of TGs and dysregulated TG metabolism are strongly associated with metabolic disorders like cardiovascular disease and diabetes. Elevated TG levels are also observed in various cancers, where TGs may contribute to cancer cell survival and growth. Consequently, inhibiting TG synthesis and storage presents a potential therapeutic approach for TG-related diseases. Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) is a key enzyme in human TG synthesis, catalyzing the esterification of diacylglycerol (DAG) with acyl-CoA to produce TGs at the endoplasmic reticulum (ER). This reaction is crucial for energy storage in tissues such as the intestine, liver, and adipose tissue. Additionally, DGAT1 protects the ER from accumulating bioactive lipids derived from excess fatty acids. DGAT1 belongs to the membrane-bound O-acyltransferase (MBOAT) family, found across all kingdoms of life. Humans possess 11 MBOATs, each mediating the acylation of various lipids or proteins. Mammalian MBOATs are saddle-shaped proteins with multiple transmembrane segments, featuring a catalytic center with conserved histidine and asparagine (or aspartate for HHAT) residues deeply embedded in the ER membrane. A tunnel through the enzyme facilitates acyl-CoA access to the catalytic site from the cytoplasm. Understanding the mechanisms of action of DGAT1 inhibitors is crucial for developing effective therapies targeting TG metabolism.

Previous research has identified several small molecule inhibitors of DGAT1, offering potential therapeutic strategies for treating metabolic disorders and certain cancers. However, a detailed understanding of the precise mechanisms by which these inhibitors interact with DGAT1 and disrupt its function has been lacking. Studies focusing on the structure and function of DGAT1 and related MBOAT enzymes have provided valuable insights into their catalytic mechanisms and substrate binding. However, a clear picture of the molecular interactions between known DGAT1 inhibitors and the enzyme itself was needed to guide the rational design of more potent and selective inhibitors. This study aims to address this gap in knowledge by employing advanced structural biology techniques such as cryo-electron microscopy (cryo-EM) to determine high-resolution structures of DGAT1 in complex with selected inhibitors.

This study utilized cryo-electron microscopy (cryo-EM) to determine the three-dimensional structures of human DGAT1 in complex with two distinct inhibitors: T863 and DGAT1IN1. Human DGAT1 was expressed in HEK293 GnTI− cells using a baculovirus expression system and purified using affinity chromatography followed by size exclusion chromatography. For cryo-EM sample preparation, purified DGAT1 was reconstituted into amphipol PMAL-C8 in the presence of each inhibitor. Cryo-EM grids were prepared using a Vitrobot and imaged using a Talos Arctica or Titan Krios electron microscope. Image processing and particle picking were performed using RELION. Initial 3D models were built based on previously published DGAT1 cryo-EM maps, followed by 3D classification and refinement. Model building and refinement were performed using PHENIX and COOT. DGAT1 activity assays were conducted using both purified DGAT1 and ACAT1-overexpressing microsomes to assess the inhibitory effects of T863, DGAT1IN1, and ATR101. Lipid droplet formation in SUM159 cells was evaluated using fluorescence microscopy after treatment with the inhibitors. Site-directed mutagenesis was used to generate ACAT1 and DGAT1 mutants for further investigation of inhibitor selectivity.

Cryo-EM structures revealed that both T863 and DGAT1IN1 bind to the acyl-CoA binding tunnel of DGAT1, competing with the natural substrate. T863 occupies the tunnel entrance, primarily interacting through hydrophobic interactions. DGAT1IN1, however, extends further into the enzyme, its amide group forming hydrogen bonds with conserved catalytic residues (His415 and Asn378). This suggests that the amide group is a critical pharmacophore for inhibiting MBOAT enzymes. Inhibitor selectivity studies using ACAT1 revealed that T863 exhibited minimal activity against ACAT1, but a single-residue mutation (Asn487Ala) in ACAT1 significantly increased its sensitivity to T863. This suggests that subtle structural differences in the acyl-CoA binding tunnel between DGAT1 and ACAT1 contribute to inhibitor selectivity. The study also showed that DGAT1IN1 displayed enhanced inhibition of ACAT1 compared to T863, further emphasizing the importance of the amide group in conferring potent inhibitory activity. The structural analysis indicated that a loop region connecting transmembrane helices TM8 and TM9 displays different conformations in DGAT1 and ACAT1, potentially creating a steric hindrance for T863 binding in ACAT1. The authors propose a classification of DGAT1 inhibitors into Type I (competing with acyl-CoA) and Type II (potentially blocking the acyl-acceptor binding site). A survey of known DGAT1 and MBOAT inhibitors revealed a shared pharmacophore comprising an amide bond flanked by two hydrophobic moieties, with the moieties determining selectivity.

This study provides the first high-resolution structural insights into the mechanisms of action of DGAT1 inhibitors. The findings highlight the importance of the acyl-CoA binding tunnel as a target for inhibitor design and reveal how subtle structural differences between related MBOAT enzymes can contribute to inhibitor selectivity. The identification of an amide group as a common pharmacophore offers a valuable starting point for the development of new and more effective MBOAT inhibitors. The two types of inhibitors, Type I (acyl-CoA competitors) and Type II (acyl-acceptor blockers), suggest multiple avenues for drug development. The discovery that a single point mutation can alter inhibitor selectivity suggests that further investigation into these structural variations between MBOAT family members could lead to the development of highly specific and potent drugs for various metabolic diseases and potentially cancer. Future studies could focus on exploring other MBOAT enzymes to understand the generalizability of the identified pharmacophore and develop broader-spectrum inhibitors.

This research provides high-resolution cryo-EM structures of human DGAT1 in complex with two inhibitors, elucidating their mechanisms of action. The study reveals an amide group as a key pharmacophore for inhibiting MBOAT enzymes and highlights the role of structural differences in determining inhibitor selectivity. These findings lay a foundation for the rational design of more effective and specific inhibitors targeting DGAT1 and other MBOAT enzymes for therapeutic applications. Future work should explore the broader applicability of this pharmacophore across the MBOAT family and investigate the potential of Type II inhibitors.

The study focused on two specific DGAT1 inhibitors and one MBOAT enzyme (ACAT1). Further investigation is needed to confirm the generalizability of the findings to other DGAT1 inhibitors and a wider range of MBOAT enzymes. The in vitro activity assays may not fully reflect the complexity of in vivo interactions. The study's findings may not be directly transferable to all organisms given the species-specific differences in enzyme structure and function.