Structure and inhibition mechanism of the catalytic domain of human squalene epoxidase

Abstract

Squalene epoxidase (SQLE), also known as squalene monooxygenase, catalyzes the stereospecific conversion of squalene to 2,3( S )-oxidosqualene, a key step in cholesterol biosynthesis. SQLE inhibition is targeted for the treatment of hypercholesteremia, cancer, and fungal infections. However, lack of structure-function understanding has hindered further progression of its inhibitors. We have determined the first three-dimensional high-resolution crystal structures of human SQLE catalytic domain with small molecule inhibitors (2.3 Å and 2.5 Å). Comparison with its unliganded state (3.0 Å) reveals conformational rearrangements upon inhibitor binding, thus allowing deeper interpretation of known structure-activity relationships. We use the human SQLE structure to further understand the specificity of terbinafine, an approved agent targeting fungal SQLE, and to provide the structural insights into terbinafine-resistant mutants encountered in the clinic. Collectively, these findings elucidate the structural basis for the specificity of the epoxidation reaction catalyzed by SQLE and enable further rational development of next-generation inhibitors.

A key mammalian cholesterol synthesis enzyme, squalene monooxygenase, is allosterically stabilized by its substrate

“…2C). This observation is consistent with the fact that stabilization of the SM catalytic domain by NB-598 has been observed using in vitro thermal shift assays (12), and with the previously reported stabilization of receptors or enzymes upon binding of their ligands in living cells (21)(22)(23)(24). Interestingly, NB-598 also up-regulated the SM-N100 regulatory domain in two different cell lines stably expressing SM-N100-ELuc (EC 50 1.3 nM) or SM-N100-GFP-V5 (EC 50 18 nM) constructs ( Fig.…”

“…Implications for Other Studies. Although the structure of the catalytic domain of SM with NB-598 was recently published, the SM-N100 region has remained refractory to crystallization (12). Yet structural analyses should provide a clearer understanding of how SM-N100 senses squalene.…”

“…2B). NB-598, which tightly binds to the active site of SM (12), up-regulated SM-ΔN100-ELuc with an EC 50 of 0.44 nM (Fig. 2C).…”

“…Despite its importance in cholesterol-accelerated degradation, the structure and function of the SM-N100 region has not been fully resolved. Thus far, biochemical analyses have revealed the presence of a reentrant loop anchoring SM to the ER membrane and an amphipathic helix required for cholesterol-accelerated degradation (7,8), while X-ray crystallography has revealed the architecture of the catalytic domain of SM (12). However, the highly hydrophobic and disordered nature of the N100 region has hampered purification and structural analysis of this region or full-length SM (12,13).…”

Unusual and Highly Bioactive Sesterterpenes Synthesized by Pleurotus ostreatus during Coculture with Trametes robiniophila Murr

“…7. Unlike the data determined for the common C 25 precursor geranylfarnesyl diphosphate (GFPP) with respect to sesterterpene biosynthesis, farnesyl-diphosphate farnesyltransferase KDQ25270 likely catalyzed geranyl pyrophosphate (GPP) and farnesyl pyrophosphate (FPP) to form the C 25 precursor, which then underwent a series of posttailoring modifications to generate postrediene A to C. KDQ25268 showed 55% identity to squalene epoxidase catalyzing the stereospecific conversion of squalene to 2,3(S)-oxidosqualene (25), which was proposed to catalyze the epoxidation reactions in the C-2, C-3, C-10, C-11, C-18, and C-19 positions. Subsequently, epoxide openings cascaded in these positions to produce postrediene A, and those steps might have been catalyzed by an epoxide hydrolase, which has often been found to catalyze the conversion of epoxides to trans-dihydrodiols (26).…”