Cocrystal structure reveals the mechanism of FSP1 inhibition by FSEN1

Abstract

FSP1 is an FAD-dependent oxidoreductase that uses NAD(P)H to regenerate the reduced forms of lipophilic quinone antioxidants, such as coenzyme Q10 and vitamin K. These quinone antioxidants function as radical scavenging agents that prevent the propagation of lipid peroxidation and the induction of ferroptosis. Although several small-molecule inhibitors of FSP1 have been developed and found to sensitize cancer cells to ferroptosis, our understanding of their molecular mechanisms remains limited and no structures of FSP1 in complex with its inhibitors have been solved. Here, we solve the cocrystal structure of FSP1 in complex with the FSP1 inhibitor FSEN1, revealing that FSEN1 binds within the FSP1 substrate-binding pocket. FSEN1 makes key interactions with a critical phenylalanine, which is absent in mouse FSP1, providing an explanation for the selectivity of FSEN1 for human FSP1. These conclusions are supported by mutagenesis of FSP1 and biochemical and cellular assays of FSP1 function. Our findings provide the first cocrystal structure of FSP1 in complex with an inhibitor, enhancing our understanding of the mechanism of FSP1 inhibition and enabling future rational medicinal chemistry efforts to advance FSP1 inhibitors as therapeutics.

Keywords: cancer; ferroptosis; inhibitor; small molecule; structure.

Fig. 1.

Overall structure of hFSP1–FSEN1 complex. (A) Crystal structure of hFSP1–NADP⁺–6-OH-FAD–FSEN1 complex. (B) A zoomed-in view of hFSP1 with FSEN1. (C and D) Structural comparison of hFSP1 in complex with NADP⁺, 6-OH-FAD, FSEN1 (C), and cFSP1 in complex with FAD and CoQ1 (PDB: 7XPI) (D).

Fig. 2.

Detailed interaction between hFSP1 and FSEN1 (A): Zoom view shows the interaction between Fsen1 and F360, T363 and S364 of hFSP1. Yellow dotted lines: π–π interaction or hydrogen bonds. (B) Zoom view shows the interaction between CoQ1 and F360 of cFSP1. Yellow dotted lines: π–π interaction. (PDB: 7XPI). (C–F): In vitro enzymatic activity of hFSP1 WT and mutants. Purified hFSP1 WT (C), F360A (D), T363A&T364A (E), and F360A&T363A&T364A (F) were incubated with CoQ1, NADPH, and different concentrations of FSEN1, and absorbance at 340 nm was monitored over time. IC50 values were determined at 1 h in FSP1 enzymatic activity assay.

Fig. 3.

Cell expressing mutants of F360 of hFSP1 are resistant to FSEN1. Dose–response of FSEN1-induced cell death in U-2 OS FSP1KO cells expressing wild-type (WT) or variant mutants of F360, T363, or S364. Cells were cotreated with 2 μM RSL3 with or without the addition of 2 μM Fer-1 (broken lines) for 24 h. Data are mean ± SD (n = 3 to 5 biological replicates).

Fig. 4.

L/F360 determines FSP1 sensitivity to FSEN1. (A) Sequence alignment of CoQ-binding domain of FSP1 from Homo sapiens (Hs), Mus musculus (Mm), Gallus gallus (Gg), Xenopus laevis (Xl), Danio rerio (Dr), Phallusia mammillata (Pm). Brick red stars: nonconserved amino acids in hFSP1 and mFSP1 among the FSEN1-contacting residues. (B–E): In vitro enzymatic activity of mFSP1 WT and mutants. Purified mFSP1 WT (B), A354T (C), L360F (D), and A354T&L360F (E) were incubated with CoQ1, NADPH, and different concentrations of FSEN1, and absorbance at 340 nm was monitored over time. IC50 values were determined at 1 h in FSP1 enzymatic activity assay.

Fig. 5.

F360 defines species specificity of FSEN1. Dose–response of FSEN1-induced cell death in U-2 OS FSP1KO cells expressing either human or mouse variants of F360, A354. Cells were cotreated with 2 μM RSL3 with or without the addition of the ferroptosis inhibitor 2 μM Fer-1 (broken lines) for 24 h. Data are mean ± SD (n = 3 to 4 biological replicates).