Defining the mechanism of action of S1QELs, specific suppressors of superoxide production in the quinone-reaction site in mitochondrial complex I

Abstract

Edited by Ruma Banerjee This work was supported by Japan Society for the Promotion of Science KAKENHI Grants JP26292060 and JP18H02147 (to H. M.) and JP18K05458 (to M. M.). The authors declare that they have no conflicts of interest with the contents of this article. This article contains Table S1, Schemes S1-S5, and Figs. S1-S2.

IACS-010759, a potent inhibitor of glycolysis-deficient hypoxic tumor cells, inhibits mitochondrial respiratory complex I through a unique mechanism

“…The radiolabeled complex I was isolated by BN-PAGE, followed by resolution on doubled SDS gel using 10 and 16% Schägger-type SDS gels (38). The labeling by [ 125 I]IACS-010759-PD1 gave a major ~30-kDa radioactive spot above a diagonal axis ( Figure 6A), which corresponds to the position of the ND1 subunit (29). We verified this assignment by Western blotting using a rabbit antibovine ND1 antibody ( Figure 6A).…”

“…Similarly, ~20% radioactivity remained in the presence of excess BAY 87-2243 (20 µM). We previously demonstrated that fenpyroximate and AQ, which predominantly bind to the 49-kDa subunit (27,28,36), do not suppress the labeling by [ 125 I]S1QEL1.1-PD1, but bullatacin that binds to the ND1 subunit (37) almost completely suppresses the labeling (29). As a reference, the suppression by bullatacin (5.0 µM) is presented in Figure 5.…”

“…To localize the binding position of S1QELs in bovine heart complex I, we previously conducted photoaffinity labeling experiments using a synthetic photoreactive derivative of S1QEL1.1 ([ 125 I]S1QEL1.1-PD1 (IC50 = 23 nM), Figure S2) and found that [ 125 I]S1QEL1.1-PD1 binds to a region in the ND1 subunit, which is under the matrix-side third loop connecting TMHs 5−6 (29). We also showed that in contrast to known quinonesite inhibitors, S1QELs do not occupy the binding cavity for ubiquinone; rather, they may indirectly modulate the ubiquinone redox reactions through inducing structural changes of the cavity by binding to the middle of ND1 (29). Thus, [ 125 I]S1QEL1.1-PD1 is also a helpful benchmark sample for investigating the binding position of IACS-010759 in complex I.…”

“…Therefore, here we investigated the action mechanism of IACS-010759 in bovine heart submitochondrial particles (SMPs) using three specific ligands as helpful benchmarks: AL1 (25,26), [ 125 I]AzQ (27,28), and [ 125 I]S1QEL1.1-PD1 (29), shown in Figure S2, the binding positions of which have been defined in complex I. To identify the binding position of IACS-010759 in complex I, we also carried out photoaffinity labeling experiments using a photoreactive IACS-010759 derivative, [ 125 I]IACS-010759-PD1 ( Figure 1).…”

“…This unique inhibitory action would be responsible for the interesting profiles of antitumor/cytotoxic activities of IACS-010759. Note that according to common terminology used in the complex I research field, we refer to the inhibitors that interfere with the terminal electron transfer step in complex I as "quinone-site inhibitors" throughout this work, though it is debatable whether all quinone-site inhibitors indeed occupy the proposed quinone binding cavity (29,30).…”