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

Abstract

The small molecule IACS-010759 has been reported to potently inhibit the proliferation of glycolysis-deficient hypoxic tumor cells by interfering with the functions of mitochondrial NADH-ubiquinone oxidoreductase (complex I) without exhibiting cytotoxicity at tolerated doses in normal cells. Considering the significant cytotoxicity of conventional quinone-site inhibitors of complex I, such as piericidin and acetogenin families, we hypothesized that the mechanism of action of IACS-010759 on complex I differs from that of other known quinone-site inhibitors. To test this possibility, here we investigated IACS-010759's mechanism in bovine heart submitochondrial particles. We found that IACS-010759, like known quinone-site inhibitors, suppresses chemical modification by the tosyl reagent AL1 of Asp¹⁶⁰ in the 49-kDa subunit, located deep in the interior of a previously proposed quinone-access channel. However, contrary to the other inhibitors, IACS-010759 direction-dependently inhibited forward and reverse electron transfer and did not suppress binding of the quinazoline-type inhibitor [¹²⁵I]AzQ to the N terminus of the 49-kDa subunit. Photoaffinity labeling experiments revealed that the photoreactive derivative [¹²⁵I]IACS-010759-PD1 binds to the middle of the membrane subunit ND1 and that inhibitors that bind to the 49-kDa or PSST subunit cannot suppress the binding. We conclude that IACS-010759's binding location in complex I differs from that of any other known inhibitor of the enzyme. Our findings, along with those from previous study, reveal that the mechanisms of action of complex I inhibitors with widely different chemical properties are more diverse than can be accounted for by the quinone-access channel model proposed by structural biology studies.

Keywords: IACS-010759; bioenergetics; cancer; chemical biology; complex I; enzyme inhibitor; hypoxia; mitochondria; photoaffinity labeling; ubiquinone.

Figure 1.

Structures of BAY 87-2243, IACS-010759, and [¹²⁵I]IACS-010759-PD1.

Figure 2.

Dose-response curves for the inhibition of electron transfer in complex I by IACS-010759. The inhibition of forward (closed circles) and reverse (open circles) electron transfer by IACS-010759 was examined. The forward electron transfer reaction (NADH oxidase activity (0.60 ± 0.05 μmol of NADH/min/mg of proteins)) was initiated by adding NADH (final concentration: 50 μm) after the incubation of SMPs with IACS-010759 for 4 min at 30 °C. The reverse electron transfer reaction (ubiquinol-NAD⁺ oxidoreduction (0.056 ± 0.004 μmol of NAD⁺/min/mg of proteins)) was initiated by adding ATP (1.0 mm) after the incubation of SMPs with IACS-010759 for 4 min at 30 °C. The mitochondrial protein concentration was set at 100 μg/ml for both assays. Values in graphs are means ± S.E. (error bars) (n = 3).

Figure 3.

Effects of IACS-010759 on alkynylation of 49 kDa-Asp¹⁶⁰ by AL1. Alkynylation of 49 kDa-Asp¹⁶⁰ in SMPs (2.0 mg of proteins/ml) was performed via LDT chemistry using AL1 (0.10 μm) in the presence of different concentrations of IACS-010759. The alkynylation can be visualized by conjugating a fluorescent tag, TAMRA-N₃, to Asp¹⁶⁰(COO)-(CH₂)₂-CCH via click chemistry after solubilizing SMPs. Bullatacin (10 μm) and BAY 87-2243 (30 μm) were used as references. Approximately 30 μg of proteins were loaded in each well. Top, gel image of SDS-PAGE analysis used for LDT chemistry; bottom, the extent of suppression by test compounds. Values in graphs are means ± S.E. (error bars) (n = 3).

Figure 4.

Effects of IACS-010759 on the specific binding of [¹²⁵I]AzQ to the 49-kDa subunit. The photoaffinity labeling of the 49-kDa subunit by [¹²⁵I]AzQ (5.0 nm) was carried out in SMPs (2.0 mg of proteins/ml) in the presence of different concentrations of IACS-010759 (up to 20 μm, 4000-fold excess). Bullatacin (5.0 μm), aminoquinazoline (5.0 μm), and BAY 87-2243 (20 μm) were used as references. Approximately 30 μg of proteins were loaded in each well. Top, gel image of SDS-PAGE analysis used for the photoaffinity labeling; bottom, the extent of suppression by test compounds. Values in graphs are means ± S.E. (error bars) (n = 3). n.s., not significant compared with control (one-way ANOVA followed by Dunnett's test).

Figure 5.

Effects of IACS-010759 on the specific binding of [¹²⁵I]S1QEL1.1-PD1 to the ND1 subunit. The photoaffinity labeling of the ND1 subunit by [¹²⁵I]S1QEL1.1-PD1 (5.0 nm) was carried out in SMPs (2.0 mg of proteins/ml) in the presence of different concentrations of IACS-010759 (up to 20 μm, 4000-fold excess), followed by the isolation of complex I by BN-PAGE and resolution of complex I subunits by SDS-PAGE. Bullatacin (5.0 μm) and BAY 87-2243 (20 μm) were used as references. Approximately 30 μg of proteins were loaded in each well. Top, gel image of SDS-PAGE analysis used for the photoaffinity labeling; bottom, the extent of suppression by test compounds. Values in graphs are means ± S.E. (error bars) (n = 3). n.s., not significant among the three (one-way ANOVA followed by Tukey's test).

Figure 6.

Photoaffinity labeling of complex I by [¹²⁵I]IACS-010759-PD1. A, SMPs (4.0 mg of proteins/ml) were cross-linked by [¹²⁵I]IACS-010759-PD1 (10 nm), followed by the purification of complex I by BN-PAGE and electroelution. Isolated complex I was resolved by doubled SDS-PAGE, and the SDS gel was subjected to silver staining, autoradiography, or Western blotting using anti-bovine ND1 antibody. All data are representative of three independent experiments. B, localization of the region labeled by [¹²⁵I]IACS-010759-PD1. The labeled ND1 subunit was digested by Lys-C or Asp-N. The digests were resolved on a 16% Schägger-type SDS gel (16% T, 6% C, containing 6.0 m urea), followed by autoradiography. Proteins equivalent to ∼50 μg of SMPs were loaded into each well. All data are representative of three independent experiments. C, schematic representation of the digestion of ND1 by Lys-C or Asp-N. The TMHs were assigned according to the structures of bovine (13) or ovine (33) complex I. Predicted cleavage sites are denoted by arrowheads and marked with the residue numbers in the mature sequences of the bovine ND1 subunit (SwissProt entry P03887).

Figure 7.

Effects of different inhibitors on the labeling of the ND1 subunit by [¹²⁵I]IACS-010759-PD1. The photoaffinity labeling of the ND1 subunit by [¹²⁵I]IACS-010759-PD1 (5.0 nm) was carried out in SMPs (2.0 mg of proteins/ml) in the presence of different inhibitors (5.0 μm each, 1000-fold), followed by the isolation of complex I by BN-PAGE and resolution of complex I subunits by SDS-PAGE. Top, gel image of SDS-PAGE analysis used for the photoaffinity labeling; bottom, the extent of suppression by test compounds. Values in graphs are means ± S.E. (error bars) (n = 3). *, p < 0.05; **, p < 0.001 compared with control (one-way ANOVA followed by Dunnett's test).

Figure 8.

The region labeled by [¹²⁵I]IACS-010759-PD1 in the ND1 subunit. A, [¹²⁵I]IACS-010759-PD1 labeled the region Asp¹⁹⁹–Lys²⁶² (brown spheres) in the ND1 subunit (brown). The third loop connecting the TMHs 5 and 6 (Asp¹⁹⁹–Ala²¹⁷) is presented in red spheres on the right. The 49-kDa and PSST subunits are shown in pink and purple, respectively. Here, we used the structural model of ovine complex I (Protein Data Bank entry 5LNK) (33) because the third loop of ND1 is disordered in the deactive state of the bovine enzyme (13). The quinone-access channel proposed in the current models was generated using MOLE with a 1.4-Å probe (RRID:SCR_018314) (61) and is shown in black. The hydrophilic domain above the dotted line shown in a was deleted in b for clarity. B, close-up of the ND1 subunit (brown). The positions of Leu⁵⁵ (a space-filling model in cyan) and the region labeled by [¹²⁵I]IACS-010759-PD1 (Asp¹⁹⁹–Lys²⁶² in red) are shown with the quinone-access channel (black).