Discovery of Bis-sulfonamides as Novel Inhibitors of Mitochondrial NADH-Quinone Oxidoreductase (Complex I)

Abstract

Mitochondrial oxidative phosphorylation (OXPHOS) is an essential cellular metabolic process that generates ATP. The enzymes involved in OXPHOS are considered to be promising druggable targets. Through screening of an in-house synthetic library with bovine heart submitochondrial particles, we identified a unique symmetric bis-sulfonamide, KPYC01112 (1) as an inhibitor targeting NADH-quinone oxidoreductase (complex I). Structural modifications of KPYC01112 (1) led to the discovery of the more potent inhibitors 32 and 35 possessing long alkyl chains (IC₅₀ = 0.017 and 0.014 μM, respectively). A photoaffinity labeling experiment using a newly synthesized photoreactive bis-sulfonamide ([¹²⁵I]-43) revealed that it binds to the 49-kDa, PSST, and ND1 subunits which make up the quinone-accessing cavity of complex I.

Figure 1

Representative inhibitors of the NADH-quinone oxidoreductase (complex I).

Figure 2

Structure of KPYC01112 (1) and the structure optimization strategy.

Scheme 1. Synthesis of the KPYC01112 Derivatives

Scheme 2. Synthesis of the ¹²⁵I-Labeled Probe [¹²⁵I]-43

Figure 3

Photoaffinity labeling of complex I by [¹²⁵I]-43. (A) SMPs (4.0 mg of protein/mL) were labeled by [¹²⁵I]-43 (10 nM), followed by the purification of complex I by BN-PAGE and electroelution. The isolated complex I was resolved by doubled SDS-PAGE, and the SDS gel was subjected to silver staining or autoradiography. (B) The subunits labeled by [¹²⁵I]-43 in bovine complex I. The 49-kDa (pink), ND1 (orange), and PSST (blue) subunits in complex I (PDB ID: 5O31) are shown. The quinone-accessing cavity is formed at the interface of the 49-kDa, ND1, and PSST subunits as indicated by a black sphere generated using MOLE with a 1.4-Å probe (https://mol.upol.cz).

Figure 4

Effects of various inhibitors (i.e., competitors) on the specific binding of [¹²⁵I]-43 to the 49-kDa subunit.