Computational and Experimental Insight into the Molecular Mechanism of Carboxamide Inhibitors of Succinate‐Ubquinone Oxidoreductase

Abstract

Succinate-ubiquinone oxidoreductase (SQR, EC 1.3.5.1), also known as mitochondrial respiratory complex II or succinate dehydrogenase (SDH), catalyzes the oxidation of succinate to fumarate as part of the tricarboxylic acid cycle. SQR has been identified as a novel target of a large family of agricultural fungicides. However, the detailed mechanism of action between the fungicides and SQR is still unclear, and the bioactive conformation of fungicides in the SQR binding pocket has not been identified. In this study, the kinetics of porcine SQR inhibition by ten commercial carboxamide fungicides were measured, and noncompetitive inhibition was observed with respect to succinate, DCIP, and cytochrome c, while competitive inhibition was observed with respect to ubiquinone. With the aim to uncover the binding conformation of these fungicides, molecular docking, molecular dynamics simulation, and molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) calculations were then performed. The excellent correlation (r(2) =0.94) between the calculated (ΔGcal ) and experimental (ΔGexp ) binding free energies indicates that the obtained docking conformation could be the bioactive conformation. The acid moiety of carboxamide fungicides inserts into the ubiquinone binding site (Q-site) of SQR, forming van der Waals (vdW) interactions with C_R46, C_S42, B_I218, and B_P169, while the amine moiety extends to the mouth of the Q-site, forming vdW interactions with C_W35, C_I43, and C_I30. The carbonyl oxygen atom of the carboxamide forms hydrogen bonds with B_W173 and D_Y91. These findings provide valuable information for the design of more potent and specific inhibitors of SQR.

Discovery of N-benzoxazol-5-yl-pyrazole-4-carboxamides as nanomolar SQR inhibitors

“…3B, 13b was a non-competitive inhibitor against SQR with respect to the substrate DCIP. These results indicate that 13b would bind to the Q-site of SQR, consistent with our previous study [11] that the carboxamide inhibitors bind to the Q-site of SQR. A similar result was obtained for commercial fungicide penthiopyrad.…”

“…The simulated binding mode 13b (Fig. 5A) showed that the carbonyl oxygen atom formed hydrogen bond with the residue of D_Y91 and B_W173 and pyrazole ring formed cation-p interaction with the residue of C_R46, which were nearly the same as the commercial carboxamide fungicides obtained in our previous work [11]. In addition, the hydrogen atom in eCHF 2 group also formed hydrogen bond with the residue of D_D90 and the phenyl ring formed hydrophobic interaction with D_Y91.…”

“…Complex II (SQR) firstly passes electrons from succinate to ubiquinone, and then complex III passes electrons from reduced ubiquinone to cytochrome c. The activity of SQR in SCR was selectively determined by using succinate and dichlorophenolindophenol (DCIP) as substrates, and the activity of only complex III in SCR was determined by using decylubiquinol (DBH 2 ) and cytochrome c as substrates, whereas the overall activity of SCR (both complex II and complex III) was determined using succinate and cytochrome c as substrates [11].…”

“…The preparation of DBH 2 from DB was carried out according to the procedure described in previous publications [11], and the concentration of DBH 2 was determined by measuring the absorbance difference between 288 and 320 nm using an extinction coefficient of 4.14 nM À1 cm À1 for the calculation [27,28].…”

“…Very recently, by integrating the molecular docking, molecular dynamics simulation and molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) calculations methods, we have successfully established the binding mode of ten commercial carboxamide fungicides for the first time [11]. The acid moiety of carboxamide fungicides inserts into the Q-site of SQR, forming vander-Waals (VDW) interactions with C_R46, C_S42, B_I218, and B_P169, while the amine moiety extending toward the mouth of the Q-site, forming VDW interactions with C_W35, C_I43, and C_I30.…”