Studies On Discovery And Kinetics Inhibition Of Complex Ⅱ And Complex Ⅲ Inhibitors

As the important "energy factory" of eukaryotic cells, mitochondrion is the place for cellular respiration. The mitochondrial respiration chain (OXPHOS electron transfer chain) is located on the inner membrane of mitochondrion and comprising of four large trans-membrane protein complexes (mitochondrial respiratory Complex Ⅰ, Ⅱ, Ⅲ and Ⅳ) as well as ubiquinone between Complex Ⅰ/Ⅱ and III and cytochrome c between Complex Ⅲ and Ⅳ. The function of mitochondrial respiration chain is biological oxidation which is coupled with phosphorylation by ATP synthesis enzyme (ATPase, called Complex V) and then complete OXPHOS producing energy molecule ATP.Succinate-ubiquinone reductase (SCR) composed by complex Ⅱ and III. Complex Ⅱ (SQR, EC1.3.5.1) catalyzes electron transfer from succinate to Q, and complex III (also known as cytochrome bcl complex, EC1.10.2.2) is a key component of the respiratory chain, in which it catalyzes electron transfer from ubiquinol (QH2) to cytochrome c (cyt c) while translocating protons across the inner mitochondrial membrane. Both complex Ⅱ and III can used to target for fungicides.First, we established the extraction and purification of succinate-ubiquinone reductase, and evaluated its purity and activity. We want to research the structural biology of SCR, obtain some of crystals, but diffraction not good and need to further optimization.Second, we developed a UV assay for SCR, SQR and QCR enzyme assay for highthroughput screening and a new enzyme assay for SCR through isothermal titration calorimetry.Third, we studied the inhibitor kinetics of complex Ⅱ inhibitors.1、Determined the activity and mode of action for ten commercial inhibitors of complex Ⅱ. We found they are non-competitive inhibitors with respect to the substrate succinate and DCIP, but competitive inhibitors with respect to substrate Q. that indicated the bind site of these inhibitors different with succinate and DCIP, but near to Q.2、We have biologically evaluated to new compounds which synthesized by our group. Partial compounds can inhibit the activity of complex Ⅱ nearly with commercial inhibitors, but the complex Ⅱ activity can not disappear by these, about30%-40%activity were remained. So we proposed and confirmed a partial noncompetitive inhibition mechanism. The Ki value of best compound was0.4464±0.0624μM.Fourth, we studied the inhibitor kinetics of complex III inhibitors.1、 Determined in detail for six commercial inhibitors of complex Ⅲ.2、 New oxazolidinedione derivatives were biologically evaluated. The further inhibitory kinetics studies against SCR revealed that the representative compound8d and famoxadone are both non-competitive inhibitors with respect to the cytochrome c, but competitive inhibitors with respect to DBH2. In addition, compound8d and famoxadone showed, respectively,35-fold and15-fold greater inhibitory activity against the porcine SCR than complex Ⅲ, indicating that these two inhibitors not only inhibited the activity of complex Ⅲ, but possibly affect the interaction between the complex Ⅱ and the complex Ⅲ.3、 Based on the method of pharmacophore-linked fragment virtual screening, we found the strobilurin derivatives which containing functional groups benzophenone and quinoline oxime morpholine were worth further research.1) ⅢIb (Ki=4.28nM) was best and partial compounds have antimalarial activity. Furthermore, if we connect two benzene ring, get Ⅲ4a (Ki=1.8987±0.0432nM) which activity will be better and show slow-binding.2) we found lots of high-activity compounds in new quinoline oxime morpholine strobilurin derivatives such as9f (Ki=0.04300nM). These high-activity compounds were slow-binding inhibitors and noncompetitive with cytochrome c but competitive with DBH2. Additionally, we determined the crystal structure of compound9e bound to the chicken complex III at2.70A resolution, providing a molecular basis for understanding its ultrapotency and confirm our results about kinetics were correct.4、We found strobilurin inhibitors have similar structure but different kinetic feature. According to the dissociation constant, we proposed a new classification which classical (Ko>0.01s-1) and non-classical (Ko≤0.01s-1) inhibitors of strobilurin. Furthermore, we proposed in the inhibition experiment,[I]> IO[E]o traditional formula can be used, but [I] comparable with [E]o, we need to use no simplification formula if we want to obtain more accurate data.In summary, this paper established the systems of extraction, purification, enzyme assay and purity evaluation of succinate-ubiquinone reductase. Furthermore, we studied the inhibitor kinetics and designed the inhibitors to target to complex Ⅱ and complex Ⅲ, elaborated in detail the partial noncompetitive and time-dependent (slow-binding) inhibition mechanism, and proposed a new classification. All these results would shed light on the research of inhibitor with high activity and new structure.