Kinetics Inhibition And Quantitative Structure-Activity Relationship (QSAR) Studies Of Phenoloxidase Inhibitors On Pieris Rapae (L.)

Phenoloxidase (PO) (EC.1.14.18.1) also known as tyrosinase, is a structure-complex and multifunctional copper-containing enzyme, which widely distributed among animals, plants, fungi, and prokaryotes. PO is thought to be involved in many biological processes of invertebrates, such as consenescence of human body, wound healing, fruits and vegetables browning and pigment formation. It is one of the key enzymes in the development process of insects, the enzyme possesses an important function in metamorphism developing and immunity system. Currently, many studies focused on this field in order to screen, design and synthesis PO inhibitors for the importance theory of PO inhibitors and its bright future. In the present paper, compounds of 5, 7, 4'-trihydroxyflavone etc. were selected to investigate the effect of natural occurring compounds on the growth of Pieris rapae larvae, compounds cupferron etc. were selected as the PO inhibitors to determine the inhibitory effects against the enzyme, besides, compounds 3-hydroxy-4-methoxy benzaldehyde thiosemicarbazone (3-H-4-MBT) etc. were designed and synthesized in our laboratory, and the kinetics inhibition of PO were studied using these compounds.Additionally, the bioactivity results were used to construct three-dimensional quantitative structure-activity relationship (3D-QSAR) models using two molecular field analysis techniques: comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA), robust and predictive 3D-QSAR models were obtained from CoMFA and CoMSIA. Furthermore, the molecular interactions between the ligands and the target were studied using a flexible docking method (FlexX) and the best scored candidates were docked flexibly.The contents and results were summarized as follows: 1. Compounds of 5, 7, 4'-trihydroxyflavone, quercetin, rutin, 5-methoxysalicylic acid and kojic acid were selected, in the present paper, to investigate the effect of natural occurring compounds on the growth of Pieris rapae larvae. Both dipping methods and ingestion methods were used in the bioassay, and the results showed that the LC50 value of the chemicals against the tested larva were estimated to be 0.226 and 0.951 g/L at 72 hours after treatment with dipping methods for 5, 7, 4'-trihydroxyflavone and quercetin, respectively, or 0.062 and 2.420 gl/L with ingestion methods, respectively. The results also indicted that all of the tested larva couldn't pupated when the concentration of 5, 7, 4'-trihydroxyflavone beyond 0.200 g/L with ingestion methods, and, the increased amount of body weight of the 5th instar Pieris rapae larva, which were dipped in the solution of 5, 7, 4'-trihydroxyflavone, quercetin, rutin, 5-methoxysalicylic acid or kojic acid, were decreased obviously compared with the control.2. The results of PO purification showed that much of the enzyme activity was in the deposition of 35% saturated (NH4)2SO4, and the enzyme was purified 3.08-fold with a recovery of 69.52%. And then, PO was purified 6.22-fold with a recovery of 42.50% when the enzyme was chromatographed on Sephadex G-100 gel filtration. The properties of PO were determined, in the present paper, the results indicated that the optimum pH was 7.0 and the enzyme with a stable activity when the pH reaction system between 6.5~7.4. The optimum temperature was 42℃, and the enzyme with a stable activity when the temperature reaction system less than 32℃. Effects of some metal ions on the PO activity were studied, the results showed that Na+ and K+ had no effects on the enzyme activity. Meanwhile, the results showed that the PO activity was enhanced by Cu2+ when the concentration at 0～0.100 mmol/L, but the activity was inhibited by the same ion when the concentration went over to 0.125 mmol/L, and the IC50 was estimated to be 0.651 mmol/L.3. The inhibitory effects on the PO activity by 4-hexylresorcino, 4-n-dodecylresorcino, cupferron, 5, 7, 4'-trihydroxyflavone and quercetin were determined, and the possible mechanism of these inhibitors were discussed also. The results showed that 4-hexylresorcino and 4-n-dodecylresorcino were reversible competitive inhibitors on PO, and the IC50 were 1.50μmol/L and 1.12μmol/L, respectively, the inhibitory constants (KI) were also determined to be 0.50μmol/L and 0.47μmol/L, respectively. Cupferron was a reversible competitive inhibitor on the enzyme, the IC50 and the inhibitory constant (KI) was 0.10 mmol/L and 0.076 mmol/L, respectively. The results also indicated that 5, 7, 4'-trihydroxyflavone and quercetin could also inhibit the PO activity, and the IC50 were estimated to be 25.65 and 43.94 mg/L, respectively.4. In the present study, the kinetic assay in air-saturated solutions and the kinetic behavior of PO from P. rapae larvae in the oxidation of L-tyrosine (a monophenol) and L-DOPA (L-3, 4-dihydroxyphenylalanine) (a diphenol) was studied. The inhibitory effects of 3-hydroxy-4-methoxybenzaldehyde thiosemicarbazone (3-H-4-MBT) and 2-hydroxybenzaldehyde (2-HBD) on the monophenolase and diphenolase activities of PO were also studied in the present paper. The results showed that 3-H-4-MBT and 2-HBD can inhibit both the monophenolase and diphenolase activities of PO. The lag period of L-tyrosine oxidation catalyzed by the enzyme was obviously lengthened and the steady-state activities of the enzyme sharply decreased in the reaction course. Inhibitor 3-H-4-MBT was found to be noncompetitively reversible compound with a KI (KI=KIS) of 0.30μmol/L and an estimated IC50 of 0.14±0.02μmol/L for monophenolase or 0.26±0.04μmol/L for diphenolase. Inhibitor 2-HBD was found to be noncompetitively reversible with a KI (KI=KIS) of 1.21 mmol/L and an estimated IC50 of 8.08±0.11μmol/L for monophenolase or 4.14±0.08μmol/L for diphenolase. In the time course of the oxidation of L-DOPA catalyzed by the enzyme in the presence of different concentrations of 3-H-4-MBT or 2-HBD, the rate decreased with increasing time until a straight line was approached. The microscopic rate constants k-0 and k+0 for the reaction of 3-H-4-MBT or 2-HBD with the enzyme were determined.5. Quantitative structure-activity relationship (QSAR) studies are important approaches in the design of pesticidal molecules. The method of Hansch-Fujita of 2D-QSAR, comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were introduced in detail in this paper, and the QSAR of PO inhibitors were carried out based on three methods above.In the present paper, the quantitative relationship between the structure of benzaldehyde derivatives, benzoic acid derivatives, benzaldehyde thiosemicarbazone derivatives and their inhibitory activities against P. rapae larvae PO were analyzed using Hansch-Fujita approach. The chemical descriptors, such as electronic parameters Hammettσ, hydrophobic parameters clogP, steric parameters MR and hydrogen bond acceptor were employed in this study. The results showed that the structure-activity relationships (SAR) of benzoic acid derivatives and benzaldehyde thiosemicarbazone derivatives were identical, and they may act on the same target site of the PO receptor. The hydrogen bond acceptor and steric parameters descriptors were the most significant factors on determining inhibitory activity of the two sets of compounds. The structure-activity relationships of benzaldehyde derivatives and benzaldehyde thiosemicarbazone derivatives indicated that the action mode on the PO receptor were different, or the action site of the two sets of compounds were different.The bioactivity results were used to construct 3D-QSAR models using two molecular field analysis techniques: CoMFA and CoMSIA. After carrying out superimposition using common substructure-based alignment, robust and predictive 3D-QSAR models were obtained from CoMFA (q2 = 0.926, r2 = 0.986, SEE = 0.250) and CoMSIA (q2 = 0.933, r2 = 0.984, F = 381.764, SEE = 0.271) with 6 optimum components. And, the steric field, hydrophobic field and hydrogen bond acceptor field were applied in the CoMSIA modle. The 3D-QSAR model built here will provide hints for the designing with novel phenoloxidase inhibitors.6. In the present study, the inhibitors were used as the ligands and the PO crystal structure (PDB: 1WX2) of S. castaneoglobisporus was employed as the target, and, the molecular interactions between the ligands and the target were studied using a flexible docking method (FlexX). The results showed that the best scored candidates were docked flexibly, and there are three interaction modes among the three compounds.The first interaction mode is the benzaldehyde thiosemicarbazone analogues interacted with the PO active site. The benzaldehyde thiosemicarbazone analogues contains a chain of atoms (H21 - N9 - C10 - N12 - H22) spatially arranged in what might be termed a"clamp"structure. The distance between both hydrogen atoms of the clamp and the carbonyl oxygen atom of Tyr98 is 1.991 ?, indicating the likely presence of a pair of hydrogen bonds. Formation of these two hydrogen bonds stabilizes the position of Tyr98, preventing Tyr98 from participating in the interaction between PO and ORF378. The second interaction mode is benzaldehyde analogues interacted with the PO active site, which is more like the first interaction mode, the hydrogen bonds were formed also. The third interaction mode is the benzoic acid derivatives interacted with the active site. Some hydrogen bonds were formed between the hydroxyl of carboxyl and oxygen of Tyr98, between the contraposition group of carboxyl and amino acid Trp184, Arg185 and Pro102.