| Pig liver esterase (PLE) which belongs to serine protease family is composed of a variety of isozymes. Pig liver esterase1(PLE1) is an important isoenzyme of the family because of higher levels. Because of the high activity and stereo selectivity, PLE becomes one of the most important enzymes in organic synthesis. However, as a drug metabolism enzyme, PLE has not been reported in drug metabolism and detoxification. According to the strong hydrolytic activity in organic chemical synthesis and powerful drug metabolism and detoxification of carboxylesterase in human and rodent animal, it is suggesting that PLE may play a role that cannot be ignored in drug metabolism of pigs and affect efficacy and toxicity of drugs. This study aims to research whether PLE1can hydrolyze clinical veterinary medicine, and the characteristics and mechanism of hydrolysis. It will provide the necessary theoretical basis for the rational use of drugs in pigs, which has important significance in theory and practice.This study was on the basis of functional expression of PLE1. First, the PLE1inhibition test in vitro was carried out to screen potential substrate from83clinical veterinary drugs. Second, inhibition constants and inhibition type of veterinary drugs were determined for further prediction of the hydrolysis of drugs by PLE1. High performance liquid chromatography (HPLC) and liquid chromatography tandem mass spectrometry (LC-MS/MS) instrument analysis methods for quantitative detection of hydrolysis by PLE1were established. Correlation analysis between inhibition constants and Michaelis constants was carried out to prove the reliability of the result. Last, according to the crystal structure of human carboxylesterase, homology model of PLE1was constructed. Docking was performed to elucidate the mechanism of PLE1catalytic hydrolysis of veterinary drugs.1. Drugs inhibited PLE1hydrolysis of p-NPAThe inhibition of83veterinary clinical drugs (chemical synthetic antibiotics, antibiotics, antifungals, anti parasite drugs, antipyretic drugs and some anesthesia drugs) on p-NPA hydrolyzed by PLE1was determined. The results showed that tylosin inhibition significance was at0.01<P<0.05, phthalylsulfathiazole and sulfisoxazole inhibition significance were at P <0.01, sulfachloropyridazine inhibition significance was at0.01<P <0.05. 2. Determination of PLE1catalytic hydrolysis on tylosin by HPLCA quantitative method for tylosin by HPLC was established to determine the concentration change of tylosin under the hydrolysis condition. The results showed that tylosin was not hydrolyzed by PLE1. Only by inhibition results can we not predict whether the veterinary drug was suitable as substrate of PLE1. Combining with determination of inhibition type was expected to provide further reference.3. Determination of inhibition type of sulfonamides on PLE1Inhibition constants (Ki values) of24sulfonamides on PLE1were determined and calculated. Different sulfonamides had different inhibition constants and Alpha value. All Alpha values of sulfonamides were more than1, suggesting that the inhibition was competitive inhibition. Namely,24sulfonamides and general substrates p-NPA combined with the same catalytic sites of PLE1suggesting that sulfonamides were likely to be hydrolyzed by PLE1.Distribution coefficients (XlogP values) of sulfonamides were obtained from PubChem Compound website. XlogP values showed a certain correlation with log Ki values. In a certain range (XlogP=0.1-1.5), with the XlogP values increasing, the log Ki values decreased first and then increased and when XlogP=1.0, the log Ki value reached the lowest value, namely the maximum inhibition, suggesting the relationship between the inhibition of sulfonamides on PLE1and the structure (XlogP value) of sulfonamides.4. Determination of PLE1hydrolysis of sulfonamides by LC-MS/MSA LC-MS/MS method for separation of sulfonamides, using0.1%formic acid and acetonitrile as the mobile phase with gradient elution was established. Full scan mode for the quantitative and qualitative analysis of sulfonamides was used. For most of sulfonamides, it showed good linear relationship in the range of1-32ppb (r≥0.999). Liquid chromatography and mass spectrum conditions were optimized to obtain liquid chromatography conditions and mass spectrum ion information. Michaelis constants (Km values) and Maximum reaction velocity (Vmax) of24sulfonamides on PLE1were determined. Concentrations of some sulfonamides had decreased significantly, suggesting obvious hydrolysis of sulfonamides by PLE1. Correlation analysis of log Km values and log Ki values indicated that positive correlation significance was at0.01<P<0.05, suggesting that the higher affinity of sulfonamides and PLE1, the more obvious inhibitory of them, and vice versa. 5. Homology modeling and molecular docking3D structures of trimer and monomer model of PLE1were constructed. Enzymes with different sulfonamides were subjected to molecular docking. The results showed that, combination of sulfonamides with active centers of liver carboxylesterase1was similar to p-NPA, the sulfur atom of sulfonamides might play the same role of carbonyl carbon atom in p-NPA, and the binding energy of sulfonamides which could inhibit PLE1was generally lower than the binding energy of substrate (p-NPA), that is, the affinity of sulfonamides was higher than the affinity of p-NPA, suggesting that sulfonamides could be hydrolyzed by PLE1. |
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