vestichИзвестия Национальной академии наук Беларуси. Серия химических наукProceedings of the National Academy of Sciences of Belarus, Chemical Series1561-83312524-2342The Republican Unitary Enterprise Publishing House "Belaruskaya Navuka"10.29235/1561-8331-2021-57-4-438-455vestich-688Research ArticleБИООРГАНИЧЕСКАЯ ХИМИЯBIOORGANIC CHEMISTRYПолучение и характеристика рекомбинантной глутатионтрансферазы Р1 человека и скрининг новых ингибиторов ферментаPreparation and characterization of recombinant human glutathione transferase P1 and screening of novel enzyme inhibitorsГилевичС. Н.GilevichS. N.

Гилевич Сергей Нилович – канд. хим. наук, вед. науч. сотрудник

ул. акад. Купревича, 5/2, 220141, Минск, Республика Беларусь

Syargey N. Gilevich – Ph. D. (Сhemistry), Leading Researcher

5/2, acad. Kuprevich str., 220141, Minsk, Republic of Belarus

gilevich@iboch.byБречкоЮ. В.BrechkaYu. V.

Бречко Юлия Владимировна – мл. науч. сотрудник

ул. акад. Купревича, 5/2, 220141, Минск, Республика Беларусь

Yuliya V. Brechka – Junior Researcher

5/2, acad. Kuprevich str., 220141, Minsk, Republic of Belarus

julia-brechko@yandex.ruИнститут биоорганической химии Национальной академии наук БеларусиInstitute of Bioorganic Chemistry of the National Academy of Sciences of Belarus202103122021574438455Copyright © Гилевич С.Н., Бречко Ю.В., 20212021Гилевич С.Н., Бречко Ю.В.Gilevich S.N., Brechka Y.V.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://vestichem.belnauka.by/jour/article/view/688

Глутатионтрансфераза Р1 человека (GSTP1) играет важную роль во второй фазе биотрансформации ксенобиотиков и в регуляции апоптотических сигнальных путей. Направленный скрининг новых ингибиторов фермента является актуальной задачей, так как избирательное подавление активности GSTP1 в опухолевых клетках может существенно повысить их чувствительность к химиотерапии. Известные методы получения рекомбинантной GSTP1 с гексагистидиновым тэгом в структуре отличаются сложностью, трудоемкостью и значительными потерями активности фермента. С целью создать простую и эффективную систему бактериальной экспрессии бестэговой GSTP1 с нативной структурой и высокой активностью, в настоящей работе проведено клонирование полноразмерного гена gstp1 в плазмидный вектор pTXB1 с последующей трансформацией клеток E. coli. Оптимизированный уровень экспрессии составил 30–32 мг фермента/л среды. С использованием глутатионсодержащих аффинных мембран из бактериального лизата выделен очищенный фермент (выход 75,7 %, удельная активность 102,6 Ед/мг белка). Гомогенность препарата подтверждена данными гель-электрофореза и масс-спектрометрии. Физико-химические и каталитические свойства рекомбинантной GSTP1 практически совпали с таковыми для нативного фермента из эритроцитов. По результатам скрининга in silico и in vitro выявлены структурные факторы и взаимодействия, определяющие эффективность ингибирования фермента карбо- и N-гетероциклическими лигандами. Установлена предпочтительная ориентация «хороших» ингибиторов в Н-сайте GSTP1. Обнаружены новые ингибиторы фермента: 1,10-фенантролин-5,6-дион, ализариновый красный С и индигокармин с величиной IC50 соответственно 31, 16 и 2,3 мкМ. Найденные соединения представляют интерес для создания новых лидерных структур с потенциальной противоопухолевой активностью.

Human glutathione transferase P1 (GSTP1) plays an important role in the second phase of xenobiotic biotransformation and in the regulation of apoptotic signal pathways. Directed screening of new enzyme inhibitors is an actual task since selective suppression of GSTP1 activity in tumor cells may substantially increase their sensitivity to chemotherapy. Known methods to obtain recombinant GSTP1 with a hexahistidine tag in the structure are complex, laborious, and suffer from significant losses of the enzyme activity. With the aim to create a simple and effective bacterial expression system for tagless GSTP1 posessing native structure and high activity, in the present work the full-length gstp1 gene was cloned into the pTXB1 plasmid vector, followed by transformation of E. coli cells. The optimized expression level amounted to 30–32 mg of the enzyme per liter of broth. Using glutathione-containing affinity membranes, the purified enzyme was isolated from bacterial lysate with the yield of 75.7 % and specific activity of 102.6 U/mg protein. The enzyme homogeneity was confirmed by gel-electrophoretic and mass-spectrometric data. Physico-chemical and catalytic properties of recombinant GSTP1 practically coincided with those of the native erythrocytary enzyme. The results of in silico and in vitro screening allowed to reveal structural factors and interactions determining the efficiency of the enzyme inhibition by carbocyclic and N-heterocyclic ligands. The preferable orientation of “good” inhibitors in the GSTP1 H-site was also established. Three strong enzyme inhibitors were found: 1,10-phenanthroline-5,6-dione, Alizarin Red S, and indigo carmine, with their respective IC50 values of 31, 16 and 2.3 μM. The new inhibitors are of certain interest for the development of novel lead structures with potential antitumor activity.

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