NMR screening of potential inhibitors of methionine γ-lyase from Citrobacter freundii

© 2014, Pleiades Publishing, Inc. Methionine γ-lyase [EC 4.4.1.11] participates in methionine catabolism in a number of bacteria and protozoa eukaryotes, including pathogenic microorganisms. The lack of this enzyme in mammals allows us consider it to be a promising target for rational antibacterial drug design. Currently, in medical practice, there are no preparations based on the inhibition of methionine γ-lyase. We present the results of a search for potential inhibitors of this enzyme using NMR screening techniques based on the identification of compounds, which are able to bind specifically to their biological target. The study included a stage of in silico virtual screening of the library of commercially available compounds and subsequent experimental selection of the leading compounds capable to interact with the enzyme. The identification of binding was carried out using saturation transfer difference (STD) spectroscopy and the WaterLOGSY technique. During the final stage, an experimental assessment of the inhibition activity of the selected compounds in the reaction of the γ elimination of L-methionine catalyzed by methionine γ-lyase was performed. Binding constants of two leading compounds were determined using the WaterLOGSY method. This study expands the structural group of potential inhibitors of methionine γ-lyase and allows us to approach the design of its inhibitors with higher efficacy

ИСПОЛЬЗОВАНИЕ ПИРИДОКСИНА ДЛЯ ПОВЫШЕНИЯ ПРОТИВООПУХОЛЕВОЙ АКТИВНОСТИ МЕТИОНИН-ГАММАЛИАЗЫ НА МОДЕЛЯХ ПЕРЕВИВАЕМЫХ ОПУХОЛЕЙ МЫШЕЙ

We presented results of monotherapy and combination therapy of transplantable murine tumor models using methionine-gamma-lyase (MGL) and pyridoxine hydrochloride. We studied MGL from Clostridium sporogenes and Citrobacter freundii. We used Lewis lung carcinoma (LLC), melanoma B16, leukemias P388 and L1210 and Fisher lymphadenosis L5178y. Neither monotherapy with MGL nor combination of MGL and pyridoxine demonstrated antitumor activity against P388 and L5178y. In the murine L1210 leukemia model, MGL C. sporogenes injected intraperitoneally in the dose of 2000 U/kg, 11 times with a 12-hour interval increased the life span of mice (ILS=22 %, р=0.035). In the LLC model, the combination of MGL C. sporogenes at a dose of 400 U/kg, i.p., 4 times with a 48-hour interval and pyridoxine at a dose of 250 mg/kg led to tumor growth inhibition (TGI=55 %, р<0.001) on the first day after the completion of treatment. Monotherapy with MGL or pyridoxine in the same regimens resulted in a 24 % TGI (р=0.263) or 21 % TGI (р=0.410), respectively. In a pair-wise comparison of treatments, MGL + pyridoxine was more effective compared to MGL used alone (р=0.061) and MGL + pyridoxine was more effective then pyridoxine alone (р=0.031). MGL from C. freundii at a dose of 200 U/kg, 4 times with a 48-hour interval plus pyridoxine at a dose of 500 mg/kg injected on day 9 after the completion of treatment led to 50 % TGI, whereas MGL monotherapy at a single dose of 400 U/kg or pyridoxine monotherapy in the same regimen showed 5 % TGI (р=0.991) and 4 % TGI (р=0.998), respectively. The pair-wise comparison showed that MGL (200 U/kg) + pyridoxine was more effective than MGL (400 U/ kg) alone (р<0.001) and pyridoxine alone (р=0.003). In the B16 model, the combination of MGL injected i.p at a dose of 2000 U/kg and pyridoxine at a dose of 300 mg/kg showed 56 % TGI on day 1after the completion of treatment (р=0.045) and 35 % TGI on day 3 (р=0.038). Pyridoxine significantly increased the anticancer effect of MGL: MGL 1000 U/kg i.p and MGL 1000 U/kg i.p. + pyridoxine 300 mg/kg led to TGI=45 % (р=0.034) on day 3 after the completion of treatment. Single maximum tolerated dose after multiple i.p. administration was defined as 2000 U/kg, simultaneous administration of pyridoxine did not increase the toxicity of MGL. In conclusion, LLC and B16 are sensitive to MGL treatment, and pyridoxine may increase the efficacy of MGL.Приведены экспериментальные данные монотерапии и комбинированной терапии моделей перевиваемых опухолей мышей препаратами метионин-γ-лиазы (МГЛ) и пиридоксина гидрохлорида. Изучены МГЛ Clostridium sporogenes и Citrobacter freundii. Использованы перевиваемые модели опухолей мышей: карцинома легкого Льюис (LLC), меланома В16, лимфолейкоз P388, лимфолейкоз L1210, лимфаденоз Фишера L5178y. На моделях P388, L5178y МГЛ не показала противоопухолевой активности ни в монорежиме, ни в сочетании с пиридоксином. На модели L1210 было получено пограничное увеличение продолжительности жизни (УПЖ) 22 %, р=0,035 при применении МГЛ C. sporogenes в дозе 2000 Е/кг 11-кратно внутрибрюшинно с интервалом 12 ч. На LLC показано, что на 1-е сут после окончания лечения одновременное внутрибрюшное (в/б) введение МГЛ C. sporogenes 400 Е/кг 4-кратно с интервалом 48 ч и пиридоксина в дозе 250 мг/кг вызывало ТРО=55 % (р<0,001), монотерапия МГЛ или пиридоксином в аналогичных дозах и режимах применения вызывала ТРО=24 % (р=0,263) и 21 % (р=0,410) соотетственно. При попарном сравнении: комбинированная терапия МГЛ + пиридоксин против монотерапии МГЛ в аналогичном режиме р=0,061, против монотерапии пиридоксином р=0,031. На LLC МГЛ C. freundii 200 Е/кг 4-кратно с интервалом 48 ч и пиридоксина в дозе 500 мг/кг одновременно на 9-е сут после окончания лечения вызывало ТРО=50 % (р=0,001), при этом монотерапия МГЛ в разовой дозе 400 Е/кг или пиридоксином в аналогичном режиме применения вызывала ТРО=+5 % (р=0,991) и 4 % (р=0,998) соответственно. При попарном сравнении: комбинированная терапия МГЛ 200 Е/кг + пиридоксин против монотерапии МГЛ 400 Е/кг в аналогичном режиме р<0,001, против монотерапии пиридоксином р=0,003. На модели меланома B16 МГЛ 2000 в/б + пиридоксин 300 мг/кг вызывает ТРО 56 % на 1-е сут (р=0,045) и 35 % на 3-и сут (р=0,038). При комбинированной терапии МГЛ + пиридоксин последний значимо повышал противоопухолевую активность МГЛ в сочетаниях: МГЛ 1000 Е/кг в/б и МГЛ 1000 Е/кг в/б + пиридоксин 300 мг/кг ТРО=45 % (р=0,034) на 3-и сут после окончания лечения. При внутривенном введении МГЛ 500 Е/кг и МГЛ 500 Е/кг + пиридоксин последний повышал эффективность лечения: максимальное ТРО 50 % на 1-е сут после окончания лечения (р=0,085, различие не достоверно) и 21 % на 3-и сут после окончания лечения ТРО 22 % (р=0,965, различие не достоверно).Разовая максимальная переносимая доза при многократном в/б введении составила 2000 Е/кг, одновременное применение пиридоксина не усугубляло токсичности МГЛ. Таким образом, LLC и меланома В16 обладают чувствительностью к терапии МГЛ. Одновременное введение пиридоксина на модели LLC и В16 достоверно повышает её эффективность

Structure of methionine gamma lyase from Clostridium sporogenes

Methionine γ-lyase (MGL) is a pyridoxal 5′-phosphate-dependent enzyme that catalyzes the γ-elimination reaction of l-methionine. The enzyme is a promising target for therapeutic intervention in some anaerobic pathogens and has attracted interest as a potential cancer treatment. The crystal structure of MGL from Clostridium sporogenes has been determined at 2.37 Å resolution. The fold of the protein is similar to those of homologous enzymes from Citrobacter freundii, Entamoeba histolytica, Pseudomonas putida and Trichomonas vaginalis. A comparison of these structures revealed differences in the conformation of two flexible regions of the N- and C-terminal domains involved in the active-site architecture

High-resolution structure of methionine gamma-lyase from Citrobacter freundii

Pyridoxal 5'-phosphate-dependent methionine gamma-lyase (MGL) is involved in the metabolism of sulfur-containing amino acids. The enzyme is a promising target in some anaerobic pathogens and is effective in cancer-cell treatment. The structure of the MGL holoenzyme from Citrobacter freundii has previously been determined at 1.9 A resolution. By modification of the crystallization procedure, the previously determined structure of C. freundii MGL has been improved to 1.35 A resolution with R and R(free) values of 0.152 and 0.177, respectively. This high-resolution structure makes it possible to analyze the interactions between the monomers in detail and to reveal the structurally invariant regions that are responsible for monomer-monomer recognition during the formation of the active enzyme. Details of the mode of cofactor binding and of the flexible regions that may be involved in substrate recognition and binding are also described

NMR screening of potential inhibitors of methionine γ-lyase from Citrobacter freundii

© 2014, Pleiades Publishing, Inc. Methionine γ-lyase [EC 4.4.1.11] participates in methionine catabolism in a number of bacteria and protozoa eukaryotes, including pathogenic microorganisms. The lack of this enzyme in mammals allows us consider it to be a promising target for rational antibacterial drug design. Currently, in medical practice, there are no preparations based on the inhibition of methionine γ-lyase. We present the results of a search for potential inhibitors of this enzyme using NMR screening techniques based on the identification of compounds, which are able to bind specifically to their biological target. The study included a stage of in silico virtual screening of the library of commercially available compounds and subsequent experimental selection of the leading compounds capable to interact with the enzyme. The identification of binding was carried out using saturation transfer difference (STD) spectroscopy and the WaterLOGSY technique. During the final stage, an experimental assessment of the inhibition activity of the selected compounds in the reaction of the γ elimination of L-methionine catalyzed by methionine γ-lyase was performed. Binding constants of two leading compounds were determined using the WaterLOGSY method. This study expands the structural group of potential inhibitors of methionine γ-lyase and allows us to approach the design of its inhibitors with higher efficacy

NMR screening of potential inhibitors of methionine γ-lyase from Citrobacter freundii

© 2014, Pleiades Publishing, Inc. Methionine γ-lyase [EC 4.4.1.11] participates in methionine catabolism in a number of bacteria and protozoa eukaryotes, including pathogenic microorganisms. The lack of this enzyme in mammals allows us consider it to be a promising target for rational antibacterial drug design. Currently, in medical practice, there are no preparations based on the inhibition of methionine γ-lyase. We present the results of a search for potential inhibitors of this enzyme using NMR screening techniques based on the identification of compounds, which are able to bind specifically to their biological target. The study included a stage of in silico virtual screening of the library of commercially available compounds and subsequent experimental selection of the leading compounds capable to interact with the enzyme. The identification of binding was carried out using saturation transfer difference (STD) spectroscopy and the WaterLOGSY technique. During the final stage, an experimental assessment of the inhibition activity of the selected compounds in the reaction of the γ elimination of L-methionine catalyzed by methionine γ-lyase was performed. Binding constants of two leading compounds were determined using the WaterLOGSY method. This study expands the structural group of potential inhibitors of methionine γ-lyase and allows us to approach the design of its inhibitors with higher efficacy

High-resolution structure of methionine gamma-lyase from Citrobacter freundii

Pyridoxal 5'-phosphate-dependent methionine gamma-lyase (MGL) is involved in the metabolism of sulfur-containing amino acids. The enzyme is a promising target in some anaerobic pathogens and is effective in cancer-cell treatment. The structure of the MGL holoenzyme from Citrobacter freundii has previously been determined at 1.9 A resolution. By modification of the crystallization procedure, the previously determined structure of C. freundii MGL has been improved to 1.35 A resolution with R and R(free) values of 0.152 and 0.177, respectively. This high-resolution structure makes it possible to analyze the interactions between the monomers in detail and to reveal the structurally invariant regions that are responsible for monomer-monomer recognition during the formation of the active enzyme. Details of the mode of cofactor binding and of the flexible regions that may be involved in substrate recognition and binding are also described