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Enterococcus faeciumを用いた細菌由来セリンヒドロキシメチルトランスフェラーゼ(SHMT)阻害剤の同定とSHMTの抗菌薬標的タンパク質としての有用性評価

牧野 祐子 東北大学

2022.03.25

概要

【研究背景】薬剤耐性 (antimicrobial resistance;AMR) の出現と新薬開発が繰り返されてきたことにより、抗菌薬の治療標的となるタンパク質の種類が減少しており、さらに新しい標的タンパク質の発見も困難になっている。本研究では、新たな抗菌薬の標的タンパク質同定を目的とした。One-carbon metabolism(1C代謝)は、原核および真核生物の細胞増殖に重要な代謝経路であり、葉酸サイクルを介して核酸合成とアミノ酸合成に関与している。セリンヒドロキシメチルトランスフェラーゼ(serine hydroxymethyl transferase;SHMT)は、テトラヒドロ葉酸(tetrahydrofolate;THF)とセリン(serine;Ser)を基質とし、5, 10-メチレン THF(5, 10-methylene THF;CH2-THF)とグリシン(glycine;Gly)を生成する反応を触媒する。近年、SHMT 阻害剤が抗がん活性や抗マラリア活性を示すことが報告されており、腫瘍や原虫分野などにおいて、SHMT が薬剤標的として注目されている。一方で抗菌薬標的としての SHMT の有用性は明らかではない。

【研究目的】細菌、ウイルス、ヒト、原虫に共通する 1C 代謝経路に関与する代謝酵素である SHMT に着目し、新しい抗菌薬の標的となるタンパク質の同定のため、抗菌薬標的として SMHT の有用性を明らかにし、細菌特異的な SHMT 阻害剤の開発および耐性の出現を抑制することのできる抗菌薬の開発に寄与することを目的とする。

【研究方法】抗菌標的としての SHMT の意義を評価するため、びまん性大細胞型 B細胞リンパ腫の治療薬として報告されている SHMT 阻害剤 1(SHMT inhibitor 1;SHIN-1)誘導体および既存の薬剤を用いて、Enterococcus faecalis、Enterococcus faecium、Staphylococcus aureus、Eschelia coli に対する抗菌活性を評価した。serine、deoxythymidine などの生理的基質を用いて、競合試験を行った。示差走査蛍光定量法および、(+)-SHIN-1 と SHMT 複合体の結晶構造解析を行い 、SHMT と阻害剤の結合に重要な相互作用の特定を行った。抗菌薬標的として SHMT の有用性を確認するため、SHMT 阻害剤と異なる作用を持つ葉酸誘導体(MTX)、ヌクレオシド誘導体(5-FdU、5-FUrd)を用いて、(+)-SHIN-1 との相乗効果を評価した。ヒト細胞への細胞毒性について評価を行った。SHMT 阻害剤および葉酸誘導体に対する耐性を誘導し、変異株を樹立し、薬剤耐性機序と交差耐性を解明した。

【研究結果】(+)-SHIN-1 は、ヒト細胞への毒性を示さない濃度で E. faecium に対して高い抗菌活性を示し、5-FdU および 5-FUrd、MTX との相乗効果を示した。E.faecium SHMT(efmSHMT)/ (+)-SHIN-1 複合体の結晶構造は、(i) (+)-SHIN-1 は SHMT に結合、すなわち、(+)-SHIN-1 が E. faecium 中で SHMT 阻害剤として働く(ii) (+)-SHIN-1 のピラゾロピラン骨格がアミノ酸 (amino acid;a.a.) の主鎖と複数の水素結合を形成する(iii) (+)-SHIN-1 のビフェニル部分およびヒドロキシ基の結合が、115-および 343-ループの固定に重要であり、efmSHMT と(+)-SHIN-1 の間の相互作用の安定化に寄与することを示した。

【結論】本研究では、新規抗菌標的として SHMT の有用性を示した。

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