Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Dalian Medical University, Dalian, China.
Department of Microbiology, College of Basic Medical Sciences, Dalian Medical University, Dalian, China.
Front Cell Infect Microbiol. 2019 Jul 17;9:251. doi: 10.3389/fcimb.2019.00251. eCollection 2019.
bifunctional enzyme GlmU is a novel target for anti-TB drugs and is involved in glycosyl donor UDP-N-acetylglucosamine biosynthesis. Here, we found that TPSA (2-[5-(2-{[4-(2-thienyl)-2-pyrimidinyl]sulfanyl}acetyl)-2-thienyl]acetic acid) was a novel inhibitor for GlmU acetyltransferase activity (IC: 5.3 μM). The interaction sites of GlmU and TPSA by molecular docking were confirmed by site-directed mutagenesis. TPSA showed an inhibitory effect on Mtb H37Ra growth and intracellular H37Ra in macrophage cells (MIC: 66.5 μM). To investigate why TPSA at a higher concentration (66.5 μM) was able to inhibit H37Ra growth, proteome and transcriptome of H37Ra treated with TPSA were analyzed. The expression of two methyltransferases MRA_0565 (Rv0558) and MRA_0567 (Rv0560c) were markedly increased. TPSA was pre-incubated with purified Rv0558 and Rv0560c in the presence of S-adenosylmethionine (methyl donor) respectively, resulting in its decreased inhibitory effect of GlmU on acetyltransferase activity. The inhibition of TPSA on growth of H37Ra with overexpressed Rv0558 and Rv0560c was reduced. These implied that methyltransferases could modify TPSA. The methylation of TPSA catalyzed by Rv0560c was subsequently confirmed by LC-MS. Therefore, TPSA as a GlmU acetyltransferase activity inhibitor may offer a structural basis for new anti-tuberculosis drugs. TPSA needs to be modified further by some groups to prevent its methylation by methyltransferases.
双功能酶 GlmU 是抗结核药物的新靶标，参与糖基供体 UDP-N-乙酰葡萄糖胺的生物合成。在这里，我们发现 TPSA（2-[5-(2-{[4-(2-噻吩基)-2-嘧啶基]硫基}乙酰基)-2-噻吩基]乙酸）是 GlmU 乙酰转移酶活性的新型抑制剂（IC：5.3 μM）。通过定点突变证实了 GlmU 和 TPSA 分子对接的相互作用位点。TPSA 对 Mtb H37Ra 的生长和巨噬细胞内的 H37Ra 具有抑制作用（MIC：66.5 μM）。为了研究为什么 TPSA 在较高浓度（66.5 μM）下能够抑制 H37Ra 的生长，我们分析了 TPSA 处理后的 H37Ra 的蛋白质组和转录组。两种甲基转移酶 MRA_0565（Rv0558）和 MRA_0567（Rv0560c）的表达明显增加。TPSA 分别与纯化的 Rv0558 和 Rv0560c 在 S-腺苷甲硫氨酸（甲基供体）存在下预孵育，导致其对 GlmU 乙酰转移酶活性的抑制作用降低。过表达 Rv0558 和 Rv0560c 的 H37Ra 的生长抑制作用降低。这表明甲基转移酶可以修饰 TPSA。随后通过 LC-MS 证实了 Rv0560c 催化的 TPSA 甲基化。因此，作为 GlmU 乙酰转移酶活性抑制剂的 TPSA 可能为新型抗结核药物提供结构基础。TPSA 需要进一步修饰以防止被甲基转移酶甲基化。
