Irreversible inhibition of the mycobacterium tuberculosis β-lactarnase by clavulanate
Irreversible inhibition of the mycobacterium tuberculosis β-lactarnase by clavulanate
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DOI：
10.1021/bi701506h
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发表时间：
2007-10-30
期刊：
影响因子：
2.9
通讯作者：
Blanchard, John S.
中科院分区：
文献类型：
--
作者：
Hugonnet, Jean-Emmanuel;Blanchard, John S.
β-内酰胺类抗生素可以抑制参与细胞壁生物合成的细菌D，D-转肽酶，但由于结核分枝杆菌对β-内酰胺类抗生素具有耐药性，因此从未被系统地用于治疗结核分枝杆菌感染。关键的耐药因素是结核分枝杆菌染色体编码的Ambler A类β-内酰胺酶BLAC的结构性产物。我们发现BLAC是一种超广谱β-内酰胺酶(ESBL)，具有高水平的青霉素酶和头孢菌素酶活性，对碳青霉烯类抗生素，包括亚胺培南和美罗培南也有一定的活性。我们用FDA批准的三种β-内酰胺酶抑制剂：舒巴坦、他唑巴坦和克拉维酸来表征该酶的抑制作用。舒巴坦对硝基呋喃具有竞争性和可逆性的抑制作用。他唑巴坦以一种时间依赖的方式抑制该酶，但由于共价酰化酶的缓慢水解，该酶的活性重新出现。相比之下，克拉维酸能迅速与酶反应，形成稳定的、无活性的水解型酶，通过质谱仪对其进行了表征。克拉维酸有可能与批准的β-内酰胺类抗生素联合使用来治疗多重耐药(MDR)和极端耐药(XDR)结核分枝杆菌菌株。
Members of the P-lactam class of antibiotics, which inhibit the bacterial D,D-transpeptidases involved in cell wall biosynthesis, have never been used systematically in the treatment of Mycobacterium tuberculosis infections because of this organism's resistance to beta-lactams. The critical resistance factor is the constitutive production of a chromosomally encoded, Ambler class A P-lactamase, BlaC in M. tuberculosis. We show that BlaC is an extended spectrum P-lactamase (ESBL) with high levels of penicillinase and cephalosporinase activity as well as measurable activity with carbapenems, including imipenem and meropenem. We have characterized the enzyme's inhibition by three FDA-approved beta-lactamase inhibitors: sulbactam, tazobactam, and clavulanate. Sulbactarn inhibits the enzyme competitively and reversibly with respect to nitrocefin. Tazobactam inhibits the enzyme in a time-dependent manner, but the activity of the enzyme reappears due to the slow hydrolysis of the covalently acylated enzyme. In contrast, clavulanate reacts with the enzyme quickly to form hydrolytically stable, inactive forms of the enzyme that have been characterized by mass spectrometry. Clavulanate has potential to be used in combination with approved P-lactam antibiotics to treat multi-drug resistant (MDR) and extremely drug resistant (XDR) strains of M. tuberculosis.