Anti-tuberculosis drug screening of alanine racemase from Mycobacterium tuberculosis and its co-crystallized with D-cycloserine

Abstract:

[Objective] The aim of this work was to investigate the inhibition mechanism of D-cycloserine (DCS) on alanine racemase from Mycobacterium tuberculosis (AlrMtb), and build up a high-throughput screening assay to screen for new inhibitors of AlrMtb. [Methods] The AlrMtb gene was cloned into pET28a vector and over-expressed in soluble form in E. coli strain BL21(DE3). The protein was purified using Ni2+-chelating chromatography followed by anion exchange chromatography. The AlrMtb protein was co-crystallized with DCS to make clear of the inhibitory mechanism. In addition, a high-throughput screening assay for new inhibitors was set up in order to obtain new anti-tuberculosis drugs that inhibited AlrMtb activity. About 384 small-molecule fragments, 792 chemicals and 2 200 components of traditional Chinese Medicine were tested in this assay where DCS was used as the positive reference. [Results] The protein crystals of AlrMtb-DCS complex diffract to 2.5 ?. The space group of the crystals is P41212 with cell parameters a=b=163.92 ?, c=57.44 ?. In the AlrMtb-DCS structure model, DCS was shown to react with PLP and formed a new molecule PMP. This process disrupted previous interaction between the C4? atom of PLP and Lysine 42 in AlrMtb and thereby changed the hydrogen bonding network in the active site. Via the high-throughput screening assay, we successfully identified two new inhibitors of AlrMtb. [Conclusion] The high-throughput inhibitor screening assay we built is an effective and powerful methodfor theidentification of new inhibitors of AlrMtb.