以抗结核活性化合物IMB-XMA0038为探针研究天冬氨酸半醛脱氢酶作为抗结核药物靶标的可行性

The tuberculosis (TB) has been resurgent globally as the emergence of drug-resistant Mycobacterium tuberculosis (MTB)，but there are no sufficient drugs to cope with these resistant strains. It’s urgent to develop new drugs to handle with this communicable disease. The approvals of bedaquiline and delamanid suggest it effective to exploit anti-resistant-tuberculosis drugs by screening inhibitors aimed at new targets. The aspartic acid metabolic pathway is distinctive for MTB, by which four amino acids (lysine, methionine, threonine and isoleucine) are synthesized. Lysine and its precursor, meso-diaminopimelic acid, are key components for the cross-linking of peptidoglycan. Hence force, the aspartic acid metabolic pathway is critical and key enzymes of this path are potential drug targets. We have demonstrated aspartate-semialdehyde dehydrogenase (ASDH), the second enzyme of this way, is essential for the growth, survival and pathogenicity of MTB. By coupling with the third type aspartate kinase (LysC) of E.coli, a high through-put screening model was constructed to search for inhibitors of ASDH. IMB-XMA0038, obtained through this model, could inhibit several clinical drug resistant MTB potently, and decrease the load of MTB H37Rv in the lung of mouse dramatically. In this study, IMB-XMA0038 will be chosen as probe to verify whether ASDH is an appropriate target for drug development. Firstly, the conformation alterations of ASDH interacted with the probe are observed trough fluorescence spectrum and ultraviolet spectrum, and the affinities between them are detected via isothermal titration calorimetry and surface plasmon resonance. Then amino acids of ASDH interacted with the probe are analyzed through molecular docking and co-crystallization. And functions of the interactions are discussed by measuring the catalytic activities, affinities of the mutated ASDHs, and measuring the growth and drug sensitivity of MTB strains with site-directed mutagenesis in ASDH. Finally, the probe compound resistant MTB strains were selected and studied by detecting mutation in the sequences of asdh and its regulatory element, and the relationship between gene mutation and drug resistance was confirmed by testing the drug sensitivity of MTB strains with mutations at the corresponding site of the resistant bacteria. Through these studies, ASDH will be proved as a new drug target of MTB.

传统抗结核药物已经不能有效治疗耐药结核病，发现和确认新靶标、并针对它们开发药物是获得抗耐药结核病药物的有效途径。研究发现，天冬氨酸半醛脱氢酶（ASDH）与结核分枝杆菌（MTB）的生长、增殖及致病性相关，是一个潜在的药物靶标。据此构建了ASDH的抑制剂高通量筛选模型，筛选得到具有体内抗结核活性的抑制剂IMB-XMA0038。本研究拟以IMB-XMA0038为探针，研究ASDH与探针结合后的构象变化及形成复合物的稳定性，分析酶与化合物相互作用的氨基酸残基，对结合位点的氨基酸进行定点突变，考察氨基酸突变对ASDH酶活性及其与IMB-XMA0038结合能力的影响，进一步考察突变ASDH对MTB生理及药物敏感性的影响；此外将分析MTB的耐药突变株asdh编码区及调控区是否发生突变并在细菌水平验证突变与耐药性的关系。通过本课题的开展，最终证明ASDH是一个药物靶标，为开发新型抗结核药物奠定基础。

结核病是由结核分枝杆菌（Mycobacterium tuberculosis, MTB)感染引起的具有高致病性和致死性的传染性疾病，随着耐药结核分枝杆菌的出现和蔓延，临床主要抗结核药物已经不能有效应对这一严峻形势，亟需开发新型抗结核药物。贝达喹啉等新型药物的成功开发为治疗耐药结核病带来了曙光，但仍然有许多耐药菌株不能被有效控制。本研究将MTB天冬氨酸半醛脱氢酶（Aspartate-semialdehyde dehydrogenase，ASDH）与大肠杆菌III型天冬氨酸激酶LysC偶联，建立靶向ASDH的抑制剂高通量筛选模型，从本单位化合物库的15万次合成化合物中筛选得到40个酶抑制剂，进而测定它们对MTB标准菌株H37Rv、临床敏感菌株、耐药菌株的抑制活性，得到IMB-XMA0038。它对各种MTB菌株均具有较强的抑制活性，且对asdh处于不同表达水平的MTB突变株表现出明显的活性差异（16倍），表明IMB-XMA0038与现有抗结核药物不具有交叉耐药性，可能是通过抑制ASDH酶活性而抑制MTB。表面等离子共振（surface plamon resonace, SPR）研究结果显示化合物与酶的亲和力Kd为3.65×10-4 M；分子对接显示化合物结合于酶的催化位点Cys130附近，通过氢键、盐键、极性作用以及疏水作用等方式与酶发生相互作用。通过构建ASDH点突变株发现Asn129，Glu224，Gly161，Ser162和Arg249突变能使菌株对化合物的敏感性出现不同程度的降低，而活性位点Cys130、Gln157和Lys227不能被置换。通过扫描电镜观察化合物对MTB细胞形态的影响，发现IMB-XMA0038能干扰MTB细胞壁的合成过程但不能破坏成熟的细胞壁。代谢组学研究表明，IMB-XMA0038能抑制菌株的三羧酸代谢、氨基酸代谢以及PE转运系统。IMB-XMA0038在SD大鼠的生物利用度约为59.3%，主要分布于肺部，不能被肝微粒体酶代谢处理，主要通过肾脏排泄及胆汁分泌排泄。化合物对小鼠的LD50值＞1000 mg/kg，在25 mg/kg剂量下给药15d能使小鼠肺部荷菌量降低1.67 log10。综上，本研究证明ASDH是一个新型抗结核药物靶标，靶向ASDH的IMB-XMA0038是一个具有研究前景的药物先导物。

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