Repurposing p97 inhibitors for chemical modulation of the bacterial ClpB-DnaK bichaperone system.
Repurposing p97 inhibitors for chemical modulation of the bacterial ClpB-DnaK bichaperone system.
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DOI：
10.1074/jbc.ra120.015413
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发表时间：
2021-01
期刊：
影响因子：
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通讯作者：
Zolkiewski M
中科院分区：
文献类型：
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作者：
Glaza P;Ranaweera CB;Shiva S;Roy A;Geisbrecht BV;Schoenen FJ;Zolkiewski M
ClpB-DNAK双环蛋白系统激活聚集的细胞蛋白，对于细菌、真菌、原生动物和植物在压力下的生存是必不可少的。AAA+ATPase ClpB是开发抗微生物药物的一个有前途的靶标，因为其活性的丧失不利于许多病原体的生存，而且在后生动物中没有发现明显的ClpB同源物。我们研究了在几种化合物存在的情况下ClpB的活性，这些化合物以前被描述为人AAA+ATPase p97的抑制因子，这是一个抗肿瘤的靶标。我们发现，p97抑制剂中效力最低的2，N4-二苯基喹唑啉-2，4-二胺(DBeQ)以Kd∼60μM与ClpB结合，并以IC50∼5μM抑制酪蛋白激活的ClpB的ATPase活性。其余的p97配体对p97的亲和力较高，不影响ClpB的ATPase。在体外，DBeQ还与具有KD∼10 0μM的DNAK相互作用，但不影响DNAK ATPase，但抑制DNAK伴侣的活性。在体外以IC50∼5μM抑制ClpB-DNAK双杂环酮系统聚集蛋白的再激活，并抑制培养的大肠杆菌的生长。DBeQ诱导的大肠杆菌增殖丧失因热休克而加剧，但在ClpB缺陷的大肠杆菌菌株中几乎被消除，这表明DBeQ在细胞中对ClpB具有显著的选择性。我们的结果为ClpB作为开发新型抗菌剂的目标提供了化学验证。我们确定DBeQ是一种很有前途的先导化合物，用于选择性靶向ClpB和/或DNAK的结构优化。
The ClpB–DnaK bichaperone system reactivates aggregated cellular proteins and is essential for survival of bacteria, fungi, protozoa, and plants under stress. AAA+ ATPase ClpB is a promising target for the development of antimicrobials because a loss of its activity is detrimental for survival of many pathogens and no apparent ClpB orthologs are found in metazoans. We investigated ClpB activity in the presence of several compounds that were previously described as inhibitor leads for the human AAA+ ATPase p97, an antitumor target. We discovered that N2,N4-dibenzylquinazoline-2,4-diamine (DBeQ), the least potent among the tested p97 inhibitors, binds to ClpB with a Kd∼60 μM and inhibits the casein-activated, but not the basal, ATPase activity of ClpB with an IC50∼5 μM. The remaining p97 ligands, which displayed a higher affinity toward p97, did not affect the ClpB ATPase. DBeQ also interacted with DnaK with a Kd∼100 μM and did not affect the DnaK ATPase but inhibited the DnaK chaperone activity in vitro. DBeQ inhibited the reactivation of aggregated proteins by the ClpB–DnaK bichaperone system in vitro with an IC50∼5 μM and suppressed the growth of cultured Escherichia coli. The DBeQ-induced loss of E. coli proliferation was exacerbated by heat shock but was nearly eliminated in a ClpB-deficient E. coli strain, which demonstrates a significant selectivity of DBeQ toward ClpB in cells. Our results provide chemical validation of ClpB as a target for developing novel antimicrobials. We identified DBeQ as a promising lead compound for structural optimization aimed at selective targeting of ClpB and/or DnaK.