Székely Rita, Wáczek Frigyes, Szabadkai István, Németh Gábor, Hegymegi-Barakonyi Bálint, Eros Dániel, Szokol Bálint, Pató János, Hafenbradl Doris, Satchell Jacqueline, Saint-Joanis Brigitte, Cole Stewart T, Orfi László, Klebl Bert M, Kéri György
Vichem Chemie Research Ltd., Herman Ottó. 15, H-1022 Budapest, Hungary.
Immunol Lett. 2008 Mar 15;116(2):225-31. doi: 10.1016/j.imlet.2007.12.005. Epub 2008 Jan 8.
The Mycobacterium tuberculosis genome encodes for eleven eukaryotic-like Ser/Thr protein kinases. At least three of these (PknA, PknB and PknG) are essential for bacterial growth and survival. PknG is secreted by pathogenic mycobacteria, in macrophages to intervene with host cell signalling pathways and to block the fusion of the lysosomes with the phagosome by a still unknown mechanism. Based on our previously published results, we have initiated a drug discovery program, aiming to improve the potency against PknG and the physiochemical properties of the initially identified hit compound, AX20017, from the class of the tetrahydrobenzothiophenes. We have established a radioactive biochemical PknG kinase assay to test the novel analogues around AX20017. We have developed lead molecules with IC50 values in nanomolar range, and demonstrated their antituberculotic effects on human macrophages. Selected leads might ultimately serve the purpose of inducing phagosomal-lysosomal fusion and therefore destroy the residence of the intracellular mycobacteria. It is unclear at this time if these "homeless" mycobacteria are getting killed by the host, but they will be at least vulnerable to the activity of antimycobacterial agents. Released mycobacteria rely on the essential function of PknB for survival, which is our second molecular kinase target. PknB is a transmembrane protein, responsible for the cell growth and morphology. We have screened our library and synthesized novel compounds for the inhibition of PknB. A pharmacophore model was built and 70,000 molecules from our synthesizable virtual library have been screened to identify novel inhibitor scaffolds for the generation of templated compound libraries. Currently, we are using a radioactive kinase assay employing GarA as the putative, physiological substrate of PknB kinase. We have identified hits and generated optimised hit compounds with IC50 values for the inhibition of PknB in the nanomolar range. Yet those promising hits are not potent enough to yield meaningful "minimum inhibitory concentrations" in mycobacterial growth assays. In the course of our future work, we will increase the potency of the next generation of PknB inhibitors in order to improve their antibacterial activity.
结核分枝杆菌基因组编码11种类真核丝氨酸/苏氨酸蛋白激酶。其中至少三种（PknA、PknB和PknG）对细菌生长和存活至关重要。PknG由致病性分枝杆菌分泌，进入巨噬细胞以干预宿主细胞信号通路，并通过一种尚不清楚的机制阻止溶酶体与吞噬体融合。基于我们之前发表的结果，我们启动了一项药物发现计划，旨在提高对PknG的效力以及最初鉴定的命中化合物AX20017（来自四氢苯并噻吩类）的理化性质。我们建立了一种放射性生化PknG激酶测定法来测试AX20017周围的新型类似物。我们已经开发出IC50值在纳摩尔范围内的先导分子，并证明了它们对人巨噬细胞的抗结核作用。选定的先导物最终可能有助于诱导吞噬体 - 溶酶体融合，从而破坏细胞内分枝杆菌的生存场所。目前尚不清楚这些“无家可归”的分枝杆菌是否会被宿主杀死，但它们至少会更容易受到抗分枝杆菌药物活性的影响。释放出来的分枝杆菌依赖PknB的基本功能来生存，这是我们的第二个分子激酶靶点。PknB是一种跨膜蛋白，负责细胞生长和形态。我们筛选了我们的文库并合成了用于抑制PknB的新型化合物。构建了一个药效团模型，并从我们可合成的虚拟文库中筛选了70000个分子，以鉴定用于生成模板化合物文库的新型抑制剂支架。目前，我们正在使用一种放射性激酶测定法，采用GarA作为PknB激酶的假定生理底物。我们已经鉴定出命中化合物并生成了优化的命中化合物，其抑制PknB的IC50值在纳摩尔范围内。然而，那些有前景的命中化合物在分枝杆菌生长测定中还不够有效，无法产生有意义的“最低抑菌浓度”。在我们未来的工作中，我们将提高下一代PknB抑制剂的效力，以改善它们的抗菌活性。
