Expression, purification, structural and biochemical characterization of the hemagglutinin-cleaving proprotein convertase PC5

蛋白转化酶家族包括9个成员，其催化结构域高度保守。PCs参与激素或生长因子前体蛋白的加工，但在感染过程中也激活病毒蛋白和细菌毒素。急性病毒性或细菌性感染性疾病短期应用PC抑制剂治疗可能是一种很有前景的治疗策略。然而，大多数PC抑制剂的选择性是有限的，因为它们在相似的范围内抑制几种PC，例如在furin和PC5的情况下。选择性PC抑制剂的开发对于治疗目的和进一步表征PC在各种疾病中的作用具有很大的兴趣。到目前为止，与furin相比，其他类键蛋白的识别基序研究较少，并且仍然知之甚少。在PC5的情况下，只有有限的数据可用于其特异性和性质。因此，本课题的目的是进一步对PC5作为抗病毒靶点进行特异性、结构、生物学和化学性质的表征，以确定其与其他PC5的差异。为了确定其选择性和进一步表征，将采用两种不同的策略。一方面，已有的PC抑制剂和底物将在酶动力学测量中筛选选择性。另一方面，需要解决PC5的实验晶体结构问题，从而实现更合理的、基于结构的药物设计。因此，PC5将在人胚胎肾细胞中表达，并使用三步色谱法纯化。建立并优化一种酶动力学测定法，以确定可用的PC抑制剂的抑制常数。同时，利用晶体学方法测定PC5的结构，将扩大高纯度酶的生产。Furin和PC5在多碱基裂解位点激活高致病性禽流感病毒H5和H7株血凝素（HA）。在共表达实验中，将研究PC5是否也能激活含有二基或三基序列基序的HA。这些结果将与短合成ha衍生底物的裂解分析进行比较。此外，在内源性条件下，PC5在HA活化中的作用必须通过小干扰rna或肽偶联磷酸二酯morpholino低聚物来阐明。在其他共表达实验中，可用抑制剂对pc5介导的HA切割的影响将被测试。这些结果的结合将有助于开发更具选择性的PC5抑制剂，其体外毒性低，适合抑制流感ha活化。未来，潜在的候选药物必须检查其药代动力学和药效学特性，以及它们的毒性和对体内疾病的治疗作用。

The family of proprotein convertases (PCs) comprises nine members, whose catalytic domains are highly conserved. PCs are involved in the processing of several precursor proteins of hormones or growth factors but also activate viral proteins and bacterial toxins during infections. The short-term treatment with PC inhibitors in acute viral or bacterial infectious diseases could be a promising therapeutic strategy. However, the selectivity profile of most PC inhibitors is limited as they inhibit several PCs in a similar range, e.g. in the case of furin and PC5. The development of selective PC inhibitors would be of great interest for therapeutically purposes and for the further characterization of the role of PCs in various diseases. So far, the recognition motif of other kexin-like PCs than furin has been less investigated and remains poorly understood. In case of PC5 only limited data are available on its specificity and properties. Therefore, the aim of this proposal is to further characterize PC5 as antiviral target regarding its specificity, structure as well as biological and chemical properties to identify differences to other PCs.For the determination of its selectivity profile and further characterization two different strategies will be applied. On the one hand already available PC inhibitors and substrates will be screened for selectivity in enzyme kinetic measurements. On the other hand experimental crystal structures of PC5 should be solved to enable a rational, structure-based design of more selective drugs. Therefore, PC5 will be expressed in human embryonic kidney cells and purified using a three-step chromatography procedure. An enzyme kinetic assay will be established and optimized for the determination of inhibition constants of available PC inhibitors. In parallel, the production of highly purified enzyme will be scaled up for the structure determination of PC5 using crystallographic methods.Furin and PC5 activate the hemagglutinin (HA) of highly pathogenic avian influenza virus H5 and H7 strains at a mulitbasic cleavage site. In co-expression experiments it will be investigated, whether PC5 can also activate HA containing di- or tribasic sequence motifs. These results will be compared to the cleavage analysis of short synthetic HA-derived substrates. Furthermore, the role of PC5 in the activation of HA under endogenous conditions has to be clarified using small-interfering RNAs or peptide-conjugated phosphordiamidate morpholino oligomers. In additional co-expression experiments the effect of available inhibitors on the PC5-mediated HA cleavage will be tested.The combination of these results will enable the development of more selective PC5 inhibitors with a low in vitro toxicity suitable for the inhibition of influenza HA-activation. In future, potential drug candidates have to be examined regarding their pharmacokinetic and pharmacodynamic properties as well as their toxicity and therapeutic effects on diseases in vivo.