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

原蛋白转换酶(PC)家族由9个成员组成，其催化结构域高度保守。PC参与了几种激素或生长因子前体蛋白的加工，但也在感染期间激活了病毒蛋白和细菌毒素。PC抑制剂对急性病毒或细菌感染性疾病的短期治疗可能是一种有前途的治疗策略。然而，大多数PC抑制剂的选择性图谱是有限的，因为它们在类似范围内抑制几个PC，例如在呋喃和PC5的情况下。选择性PC抑制剂的开发将对治疗目的和进一步表征PC在各种疾病中的作用具有重要意义。到目前为止，对除呋喃以外的其他类可信PC的识别基序研究较少，对其了解也很少。在PC5的情况下，只有有限的数据可用来了解其特性和特性。因此，这项建议的目的是进一步表征PC5作为抗病毒靶标的特异性、结构以及生物学和化学性质，以确定其与其他PC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，将在酶动力学测量中筛选已有的PC抑制剂和底物的选择性。另一方面，应解决PC5的实验晶体结构，以便能够合理地、基于结构设计更具选择性的药物。因此，PC5将在人类胚胎肾脏细胞中表达，并通过三步层析程序进行纯化。将建立和优化一种酶动力学分析方法，用于测定现有PC抑制剂的抑制常数。同时，将扩大生产高纯度的酶，用于结晶学方法确定PC5的结构。Furin和PC5在多碱基切割位点激活高致病性禽流感病毒H5和H7毒株的血凝素(HA)。在共表达实验中，将研究PC5是否也能激活含有二或三个碱基序列基序的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，PC5在内源性条件下激活HA中的作用必须通过小干扰RNA或多肽连接的磷二酸吗啉低聚物来阐明。在额外的共表达实验中，将测试可用的抑制剂对PC5介导的HA裂解的影响。这些结果的结合将使开发出更具选择性、体外毒性低的适合于抑制流感HA激活的PC5抑制剂。未来，潜在的候选药物必须检查它们的药代动力学和药效学特性，以及它们在体内的毒性和治疗疾病的效果。

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.