Project Details
Projekt
Expression, purification, structural and biochemical characterization of the hemagglutinin-cleaving proprotein convertase PC5
Applicant
Dr. Kornelia Hardes
Subject Area
Pharmacy
Biological and Biomimetic Chemistry
Virology
Biological and Biomimetic Chemistry
Virology
Term
from 2016 to 2019
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 309089047
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.
DFG Programme
Research Grants
