Entwicklung, Synthese und Charakterisierung neuer Inhibitoren der West-Nil-Virus NS2B-NS3-Protease
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Abstract
New P1-P3 residues with acylated diaminobutyric- and diaminopropionicacid-derivates
New substrate analogue inhibitors should be obtained by variation of the P1-P3 residues. Therefore the lead structure Phac-Lys-Lys-Arg-NH2 was modified with α,γ-diaminobutyric- (Dab) or α,β-diaminopropionic acid (Dap). These residues could afterwards be coupled with various additional amino acids. The most effective compound of this series possesses Dap(Gly) in P2-position and has a Ki of 12.2 µM.
Ketones as inhibitors for the NS2B-NS3 protease
Ketones can be used as transition-state-inhibitors for serine proteases. By using a Weinreb amide as an intermediate two ketones were synthesized. It appeared that the peptidic ethyl ketone derivate is a potent WNV inhibitor with a Ki of 1.07 µM. The steric more demanding benzyl ketone is 24 times less potent than the ethyl ketone.
Cell-penetrating cyclic peptides as inhibitors for the NS2B-NS3 protease
To address the NS2B-NS3 protease inhibitors have to get into the cytosol. In order to achieve this, inhibitors can be coupled with cell-penetrating peptides. Two of such inhibitors were synthesized. The more effective inhibitor contains a cell penetrating sequence (Phe-2Nal-Arg4-Glu) and an inhibitor sequence with five DArg residues. The Ki of this compound is 0.5 µM for the WNV protease and 2.9 µM for the Den2 protease. Unfortunately only small amounts of this compound got obtained, so that a testing in cell culture was not possible. But from similar cyclic furin inhibitors it is known, that the effectiveness of such compounds is relatively low for WNV und Den2 cell cultures.
Cholesteryl- and Dihydrocholesteryl derivates
To obtain a higher cell permeability Cholestryl- and Dihydrocholesteryl residues were coupled to the lead structure 4-AMe-Phac-Lys-Lys-Arg-NH2. In addition 0-3 ethylene glycol linker were inserted between the inhibitor and the Cholesteryl residue, this should make the inhibitor more flexible. It turned out, that the linkers did not have any effect on the inhibition. All IC50 values were around 0.6-0.7 µM for the WNV protease. During cell culture testing it became apparent, that these compounds were comparatively toxic. Because of that they could only be tested in a concentration of 5 µM. The inhibition at this concentration was smaller than by the reference substance Ribavirin.
Inhibitors with an additional P5 residue
By connection of an additional P5 residue to with a p-aminomethyl group substituted P4 PhAc residue of the lead structure the affinity of the compounds should be further improved. All synthetized inhibitors of this type showed comparable Ki values of about 1.8-2.6 µM. After modeling of the binding mode in silico, this issue could probably be explained. The additional P5 residue does not come in direct contact with the protease, in contrary it sticks out into the solvent. Therefore the P5 residue has maximally little influence on the affinity. Other P5 modifications in para position of the P4 residue are probably not very useful. Maybe the coupling could have more effect in meta or ortho position of the P4 PhAc group.
Docking studies for acquisition of new inhibitor fragments
With the help of the work group Kolb docking experiments with the WNV protease (PDB: 2YOL) were conducted. Four potential inhibitor fragments were identified and their percentage inhibition on the WNV protease at a concentration of 625 µM was determined. Two fragments showed a certain inhibitory effect with 17% and 12%. Five inhibitors were synthesized from these fragments. However these compounds were very weak. The best derivate has a Ki value of 60.4 µM. The fact that the individual fragments showed some inhibition but the final inhibitors were not effective, suggests that the linkage of the fragments needs to be improved.
Inhibition of the WNV-protease by zinc ions
By enzyme kinetic measurements a Ki value of 31 µM could be determined for zinc ions. It was proved that the inhibition is provided by the zinc ion and not the corresponding anion. Zinc chloride, zinc nitrate and zinc acetate showed the same inhibition. Other metal cations have no effect on the WNV protease. Also zinc ions have no effect on the Den2 protease. With complexing agents like EDTA the inhibition can be suspended. The position of the intersection point in the Lineweaver-Burk plot suggests that it must be a non-competitive inhibition. Further it could be shown, that the supplement of zinc ions to an enzyme kinetic measurement of a competitive inhibitor can decrease the IC50 value of this inhibitor. The testing of zinc ions in cell culture did not show any effect on the replication of the virus. Maybe only an insufficient concentration of zinc ions is assimilated intracellular.
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Dates
Created: 2018Issued: 2018-09-24Updated: 2018-09-24
Faculty
Fachbereich Pharmazie
Publisher
Philipps-Universität Marburg
Language
ger
Data types
DoctoralThesis
Keywords
West-Nil-Virus
DFG-subjects
Pharmazie
DDC-Numbers
615
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Epp, Anton: Entwicklung, Synthese und Charakterisierung neuer Inhibitoren der West-Nil-Virus NS2B-NS3-Protease. : Philipps-Universität Marburg 2018-09-24. DOI: https://doi.org/10.17192/z2018.0244.
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This item has been published with the following license: In Copyright