Authors:	Knaff, Philip Maximilian
Title:	Development of peptidomimetic substrates and inhibitors for transmembrane serine proteases
Online publication date:	22-Aug-2023
Year of first publication:	2023
Language:	english
Abstract:	A new strategy for the treatment of cancer is the concept of hollow nanocarriers which function as a delivery vehicle for drugs and allow increased drug efficiency. For targeted release of the drug to the site of action, nanocarriers can be functionalized with a smart moiety. In the scope of the "HEPSIN-NANOLYSIS" project, this moiety comprises of a peptide which act as a proteolytically degradable substrate for the serine protease hepsin. As a pericellular-active protease, hepsin is easily accessible and its high overexpression in androgen-independent prostate cancer makes it of particular interest. The peptide substrate known in literature with the amino acid sequence RQLR↓VVGG lacks specificity towards hepsin, which limits the applicability of this therapeutic concept due to the large number of proteases present in blood. To tackle this issue, a methodology for finding and optimizing novel cleavable substrates is developed in the first study of chapter A. This methodology included the in silico preparation of combinatorial peptide libraries targeting the P- and P′-site and molecular docking of the respective library in the catalytic active sub site cavity of hepsin. Calculation of binding scores and analysis of binding modes allowed the selection of peptide combinations which were used to improve the reported substrate RQLR↓VVGG. Selected substrate candidates were synthesized as IQF substrates using the FRET system Mca/Dnp and enzymatically tested. In vitro screening against hepsin revealed catalytic efficiencies up to 2.3-fold higher than the literature known substrate. The stability in body fluids could be further improved through introduction of D-amino acids on a selected substrate. With this strategy, two substrate combinations were identified with resistance to degradation by serum or plasma while maintaining cleavability to hepsin. The enzymatic activity of hepsin may not only be used as stimulus for triggered drug release, but it also presents an attractive drug target through its ability to promote the formation of cancer metastasis through cell migration. Therefore, the above-mentioned approach was used to design covalent peptidomimetic inhibitors for this serine protease. In this regard, eight top scoring peptide combinations targeting the P-site were connected with electrophilic serine traps. It was shown that the peptide combinations result in hepsin inhibitors having high potency and good off-target selectivity against coagulation proteases. Lead candidates were stable for up to 10 days in serum, plasma and medium. Further, the influence of the serine trap and truncated versions on a selected candidate were investigated which allowed deeper insides into structure-activity relationships. With this strategy, a variety of candidates with high antiproteolytic activity could be identified with new structures which may offer a promising scaffold for further drug development. Our strategy to prevent viral infections synergizes with potential anticancer therapeutics, too. The transmembrane serine proteases 2 (TMPRSS2) primes the SARS-CoV-2 spike protein and drives viral entry into human cells. Therefore, TMPRSS2 represents an interesting target for COVID-19 therapy and in analogy to hepsin, peptidomimetic TMPRSS2 inhibitors were developed. Molecular docking studies were used to identify tripeptidomimetic binders of the TMPRSS2 catalytic site, which were then connected to an electrophilic serine trap to create covalent and stable inhibitors. The synthesized compounds inhibited TMPRSS2 and selected coagulation proteases with good off-target selectivity and lead candidates were stable in cell culture medium, blood serum and plasma for at least ten days. Selected compounds were shown to prevent SARS-CoV-2 pseudoparticle and genuine SARS-CoV-2 wildtype infection in cell culture with comparable efficiency as camostat mesylate. Notable, the compounds also inhibited infection of SARS-CoV-2 variants of concern carrying spike mutations D614G, B.1.1.7 and B.1.315. The results highly motivate further development of the best antiviral TMPRSS2 inhibitors with regard to pharmacokinetic analysis and the potential route of application e.g. in form of an inhalative spray.
DDC:	500 Naturwissenschaften 500 Natural sciences and mathematics 540 Chemie 540 Chemistry and allied sciences 570 Biowissenschaften 570 Life sciences 610 Medizin 610 Medical sciences
Institution:	Johannes Gutenberg-Universität Mainz
Department:	FB 09 Chemie, Pharmazie u. Geowissensch.
Place:	Mainz
ROR:	https://ror.org/023b0x485
DOI:	http://doi.org/10.25358/openscience-6419
URN:	urn:nbn:de:hebis:77-openscience-bab4d766-6d43-43fb-a0cf-3d4c147662700
Version:	Original work
Publication type:	Dissertation
License:	In Copyright
Information on rights of use:	http://rightsstatements.org/vocab/InC/1.0/
Extent:	XI, 211 Seiten ; Illustrationen, Diagramme
Appears in collections:	JGU-Publikationen