Entdeckung der potente Inhibitoren der β-Tryptase durch Protein Oberflächenerkennung

Jiang, Qian-Qian

Human β-tryptase is the predominant serine protease in human mast cells and is known to be involved in the pathogenesis of asthma and other allergic and inflammatory disorders. It has a tetrameric structure consisting of four monomers with the A2B2 arrangement pointing towards to a central pore and four active sites buried inside the cavity. This thesis focused on the discovery of novel and potent inhibitors of β-tryptase. Not like the classical active site inhibitors of β-tryptase as mostly described in the literature, the multivalent ligands were designed by using new structures and new approaches to inhibit β-tryptase through protein surface recognition. The ligands featuring the basic amino residues were used to bind to the acidic hot spots around the entrance to the central pore, thus blocking the access to the active sites. Besides the proteinogenic amino acids, the guanidiniocarbonyl pyrrole (GCP), as an artificial arginine analog moiety was used as the tailor-made binding motif to investigate its influence on protein surface binding and enzyme inhibition. The inhibitors were shown to efficiently bind to the protein surface through the electrostatic interactions and hydrogen bonding. The thesis contains three different parts. The first part of this thesis was based on the previous work concerning the enzyme inhibition by designing a new class of inhibitors. A series of six new tetravalent ligands with two different sets of arms was synthesized by using solid phase peptide synthesis (SPPS) and an orthogonal protecting group strategy. The design of these ligands was encouraged by the tetrameric structure of β-tryptase with its A2B2 arrangement of the four monomers. Furthermore, a new docking study revealed the presence of two different binding sites on the protein surface. Additionally, the GCP was now introduced into the arms of the ligands to investigate its influence on the inhibition of β-tryptase. Furthermore, two ligands with four identical arms also containing the GCP group were additionally synthesized to study the influence of the GCP moiety on the inhibition properties compared to related ligands identified by the previous work in this group. The best ligand from this new series (RWKG)2(GCP-LFG)2 indeed contains the GCP group and with a Ki-value of 67 nM is two orders of magnitude more efficient than the analogous ligand (RWKG)2(RLFG)2 derived solely from proteinogenic amino acids. Hence, four-armed ligands with two different arms are indeed efficient inhibitors for β-tryptase and the artificial GCP group can improve the binding affinity of this type of ligand to the protein, demonstrating the advantage of tailor-made binding motifs to increase affinity. In the second part of this thesis, dynamic combinatorial chemistry was used as a new method for the identification of inhibitors of β-tryptase. Pre-equilibrated dynamic combinatorial libraries based on acyl hydrazone interchange of peptide-derived hydrazides and di- and tri-aldehydes have been used to discover potent inhibitors of β-tryptase. Following a deconvolution strategy the comparison of the inhibitory activity of the full library with different sub-libraries in which individual building blocks were missing showed that the most active library members contain a central rigid aromatic scaffold with three attached cationic peptide arms containing also a tailor-made GCP oxoanion binding motif attached to a lysine side chain. The most potent hydrazones with peptide arms GKWR and GKWK(GCP), respectively were shown to inhibit β-tryptase in a reversible and non-competitive way with low nanomolar affinity (Ki ca. 10-20 nM) and high selectivity against other related serine proteases (e.g. trypsin, chymotrypsin). These hydrazones are one order of magnitude more efficient than related tetravalent inhibitors derived from a flexible lysine trimer scaffold with four attached peptide arms of the same sequence GKWR (Ki = 170 nM) obtained from the previous work in this group. Molecular mechanics calculations showed that this type of inhibitors can bind to the acidic hot spots around the entrance to the central pore of the enzyme, preventing the substrate from reaching the active sites and thus shutting down the enzyme activity. The last part of this thesis was concerned with the development of novel inhibitors of β-tryptase by using gold nanoparticles (Au NPs) as scaffold based on protein surface recognition event. A series of ligands featuring thiolalkylated triethylene glycol (TEG) chain terminating with different recognition motifs and the artificial arginine analog GCP group were synthesized via microwave-assisted SPPS for the conjugation of gold nanoparticles. The gold nanoparticles were prepared by laser ablation. The monovalent and bivalent conjugated Au NPs with one or two types of ligands were prepared by ex situ and in situ conjugation. The conjugates were evaluated by UV/Vis, disc centrifugation, DLS and TEM and shown to have good long-term stability. The conjugates could efficiently inhibit β-tryptase with lower micromolar affinity (IC50 = 1-3 μM). Therefore, we could demonstrate that we are able to prepare and analyze the peptide conjugated Au NPs and apply these conjugates in enzyme inhibition.

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Jiang, Qian-Qian: Entdeckung der potente Inhibitoren der β-Tryptase durch Protein Oberflächenerkennung. 2013.

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