Development of enhanced furin inhibitors with reduced toxicity as potential broad spectrum antiviral drugs

At the beginning of the twentieth century, the focus of peptide research was largely on human signalling hormones. A breakthrough in the field of peptide therapeutics was the first medicinal application of insulin isolated from animal pancreas, which revolutionized the treatment of type 1 diabetes. Since then, more than 80 peptide drugs have been used for a variety of diseases, including diabetes, cancer, osteoporosis, multiple sclerosis, HIV infection, and chronic pain. The production of synthetic therapeutic peptides has become possible with the development of solid-phase peptide synthesis by Merrifield in the year 1963. This milestone has provided access to pure and sufficient amounts of peptides enabling countless research projects to determine the functional and structural bioactive properties of peptides. In the context of drug discovery, this thesis focused on the development of synthetic inhibitors of the Ca2+-dependent type-I transmembrane protein furin, which belongs to the proprotein convertase (PC) family of serine endoproteases. It is ubiquitously expressed in vertebrates and invertebrates and activates a large number of proprotein substrates in the secretory pathway, including prohormones, proenzymes, and proforms of receptors or extracellular matrix proteins. Furin also plays a crucial role in tumorigenesis, neurodegenerative disorders, and diabetes or arteriosclerosis as well as in many bacterial and viral diseases. It cleaves numerous precursors of bacterial toxins such as Pseudomonas exotoxin A, Shiga-, and diphtheria toxin. Some enveloped viruses possess surface proteins that contribute to the fusion between viral and host cell membranes and have to be activated by furin or related PCs. Examples of such glycoproteins include those from highly pathogenic avian Influenza viruses, Measles, Ebola, and Flaviviruses such as Dengue-, West Nile-, and Zika-virus. Recent studies revealed that furin is implicated in the activation of the spike (S) protein of the new SARS-CoV-2. Given its implications in a variety of biological processes and diseases, furin emerged as a target of paramount importance in drug discovery. To suppress the propagation cycle of furin-dependent viruses, novel peptidomimetic furin inhibitors were developed in this work. Based on the preferred multibasic cleavage motif of furin (–Arg–X–Lys/Arg–Arg↓–) and on previous work from our group, novel inhibitors of furin were synthesized by a combination of solid-phase peptide synthesis and solution synthesis. In a first manuscript, several series of new macrocyclic furin inhibitors have been designed and synthesized. The development of cyclic peptides is a widely used strategy to improve the stability and bioavailability of peptide drugs. The cyclization was performed between different positions of these substrate-analogue inhibitors and by incorporating different linker segments. The inhibitory potency of these compounds was determined in enzyme kinetic assays with soluble human furin. In cooperation with other groups the binding mode of selected cyclic inhibitors in complex with furin was determined and their antiviral potency was tested in cells infected with Respiratory syncytial virus (RSV). A second paper describes the optimization of a non-cyclic inhibitor starting from a previously designed linear inhibitor MI-1148 that inhibits furin with a Ki value of 5 pM but suffers from a considerably toxicity in mice and rats. Additional studies with structurally related compounds suggested that the severe toxicity of these compounds is caused by the multibasic character of this inhibitor type and not by the inhibition of the host protease furin. By replacing the arginine residues (pKa of side chain 13.5) with the noncanonical amino acid canavanine (pKa 7.0), a new series of potent furin inhibitors was prepared. The best compound MI-1851 inhibits furin with a Ki value of 10.1 pM. Furthermore, the toxicity of this compound was significantly reduced. In cell culture studies, the novel canavanine-containing compounds also showed a significant antiviral activity against furin-dependent viruses, such as RSV, WNV, and Dengue-2 virus, and most importantly, the current SARS-CoV-2 virus. In a third co-authored paper, this inhibitor MI-1851 was also used to demonstrate an antiviral effect on the new SARS-CoV-2. Moreover, through a series of newly synthesized FRET-substrates derived from the sequence of the potential S1/S2 cleavage site of the SARS-CoV-2 spike protein, it was possible to prove the SARS-CoV-2 S protein activation at this sequence by furin. In a fourth paper, the synthesis and characterization of a small series of 11 new fluorescence substrates are described, which were tested with five basic PCs including furin, PC1, PC2, PC5A, and PC7. For four of the tested PCs, improved substrates have been identified, which are more efficiently cleaved than the known reference substrates. The improved furin substrates enabled kinetic measurements at significantly reduced enzyme concentrations, thereby avoiding tight-binding conditions. At these reduced furin concentrations, a slow-binding behaviour was observed for the first time with inhibitor MI-1148, which enabled the determination of its individual kon and koff rate constants. The new inhibitors and substrates may help further elucidate the role of furin and related PCs in physiology and disease