Peptidomimetics in Drug Development: Substrates, Titrants, and Inhibitors to Study and Combat SARS-CoV-2 and Proteolysis Targeting Chimeras as ¹⁸F-Tracers

Abstract

This thesis provides a profound contribution to the application spectrum of peptidomimetics in the drug discovery process. It demonstrates the exploration of the main protease (M^pro) of SARS-CoV-2 through substrates and titrants, the development of promising M^pro inhibitors for the treatment of COVID-19, and the design of a proteolysis targeting chimera (PROTAC) as an ¹⁸F-tracer. Initially, a literature review of coumarin-labeled probes for serine and cysteine proteases was conducted, establishing a basis for the investigations on M^pro. The review highlights the application of various coumarin analogs in fluorogenic substrates, internally quenched fluorescent substrates, as well as activity-based probes and discusses the advantages and limitations of these tool compounds in terms of substrate mapping and the imaging of enzymatic activities in biological systems. This information was applied to develop a coumarin-based fluorogenic substrate for SARS-CoV-2 M^pro, enabling the creation of a high-throughput screening assay. The identification of a hit compound class led to the design of azapeptide nitriles as potent M^pro inhibitors. Subsequently, these azanitriles were subjected to a systematic active-site scanning resulting in the identification of suitable P1-P4 fragments. The combination of fragment-based with structure-guided drug design led to the development of macrocyclic azapeptide nitriles as extraordinarily potent M^pro inhibitors with an improved selectivity profile. Selected M^pro inhibitors showed promising antiviral activity and sufficient in-vitro plasma stability. Additionally, an active-site titrant was conceived, enabling the quantification of the enzymatically active portion of M^pro. This facilitated the detailed kinetic characterization of two known substrates and two newly designed coumarin-based fluorogenic substrates, allowing for a comparison of their applicability in M^pro inhibition assays. In another project, PROTAC and positron emission tomography (PET) technologies were combined to develop a peptidic PET PROTAC as an ¹⁸F-tracer targeting cyclin-dependent kinase 6 (CDK6). A “cold” ¹⁹F-PROTAC-tracer was designed and synthesized demonstrating sufficient CDK6 degradation. The required precursor molecule, to be converted to the respective “hot” ¹⁸F-radiotracer in a late-stage fluorination, was made accessible by using click chemistry. The precursor’s suitability for the radiolabeling was demonstrated.