Development of Molecular Chimeras Targeting the NLRP3 Inflammasome and HDAC6

Abstract

In recent years, the development of chimeric molecules inducing the proteasomal degradation of various proteins of interest (POIs) gained enormous attention in medicinal chemistry. Representatives of this new drug modality are commonly referred to as proteolysis targeting chimeras (PROTACs) and are equipped with both, a ligand for a selected POI and a ligand for an E3 ubiquitin ligase, while being interconnected with a linker. This study aimed at the design and synthesis of degrader molecules targeting either nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing protein 3 (NLRP3), a key player in innate immunity, or histone deacetylase 6 (HDAC6), which is associated with a myriad of diseases in the fields of oncology, autoimmunity and neurology. <br /> For the development of a diversified NLRP3 degrader portfolio, diaryl sulfonylurea ligands were prepared, inspired by the most well-characterized NLRP3 inhibitor MCC950. They were conceptualized in order to implement varying exit vector strategies for linker attachment, while considering the herein disclosed structure-stability relationships. Connection of these building blocks with established ligands for the E3 ligases cereblon (CRBN), von Hippel-Lindau (VHL) and inhibitor of apoptosis protein (IAP) was achieved by utilizing individually bifunctionalized linkers, ultimately affording a series of 29 molecular chimeras. Among these, a more advanced subset was accomplished via a final copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), a straightforward type of ‘click chemistry’. The compounds were subjected to initial physicochemical, pharmacokinetic and biophysical experiments. Thermal stability measurements of the addressed proteins, i.e. NLRP3, CRBN and VHL, upon degrader treatment, provided first insights into binary complex formation behavior. <br /> Another project focused on the design of fluorescent and biotin probes targeting NLRP3. For this purpose, an expandable NLRP3 ligand was either conjugated with a coumarin fluorophore or a biotin moiety. The prepared tool compounds were analyzed by means of surface plasmon resonance spectroscopy, regarding their affinity to NLRP3. Their ability to inhibit the NLRP3 inflammasome was evaluated in a cytokine release assay. One fluorescent probe was furthermore investigated in terms of confocal microscopy in order to label NLRP3 in cells. <br /> The last project of this thesis was directed at the development of the first non-hydroxamate selective HDAC6 degraders. By the introduction of a pyrimidine-linked difluoromethyl-1,3,4-oxadiazole warhead, a set of six potent HDAC6 degraders recruiting the ligases CRBN or VHL was obtained. Two PROTACs were investigated in more detail, regarding their degradation selectivity profile. Eventually, the assumed proteolytic degradation mechanism was verified by complementing control experiments, including the application of tailored chemical negative controls. <br /> Taken together, this thesis comprises the development of novel molecular chimeras targeting the NLRP3 inflammasome and HDAC6.