Exploring Covalently Acting Natural Products: Synthesis of Gymnastatin Family of Alkaloids and Development of Nimbolide- and Bardoxolone-Based Protein Degraders

Natural products have long been a rich source of medicinal compounds and inspirations for drug discovery. Among these, molecules that interact with their targets through covalent bonds are highly attractive in biological applications. This dissertation describes our studies on the biosynthetic relationship and chemical synthesis of potentially covalently acting natural products of gymnastatin family, as well as the application of the cysteine-targeting limonoid natural product nimbolide and triterpene derivative bardoxolone for targeted protein degradation.Gymnastatins are a family of cytotoxic natural products derived from tyrosine, which possess a variety of electrophilic functional groups. Through bio-inspired strategies, the total syntheses of gymnastatin G, dankastatin B and dankastatin C was achieved. The previously presumed biomimetic strategy failed to provide the desired stereochemistry during formation of the bicyclo[3.3.1]nonane core of gymnastatin G, complicating our understanding of the biosynthesis of gymnastatins. In addition, the connection between gymnastatin G and a related natural product aranorosin was established through a simple chemical transformation. These results provide further insight into the biosynthesis and chemical reactivity of gymnastatins (Chapter 1). In Chapter 2, we describe the use of nimbolide as a novel E3 ligase recruiter in targeted protein degradation (TPD) applications. While TPD and proteolysis-targeting chimeras (PROTACs) have arisen as powerful therapeutic modalities, the lack of E3 ligase recruiters remains one of the major limitations of current TPD strategies. Extending our recent discovery of nimbolide as a covalent recruiter for the E3 ligase RNF114, we show the broader utility of this E3 ligase recruitment by developing a nimbolide-based PROTAC BT1, which selectively degrades oncogenic fusion protein BCR-ABL over c-ABL in leukemia cancer cells. The selectivity profile of BT1 is opposite to existing cereblon or VHL-recruiting BCR-ABL degraders of similar linker composition. This work further establishes nimbolide as an additional general E3 ligase recruiter for use in PROTACs and demonstrates the importance of expanding the arsenal of E3 ligase recruiters. In Chapter 3, an unexplored mode of E3 ligase recruitment, namely, covalent, reversible E3 ligase recruitment was explored. Efficient proteasome-mediated degradation of BRD4 was achieved by a bifunctional small molecule linking the KEAP1-Nrf2 activator bardoxolone to a BRD4 inhibitor JQ1, which further expanded the TPD toolbox to include bardoxolone as an easily prepared E3 ligase recruiter and further highlighted the power of natural products in such applications

Chemical Investigations of Gymnastatin and Dankastatin Alkaloid Biosynthesis

Electrophilic natural products have provided fertile ground for understanding how nature inhibits protein function using covalent bond formation. The fungal strain Gymnascella dankaliensis has provided an especially interesting natural library of halogenated cytotoxic agents derived from tyrosine which feature an array of reactive functional groups. Herein we explore biosynthetic relationships between architecturally complex gymnastatin and dankastatin members, finding factors that favor formation of a given scaffold from a common intermediate. Additionally, we find that multiple natural products can be formed from aranorosin, a non-halogenated natural product also produced by gymnascella sp. fungi, using simple chloride salts. Finally, growth inhibitory activity of multiple members against challenging human triple negative breast cancer cells is reported

Stimulation of Tetrabromobisphenol A Binding to Soil Humic Substances by Birnessite and the Chemical Structure of the Bound Residues

Studies have shown the main fate of the flame retardant tetrabromobisphenol A (TBBPA) in soils is the formation of bound residues, and mechanisms on it are less-understood. This study investigated the effect of birnessite (δ-MnO₂), a naturally occurring oxidant in soils, on the formation of bound residues. ¹⁴C-labeled TBBPA was used to investigate the pH dependency of TBBPA bound-residue formation to two soil humic acids (HAs), Elliott soil HA and Steinkreuz soil HA, in the presence of δ-MnO₂. The binding of TBBPA and its transformation products to both HAs was markedly increased (3- to 17-fold) at all pH values in the presence of δ-MnO₂. More bound residues were formed with the more aromatic Elliott soil HA than with Steinkreuz soil HA. Gel-permeation chromatography revealed a uniform distribution of the bound residues within Steinkreuz soil HA and a nonuniform distribution within Elliott soil HA. ¹³C NMR spectroscopy of ¹³C-TBBPA residues bound to ¹³C-depleted HA suggested that in the presence of δ-MnO₂, binding occurred via ester and ether and other types of covalent bonds besides HA sequestration. The insights gained in this study contribute to an understanding of the formation of TBBPA bound residues facilitated by δ-MnO₂

Chemoproteomics-Enabled Ligand Screening Yields Covalent RNF114-Based Degraders that Mimics Natural Product Function

The translation of natural product function to fully synthetic small molecules has remained an important process in medicinal chemistry for decades resulting in numerous FDA-approved medicines. We recently discovered that the terpene natural product nimbolide can be utilized as a covalent recruiter of the E3 ubiquitin ligase RNF114 for use in targeted protein degradation (TPD) ¬– a powerful therapeutic modality within modern day drug discovery. Using activity-based protein profiling-enabled covalent ligand screening approaches, we herein realize the discovery of fully synthetic RNF114-based recruiter molecules that can also be exploited for PROTAC applications, and demonstrate their utility in degrading oncology targets such as BRD4 and BCR-ABL in cells. The identification of simple and easily manipulated drug-like scaffolds that can mimic the function of a complex natural product is beneficial in further expanding the toolbox of E3 ligase recruiters, an area of great importance in drug discovery and chemical biology