Boronic acids have been shown to interact with biological molecules including carbohydrates and reactive oxygen species (ROS). This variety of biological targets is due to the unique reactivity of boron that, within a physiological pH range, can adopt two electronically and structurally distinct forms that differ in Lewis acidity. This type of reactivity can be exploited for the design of novel biomedical tools and strategies for biological-based therapeutics.

The development of therapeutic proteins is generating considerable interest. This interest is due to the high selectivity of proteins for a target and the high enzymatic activity a protein-based therapeutic can possess. There are, however, a number of challenges that need to be met. The delivery of peptides and proteins to the site of action is notoriously difficult. This challenge is due, in part, to the difficulty of molecules of peptidic size and complexity to reach the site of action, primarily the cytosol of a cell, and still maintain function. Although there is considerable effort to develop better ways to facilitate uptake into the cytosol, many of these strategies have serious limitations related to toxicity or physiological stability.

In chapter 2, I describe the development of a discreet, small-molecule boronic acid-based delivery strategy for the delivery of native protein into the cytosol of a cell. This was accomplished by appending a specialized boronic acid directly to solvent-exposed lysine residues of a protein of interest via an immolative linker possessing esterase sensitivity. The boronic acid, benzoxaborole, has enhanced affinity for the non-reducing saccharides that are prevalent on the surface of a cell. By increasing the number and affinity of interactions with the cell surface, this strategy was shown to significantly improve cellular uptake of both green fluorescent protein (GFP) and a cytotoxic variant of RNase A. Most importantly, these boronic acid appendages are quantitatively cleaved when exposed to endosomal and cytosolic esterases, releasing completely native, unmodified protein.

Another challenge with biologic therapeutics is overcoming the potential pleiotropic nature of the particular protein or enzyme. This challenge is explicit in the use of angiogenin (ANG) as a potential treatment of amyotrophic lateral sclerosis (ALS). ANG has been shown to be a powerful neuroprotectant and recovery agent in mouse models for ALS. Nonetheless, treatment with wild-type angiogenin can also promote undesired angiogenesis and tumor growth in the systemic system.

In chapter 3, I highlight a specific masking strategy that silences the catalytic activity of ANG under normal physiological conditions. When exposed to reactive oxygen species (ROS) leading to oxidative stress, this mask is removed, and catalytic activity is reconstituted. Oxidative stress is a key component in a number of disease states including cancer, cardiomyopathy, inflammation, diabetes, and many neurodegenerative diseases.

In ALS, the up-regulation of ROS in the central nervous system (CNS) is primarily responsible for the motor neuron degeneration characteristic of the disease. The presence of a boronic acid-masking agent engenders ANG with pro-drug characteristics, which not only expands the potential use of this protein as a therapeutic for ALS, but also offers a novel way of fine-tuning enzyme activity in a controlled fashion.

Boronic acids have also been utilized as important functional groups in medicinal chemistry. For example, VelcadeRTM (bortezomib), is a first-in-class boronic acid-containing protease inhibitor and the first boronic acid drug to be approved by the FDA. Another potential target is the homotetrameric protein transthyretin (TTR). The misfolding of TTR leads to amyloid fibril formation in the peripheral nervous system or cardiac tissue, leading to several known human amyloidogenic diseases. Current treatment for TTR amyloidosis requires a high-risk and costly liver transplant to remove the primary source of a deleterious TTR variant. Alternative strategies use small-molecules to stabilize the TTR quaternary structure and thereby preventing fibril formation.

In chapter 4, I discuss the development and characterization of a targeted library of small-molecule boronic acids that bind to the thyroxin (T 4) binding pockets and strongly inhibit fibril formation in both wild-type and a common disease variant, V30M--TTR. Structural data has shown that two molecules from this library bind in a covalent fashion with polar residues within the T4 binding pocket. These are the first boronic acids to interact covalently with a non-catalytic residue within a protein-binding site and opens new avenues for the use of boronic acids in the design of small-molecule therapeutics.