Abstract
ConspectusProximity-induced chemistry (PIC) refers to the transient reactivity between two or more molecules upon physical closeness which are otherwise unreactive. Harnessed by nature to control fundamental biological processes such as transcription and signal transduction, PIC increases the probability of correctly oriented, effective collisions, facilitating fundamental cellular processes. Within the field of chemical biology, PIC has been employed for several clinically relevant purposes, including the degradation of aberrant biomolecules and construction of protein therapeutics. This Account focuses on the application of PIC strategies for the development of site-specific bioconjugation techniques, termed proximity-assisted bioconjugation (PAB). Site-specific bioconjugation refers to the precise modification of biomolecules to generate homogeneous products. Such techniques are vital for the development of protein therapeutics including antibody-drug conjugates (ADCs), the investigation of the biological mechanisms of post-translational modifications (PTMs), and the visualization of biomolecular interactions in vitro and in vivo. While numerous strategies have been developed, many suffer from poor yields, limited product stability, demanding experimental procedures, and/or a lack of regioselectivity. Thus, PIC principles have been implemented to address these limitations, leading to the development of PAB strategies which achieve precise, regioselective modification of biomolecules. In this Account, we describe the development of PAB techniques within our group at the University of Cambridge and Instituto de Medicina Molecular (iMM) over the past five years. Our journey with PAB began serendipitously while investigating maleic acid derivatives for cysteine bioconjugation. Here, we discovered the secondary participation of proximal lysines on Trastuzumab-V205C and Gemtuzumab-V205C, conjugatable THIOMAB antibodies commonly used in ADCs, leading to the formation of distinct bioconjugate products relative to IgGs without such lysines. Further investigation into the proximal lysine (K207) of Trastuzumab-V205C revealed that residue 207 could be harnessed directly or mutated to precisely tune the stability of ADCs due to proximity interactions between K207 and covalent modifications of C205. Considering that two Trastuzumab drug conjugates are approved for clinical use, these findings have contributed to the evolving understanding of the chemical landscape of this antibody and help inform future ADC design and development. Further, we describe efforts from our group to develop two distinct PAB approaches: regioselective lysine acetylation of histone H3 and phage display-compatible peptide cyclization. These strategies combine induced-proximity with traditional bioconjugation techniques to enable regioselective modification of biomolecules which are historically difficult to selectively modify. These methods are readily adaptable to related systems and serve as representative examples of how to successfully develop PAB strategies for desired applications. In short, this Account highlights our group's contributions to and insights on PAB methodologies wherein we illustrate how PIC can be thoughtfully applied to bioconjugation techniques for various aims including regioselective bioconjugation and enhanced bioconjugate stability. We expect that PAB approaches will continue to diversify bioconjugation applications and greatly expand the toolkit of chemical biologists.