Biospecific Chemistry via Latent Bioreactive Uaa

Biospecific chemistry enabled by latent bioreactive Uaa via proximity-enabled bioreactivity

Chemical biology has afforded innovative chemistry-based approaches for studying biology, yet on the macromolecular level these chemistries are largely bio-orthogonal or limited to in vitro settings. To obtain physiologically relevant results, we are developing biospecific chemistry in live systems for biological studies in vivo.

Figure 2.1. Biospecific chemistry for introducing covalent linkages between biomacromolecules.

Proximity-enabled bioreactivity: Protein side chains can spontaneously form a covalent linkage via cysteines only. We broke this natural barrier by adding new covalent bonds into proteins in live cells. We have designed a Uaa to react with a natural amino acid through proximity-enabled bioreactivity: The reactivity of the bioreactive Uaa is fine-tuned so that it remains intact inside cells and through protein translation; only when the Uaa is placed proximal to the target natural amino acid residue will the reaction occur specifically forming a new covalent bond.

Figure 2.2. Generation of new covalent bonds within and between proteins through proximity-enabled bioreactivity. Bioreactive Uaa F_fact was designed to react with Cys only when they are in proximity.

We have designed and encoded a suite of latent bioreactive Uaas capable of targeting various natural amino acids, enabling the generation of covalent bonds within and between proteins. We demonstrated that the resultant new bond enables infinite binding affinity to proteins, enhances optical properties challenging to engineer, and allows us precisely probe ligand-GPCR interaction in live mammalian cells. This new concept of proximity-enabled bioreactivity thus opens the door to genetically encoding a new class of Uaas, the latent bioreactive Uaas, which is affording novel avenues toward generating protein properties previously inaccessible, with broad applications in biological studies, protein therapeutics, and synthetic biology.

In addition to covalently targeting proteins, we recently demonstrated that proximity-enabled bioreactivity can also be applied to covalently target RNAs and carbohydrates.

Optical nano-switch for molecular opto-biology: The ability to control protein function with light provides excellent temporal and spatial resolution for precise investigation in situ. We have developed a nano-switch technology for optical control of proteins in their native settings with general applicability and ultra-specificity. A photo-reversible Uaa is genetically incorporated into the target protein, which reacts with a nearby natural amino acid to build a nano-bridge in situ via proximity-enabled bioreactivity. Our method has minimal perturbation to proteins under study, can be generally applied to proteins, and has high resolution that is specific for desired subunits, domains, and even single residues.

Figure 2.3. Photoswitchable click amino acids (PSCaas) react with Cys to build optical nanoswitch onto proteins in situ, which allows reversible optical modulation of protein structure and function with high resolution up to the amino acid residue level.

GECX for chemical cross-linking in vivo: Identification of weak and transient interactions among biomolecules in the native settings is critical for understanding biology and pathophysiology but remains highly challenging. Using the principle of Genetically Encoded Chemical Cross-linking (GECX), we succeeded in selectively cross-linking protein-protein, protein-RNA, and protein-carbohydrate interactions in live cells with single unit resolution. GECX integrates the advantages of genetic encoding to achieve live cell compatibility and of chemical cross-linking to achieve spontaneous reactivity and residue specificity. GECX is able to capture low-affinity binding, elusive interactions, and low abundant events in live cells for identification, and to selectively build covalent linkages among biomolecules for rational engineering.

Figure 2.4. GECX to capture protein−protein interactions in live cells for subsequent identification by MS.

PERx for developing covalent protein drugs: While small molecule covalent drugs have seen significant success, the potential of covalent protein therapeutics remains largely unexplored. We developed a versatile platform technology called Proximity-enabled Reactive Therapeutics (PERx) for creating covalent protein drugs. We have demonstrated that PERx-capable protein therapeutics exhibit enhanced potency and efficacy compared to traditional protein drugs in applications such as cancer immunotherapy, SARS-CoV-2 neutralization, and targeted radionucleotide therapy. The widespread adoption of PERx is heralding a new era in covalent biologic treatments.

Figure 2.5. Principle of proximity-enabled reactive therapeutics (PERx) for developing covalent protein drugs.

References

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