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The proximity-enabled sulfur(vi) fluoride exchange (SuFEx) reaction generates specific covalent linkages between proteins in cells and in vivo, which opens innovative avenues for studying elusive protein-protein interactions and developing potent covalent protein drugs. To exploit the power and expand the applications of covalent proteins, covalent linkage formation between proteins is the critical step, for which fundamental kinetic and essential properties remain unexplored. Herein, we systematically studied SuFEx kinetics in different proteins and conditions. In contrast to in small molecules, SuFEx in interacting proteins conformed with a two-step mechanism involving noncovalent binding, followed by covalent bond formation, exhibiting nonlinear rate dependence on protein concentration. The protein SuFEx rate consistently changed with protein binding affinity as well as chemical reactivity of the functional group and was impacted by target residue identity and solution pH. In addition, kinetic analyses of nanobody SR4 binding with SARS-CoV-2 spike protein revealed that viral target mutations did not abolish covalent binding but decreased the SuFEx rate with affinity decrease. Moreover, off-target cross-linking of a SuFEx-capable nanobody in human serum was not detected, and the SuFEx-generated protein linkage was stable at cellular acidic pHs, suggesting SuFEx suitability for in vivo usage. These results advanced our understanding of SuFEx reactivity and kinetics in proteins, which is invaluable for ongoing exploration of SuFEx-enabled covalent proteins for basic biological research and creative biotherapeutics.

Molecularly targeted radionuclide therapies (TRTs) struggle with balancing efficacy and safety, as current strategies to increase tumor absorption often alter drug pharmacokinetics to prolong circulation and normal tissue irradiation. Here we report the first covalent protein TRT, which, through reacting with the target irreversibly, increases radioactive dose to the tumor without altering the drug's pharmacokinetic profile or normal tissue biodistribution. Through genetic code expansion, we engineered a latent bioreactive amino acid into a nanobody, which binds to its target protein and forms a covalent linkage via the proximity-enabled reactivity, cross-linking the target irreversibly in vitro, on cancer cells, and on tumors in vivo. The radiolabeled covalent nanobody markedly increases radioisotope levels in tumors and extends tumor residence time while maintaining rapid systemic clearance. Furthermore, the covalent nanobody conjugated to the α-emitter actinium-225 inhibits tumor growth more effectively than the noncovalent nanobody without causing tissue toxicity. Shifting the protein-based TRT from noncovalent to covalent mode, this chemical strategy improves tumor responses to TRTs and can be readily scaled to diverse protein radiopharmaceuticals engaging broad tumor targets.

The introduction of new covalent bonds into proteins is affording novel avenues for protein research and applications, yet it remains difficult to generate covalent linkages at all possible sites and across diverse protein classes. Herein, we genetically encoded meta-fluorosulfate-L-tyrosine (mFSY) to selectively react with lysine, tyrosine, and histidine via proximity-enabled SuFEx reaction. mFSY was able to target residues that were elusive for previous unnatural amino acids, and permitted engineering of various proteins including affibody, nanobody, and Fab into covalent binders that irreversibly cross-linked EGFR and HER2. mFSY is thus valuable for developing covalent proteins for biological research, synthetic biology, and biotherapeutics.

Genetically introducing novel chemical bonds into proteins provides innovative avenues for biochemical research, protein engineering, and biotherapeutic applications. Recently, latent bioreactive unnatural amino acids (Uaas) have been incorporated into proteins to covalently target natural residues through proximity-enabled reactivity. Aryl fluorosulfate is particularly attractive due to its exceptional biocompatibility and multitargeting capability via sulfur(VI) fluoride exchange (SuFEx) reaction. Thus far, fluorosulfate-l-tyrosine (FSY) is the only aryl fluorosulfate-containing Uaa that has been genetically encoded. FSY has a relatively rigid and short side chain, which restricts the diversity of proteins targetable and the scope of applications. Here we designed and genetically encoded a new latent bioreactive Uaa, fluorosulfonyloxybenzoyl-l-lysine (FSK), in E. coli and mammalian cells. Due to its long and flexible aryl fluorosulfate-containing side chain, FSK was particularly useful in covalently linking protein sites that are unreachable with FSY, both intra- and intermolecularly, in vitro and in live cells. In addition, we created covalent nanobodies that irreversibly bound to epidermal growth factor receptors (EGFR) on cells, with FSK and FSY targeting distinct positions on EGFR to counter potential mutational resistance. Moreover, we established the use of FSK and FSY for genetically encoded chemical cross-linking to capture elusive enzyme-substrate interactions in live cells, allowing us to target residues aside from Cys and to cross-link at the binding periphery. FSK complements FSY to expand target diversity and versatility. Together, they provide a powerful, genetically encoded, latent bioreactive SuFEx system for creating covalent bonds in diverse proteins in vitro and in vivo, which will be widely useful for biological research and applications.

Site-specific modification of proteins with functional molecules provides powerful tools for researching and engineering proteins. Here we report a new chemical conjugation method which photocages highly reactive but chemically selective moieties, enabling the use of protein-inert amines for selective protein modification. New amino acids FnbY and FmnbY, bearing photocaged quinone methides (QMs), were genetically incorporated into proteins. Upon light activation, they generated highly reactive QM, which rapidly reacted with amine derivatives. This method features a rare combination of desired properties including fast kinetics, small and stable linkage, compatibility with low temperature, photocontrollability, and widely available reagents. Moreover, labeling via FnbY occurs on the β-carbon, affording the shortest linkage to protein backbone which is essential for advanced studies involving orientation and distance. We installed various functionalities onto proteins and attached a spin label as close as possible to the protein backbone, achieving high resolution in double electron-electron paramagnetic resonance distance measurements.

The article explores the evolving landscape of gender dysphoria treatment in children and adolescents, focusing on social, legal, and medical transitions. It highlights the historical rarity of gender dysphoria and the recent surge in referrals, particularly among post-pubertal individuals assigned female at birth. It includes the introduction of puberty blockers and hormonal treatments, influenced by Dutch protocols and subsequent guidelines.

The article focuses on addressing the mental health crisis among children, adolescents, and young people in Europe, exacerbated by events like the COVID-19 pandemic. It discusses the impact on mental health, including increased anxiety, depression, and other issues, and highlights the European Union's response with comprehensive approaches and funding initiatives to improve mental health care accessibility, quality, and integration into broader policy frameworks.