RESUMO
The complex social ecosystem regulates the spectrum of human behavior. However, it becomes relatively easier to understand if we disintegrate the contributing factors, such as locality and interacting partners. Interestingly, it draws remarkable similarity with the behavior of a residue placed in a social setup of functional groups in a protein. Can it inspire principles for creating a unique environment for the precision engineering of proteins? We demonstrate that localization-regulated interacting partner(s) could render precise and traceless single-site modification of structurally diverse native proteins. The method targets a combination of high-frequency Lys residues through an array of reversible and irreversible reactions. However, excellent simultaneous control over chemoselectivity, site selectivity, and modularity ensures that the user-friendly protocol renders acyl group installation, including post-translational modifications (PTMs), on a single Lys. Besides, it offers a chemically orthogonal handle for the installation of probes. Also, a purification protocol integration delivers analytically pure single-site tagged protein bioconjugates. The precise labeling of a surface Lys residue ensures that the structure and enzymatic activities remain conserved post-bioconjugation. For example, the precise modification of insulin does not affect its uptake and downstream signaling pathway. Further, the method enables the synthesis of homogeneous antibody-fluorophore and antibody-drug conjugates (AFC and ADC; K183 and K249 labeling). The trastuzumab-rhodamine B conjugate displays excellent serum stability along with antigen-specific cellular imaging. Further, the trastuzumab-emtansine conjugate offers highly specific antiproliferative activity toward HER-2 positive SKBR-3 breast cancer cells. This work validates that disintegrate theory can create a comprehensive platform to enrich the chemical toolbox to meet the technological demands at the chemistry, biology, and medicine interface.
Assuntos
Ecossistema , Lisina , Humanos , Lisina/química , Proteínas/química , Trastuzumab/química , CatáliseRESUMO
The maintenance of machinery requires its operational understanding and a toolbox for repair. The methods for the precision engineering of native proteins meet a similar requirement in biosystems. Its success hinges on the principles regulating chemical reactions with a protein. Here, we report a technology that delivers high-level control over reactivity, chemoselectivity, site-selectivity, modularity, dual-probe installation, and protein-selectivity. It utilizes cysteine-based chemoselective Linchpin-Directed site-selective Modification of lysine residue in a protein (LDMC-K). The efficiency of the end-user-friendly protocol is evident in quantitative conversions within an hour. A chemically orthogonal C-S bond-formation and bond-dissociation are essential among multiple regulatory attributes. The method offers protein selectivity by targeting a single lysine residue of a single protein in a complex biomolecular mixture. The protocol renders analytically pure single-site probe-engineered protein bioconjugate. Also, it provides access to homogeneous antibody conjugates (AFC and ADC). The LDMC-K-ADC exhibits highly selective anti-proliferative activity towards breast cancer cells.
Assuntos
Cisteína , Imunoconjugados , Cisteína/química , Imunoconjugados/química , Lisina/química , Engenharia de Proteínas , Proteínas/químicaRESUMO
Amphiphilic assemblies made from diverse synthetic building blocks are well known for their biomedical applications. Here, we report the synthesis of gemini-type amphiphilic molecules that form stable assemblies in water. The assembly property of molecule M2 in aqueous solutions was first inferred from peak broadening observed in the proton NMR spectrum. This was supported by dynamic light scattering and transmission electron microscopy analysis. The assembly formed from M2 (M2agg) was used to solubilize the hydrophobic drugs curcumin and doxorubicin at physiological pH. M2agg was able to effectively solubilize curcumin as well as protect it from degradation under UV irradiation. Upon solubilization in M2agg, curcumin showed excellent cell permeability and higher toxicity to cancer cells over normal cells, probably because of enhanced cellular uptake and increased stability. M2agg also showed pH-dependent release of doxorubicin, resulting in controlled toxicity on cancer cell lines, making it a promising candidate for the selective delivery of drugs to cancer cells.