High binding affinities of GAG toward extracellular regulatory proteins are governed by recognition diversity, sulfation pattern, length, and anomeric functionalization.
Protein-templated fragment ligations have been established as a powerful method for the assembly and detection of optimized protein ligands. Initially developed for reversible ligations, the method has been expanded to irreversible reactions enabling the formation of super-additive fragment combinations. Here, protein-induced Mannich ligations are discovered as a biocatalytic reaction furnishing inhibitors of the transcription factor STAT5. STAT5 protein catalyzes multicomponent reactions of a phosphate mimetic, formaldehyde, and 1H-tetrazoles yielding protein ligands with greatly increased binding affinity and ligand efficiency. Reactions are induced under physiological conditions selectively by native STAT5 but not by other proteins. Formation of ligation products and (auto-)inhibition of the reaction are quantified and the mechanism is investigated. Inhibitors assembled by STAT5 block specifically the phosphorylation of this protein in a cellular model of acute myeloid leukemia (AML), DNA-binding of STAT5 dimers, expression of downstream targets of the transcription factor, and the proliferation of cancer cells in mice.
Sulfated saccharides are an essentialp art of extracellularm atrices, and they are involvedi nalarge number of interactions. Sulfated saccharide matricesi no rganismsa ccumulate heavy metal ions in addition to othere ssentialm etal ions. Accumulation of heavy metal ions alters the function of the organisms and cells, resulting in severea nd irreversible damage. The effect of the sulfation pattern of saccharides on heavym etal binding preferences is enigmatic because the accessibility to structurally definedsulfated sac-charides is limited and because standard analytical techniquescannot be used to quantify these interactions.Wedeveloped an ew strategy that combines enzymatic and chemicals ynthesis with surfacec hemistry and label-free electrochemical sensing to study the interactions between well-defined sulfated saccharides and heavy metal ions. By using these tools we showedt hat the sulfation pattern of hyaluronic acid governs their heavy metal ions binding preferences.
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