The transcription factor STAT5 catalyzes Mannich ligation reactions yielding inhibitors of leukemic cell proliferation

Abstract

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.

Direct Targeting Options for STAT3 and STAT5 in Cancer

“…Although tetrazoles were not active against STAT5B (>10 mM), the ligation products showed substantial activity, which the authors attribute to super-additive binding interactions. Intracellular physiological concentrations of formaldehyde also emphasize the viability of this molecule as a STAT inhibitor and the utility of bio-catalytic Mannich reactions [105]. A study by Juen et al reported the optimization of the STAT5 inhibitor, Cpd17f (Figure 3v) [103].…”

Recent advances in synthetic and medicinal chemistry of phosphotyrosine and phosphonate-based phosphotyrosine analogues

“…36 Recently Rademann and co-workers demonstrated that α-ketophosphonates, and particularly 4-phosphonocarbonyl phenylalanine (pcF), can also be used as photoactive enzymatically stable phosphotyrosine mimetics targeting, deactivating and labelling phosphotyrosine binding proteins. [81][82][83] For example, pcF was incorporated into the sequence of a STAT5 SH2 domaindirected peptide, labelled with 5,6-carboxyfluorescein (CF) for binding studies and protein detection. 81 The resulting peptide 5-CFGpcFLSLPPW-NH 2 exhibited 20-fold reduced binding affinity in comparison to a native phosphotyrosinecontaining peptide.…”

“…This approach was utilised to pull-down STAT5 from cell lysates using a similar pcF-containing, but dual-labelled (carboxyfluorescein and biotin) STAT5-SH2binding peptide, and was applied to confirm selectivity of the studied inhibitors. 82 To integrate pcF into peptides, Horatscheck et al developed building block 10 for Fmoc SPPS (Fig. 9B).…”

How Proteins Catalyze Ligand Formation: Protein-Templated Fragment Ligation Employed in the Validation of Cancer Targets

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Fragment-based drug discovery has been established as a powerful method for the assembly of optimized protein ligands. We employed protein-templated ligations, initially for the site-directed detection of low-affinity fragments and subsequently for the identification of potent fragment combinations that are useful as chemical tools [1][2][3][4].Reversible and irreversible reactions have been employed for ligand construction. Here, we will reflect on the reaction scope of protein-catalyzed ligand formation and consider the thermodynamic and kinetic implications of the reactions.

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“…Fragment-based drug discovery has been established as a powerful method for the assembly of optimized protein ligands. We employed protein-templated ligations, initially for the site-directed detection of low-affinity fragments and subsequently for the identification of potent fragment combinations that are useful as chemical tools [1][2][3][4].…”