Cofactor Analogues as Active Site Probes in Lysine Acetyltransferases

Abstract

Lysine acetyltransferases (KATs, also termed histone acetyltransferases, HATs) catalyze the acetylation of substrate lysine residues by employing the cofactor acetyl-coenzyme A (AcCoA), thereby providing a dynamic control mechanism of protein function. Because of their major involvement in cell development and homeostasis, small-molecule modulators of KAT activity are urgently needed to assess their therapeutic potential and for probing their underlying biology. Recent advances in the field suggest that targeting the cofactor binding site represents a promising strategy for identifying potent and selective ligands. Here, we present the synthesis of two functional cofactor-based chemical probes and their usage as mechanistic tools in a broadly applicable assay platform. A fluorescence polarization (FP)-based binding assay was combined with biolayer interferometry competition analysis and a FP competition activity immunoassay to enable easy, reliable, and profound evaluation of ligands that target the KAT cofactor binding site.

Harnessing Ionic Selectivity in Acetyltransferase Chemoproteomic Probes

“…In addition to acetyltransferases, we identified several CoA binders and AT interactors that show selective enrichment by probes 1 or 2 . While we caution that the direct interaction of our probes with many of these targets remains to be validated, a recent study has demonstrated that similar Ahx-CoA analogues can be readily converted into fluorescence polarization probes for small molecule inhibitor screening . This suggests that the potential for the chemoproteomic probe–enzyme pairs described may provide a universal entry into biological profiling, functional enzyme analysis, and inhibitor development, as has been demonstrated for other probe classes. − Finally, we note several limitations of our approach as currently constituted.…”

“…Capture resin 1 consists of CoA tethered through a thioether linkage to a series of four 6-aminohexanoic acid (Ahx) linkers. Similar to Lys-CoA, Ahx-CoA analogues have been found to bind a wide variety of acetyltransferases with good affinity while also providing a straightforward handle for functionalization, enabling them to be used as a general scaffold for the development of fluorescence polarization assays. , In addition to facilitating our physiochemical discernment strategy (vide infra), we hypothesized that characterizing their proteome-wide binding properties might expand the applications of these novel CoA analogues. Capture resin 2 is near identical in structure, containing a single substitution of the first Ahx residue (adjacent to the CoA) with an aspartate.…”

Effect of lysine side chain length on histone lysine acetyltransferase catalysis

“…KATs have been found to catalyze the transfer of sterically more demanding acyl moieties, including propionyl, crotonyl, and butyryl, both in vivo and in vitro, from their respective acyl CoA cosubstrate, with differences in cosubstrate acceptance among families 7,8,[34][35][36] . The promiscuity of AcCoA led to the development of several chemical probes for KATs as well as a click-based labelling procedure for the detection of KATs substrates, employing synthetic CoA surrogates and engineered KATs [37][38][39] . Although in the recent years the investigation of epigenetic enzymes' susceptibility towards lysine chemical modification gathered great attentions, especially with regards to histone lysine methyltransferases (KMTs), histone lysine demethylases (KDMs) and KDACs, we are currently lacking basic molecular knowledge in the context of histone lysine acetylation [40][41][42][43][44][45][46][47] .…”

Funktionalisierte Cofaktor‐Analoga für die Erforschung von Interaktomen und darüber hinaus

“…Dies verdeutlicht die Vielseitigkeit der Methodik bei der Suche nach neuen Inhibitoren. [97] In fluoreszierenden Cofaktor-Analoga werden häufig Linker verwendet, um die Störung der Bindung an deren Zielprotein(e) zu minimieren. So wurde vor kurzem festgestellt, dass bei Rhodamin-SAM-Analoga ein Linker notwendig ist, damit diese von der Methyltransferase M.TaqI erkannt werden und somit DNA-Ziele des Enzyms markiert werden können.…”