OleD Loki as a Catalyst for Hydroxamate Glycosylation

Abstract

Herein we describe the ability of the permissive glycosyltransferase (GT) OleD Loki to convert a diverse set of >15 histone deacetylase (HDAC) inhibitors (HDACis) into their corresponding hydroxamate glycosyl esters. Representative glycosyl esters were subsequently evaluated in assays for cancer cell line cytotoxicity, chemical and enzymatic stability, and axolotl embryo tail regeneration. Computational substrate docking models were predictive of enzyme-catalyzed turnover and suggest certain HDACis may form unproductive, potentially inhibitory, complexes with GTs.

Keywords: HDAC; glucosylation; glycosyltransferase; histone deacetylase.

Figure 1.

A) Schematic of the colorimetric screen. B) Reaction progress as monitored via ΔA₄₁₀ using the standard assay format (see panel A; 1 mm hydroxamate, 2 mm CINP β-d-Glc, 0.1 mm UDP, 25 mm Tris pH 8.0, 5 mm MgCl₂, 0.25 μm OleD Loki, 30 μL total volume, 30 °C, 1 h). Vehicle alone (no acceptor) served as the negative control and a well-characterized OleD substrate (4-MeUmb) as the positive control. Assays were conducted in triplicate. GT, OleD Loki; ClNP β-d-Glc, 2-chloro-4-nitrophenol β-d-glucose; UDP, uridine diphosphate. The bars in red represent the compounds selected for scale-up and characterization. Hydroxamate structures are illustrated in Figure 2.

Figure 2.

HDACis tested as putative OleD Loki substrates. Compounds in black were identified as hits in the colorimetric screen and subsequently confirmed by LC–MS. Percent conversion (in parentheses) was determined via HPLC peak integration. Compounds in grey lacked turnover.

Figure 3.

Representative hydroxamate glycosyl ethers selected for scale-up and further study.

Figure 4.

Comparative cytotoxicity of representative hydroxamates and hydroxamate glycosyl ethers toward A) human colorectal (HTC116) and B) non-small cell lung (A549 cells) cancer cell lines.

Figure 5.

The predicted binding OleD binding models for A) abexinostat (high turnover, Figures 1 and 2, 1), B) tepoxalin (moderate turnover, Figures 1 and 2), C) MC1568 (low turnover, Figures 1 and 2) and D) tubacin (no turnover, Figures 1 and 2) based on the wild-type OleD-erythromycin ligand-bound crystal structure (PDB ID: 2IYF). Yellow dashed lines reflect putative key binding/catalytic contacts (distances in Å), including that of the ligand hydroxamate with the putative OleD active site base (His19 side chain of the active-site H19–D110 acid–base pair).