Screening-based discovery of drug-like O-GlcNAcase inhibitor scaffolds

Abstract

O-GlcNAcylation is an essential posttranslational modification in metazoa. Modulation of O-GlcNAc levels with small molecule inhibitors of O-GlcNAc hydrolase (OGA) is a useful strategy to probe the role of this modification in a range of cellular processes. Here we report the discovery of novel, low molecular weight and drug-like O-GlcNAcase inhibitor scaffolds by high-throughput screening. Kinetic and X-ray crystallographic analyses of the binding modes with human/bacterial O-GlcNAcases identify some of these as competitive inhibitors. Comparative kinetic experiments with the mechanistically related human lysosomal hexosaminidases reveal that three of the inhibitor scaffolds show selectivity towards human OGA. These scaffolds provide attractive starting points for the development of non-carbohydrate, drug-like OGA inhibitors.

Fig. 1

(A) Prestwick library screen against CpOGA. The scatter-plot shows on the horizontal axis the remaining activity of CpOGA in presence of the compounds (measured by the liberation of 4MU) and on the vertical axis the intrinsic absorbance/fluorescence of the compounds at the excitation and emission wavelength (360 nm/460 nm). Points in the lower left corner indicate compounds that do not absorb at the excitation wavelength, yet do exhibit reduced fluorescence and thus reduce CpOGA activity. Six compounds are labelled in coloured dots, according to the legend in the graph. (B) Chemical structures of compounds selected with the aid of the Prestwick screening data: (a) N⁶-methyladenine, (b) diprophylline, (c) acetazolamide, (d) semustine, (e) streptozotocin (STZ), (f) ketoconazole and (g) buspirone. (C) Diprophylline is a competitive inhibitor of CpOGA. Steady-state kinetic data (triplicates) using 0.2 nM CpOGA, 0–25 μM substrate (4MU-GlcNAc), 0–110 μ inhibitor and 7 min reaction time were fitted using the standard equation for competitive inhibition in the GraFit program . (D) Dose–response curves of the selected compounds against hOGA. Data obtained with the 4MU-GlcNAc assay (2 nM hOGA, 80 μM 4MU-GlcNAc, 0.7 nM–100 μM inhibitor, 60 min reaction time) from buspirone, diprophylline and acetazolamide, semustine, N⁶-methyladenine and ketoconazole were fitted using the standard IC₅₀ equation in the GraFit program .

Fig. 2

(A) Stereo figure of diprophylline in the active site of CpOGA. The CpOGA active site is shown with residues contributing to diprophylline binding and/or surrounding the compound, as sticks with grey carbon, red oxygen and blue nitrogen atoms. Diprophylline is depicted in sticks with green carbon, red oxygen, blue nitrogen atoms. Hydrogen bonds between the ligand and the protein are indicated by black dashed lines. Unbiased ∣F_o∣ − ∣F_C∣, ϕ_calc electron density (2.75 σ) for diprophylline is shown in cyan. (B) Stereo view of the superimposed crystallographically determined complexes of CpOGA with diprophylline (colour scheme as in panel (A) and STZ (PDB entry 2VUR, depicted in sticks with cyan carbon, red oxygen and blue nitrogen atoms). Hydrogen bonds between the protein and diprophylline are indicated by black dashed lines.