sp²-Iminosugars targeting human lysosomal β-hexosaminidase as pharmacological chaperone candidates for late-onset Tay-Sachs disease

Figures & data

Figure 1. Structures of the HexA inhibitors DNJNAc, NHAc-CAS, PUGNAc, NAG-thiazoline, pymetahmine, M31850, DMH-DNJNAc, LABNAc, Gal-PUGNAc and Gal-NAG-thiazoline, and of the sp²-iminosugars NOI-NJ, pFPhT-DGJ and 6S-NBI-DGJ.

Figure 2. Schematic representation of the proposed pharmacological chaperone strategy tackling TSD causative misfolded HexA, based on active-site directed sp²-iminosugars (a generic bicyclic amphiphilic structure with a hydrophobic R substituent is depicted).

Scheme 1. Synthesis of the DGJNAc thioureas 1–4 and the corresponding bicyclic isothioureas 5–8.

Table 1. IC₅₀ or K_i values for sp²-iminosugars 1–8 human lysosomal hexosaminidases and recombinant human O-GlcNAcase.^a

	Human enzymes
	IC50 (µM)	Ki (µM)
Compound	Total Hex^b	HexA^b pH 7/pH 5	OGA
DGJNAc thioureas
1	>100	4.7/33.7	>100
2	>100	>100/>100	>100
3	>100	>100/>100	>100
4	10.9	0.91/9.4	>100
DGJNAc isothioureas
5	2.6	0.99/9.8	>100
6	1.3	0.58/6.4	>100
7	2.2	0.66/6.9	25
8	0.33	0.41/3.9	>100

^aIC₅₀ and K_i data are the mean of three independent determinations; errors are in the range of 5–10% and are omitted for clarity. Inhibition was competitive in all cases. No inhibition was observed for any compound at 200 μM on lysosomal α-glucosidase, α- and β-galactosidases and α- and β-mannosidases in cell lysates.

^bDetermined in human fibroblast lysates.

Figure 3. Predicted binding modes of phenylthiourea 4 (A; magenta sticks) and protonated octyliminothiazolidine 6 (B; orange sticks) to HexA (coordinates from PDB ID 2GK1; tan sticks) from the molecular docking experiments. Hydrogen atoms from ligands are not shown for clarity. Hydrogen bonds are represented as black dashed lines, and hydrophobic interactions as red dashed lines.

Figure 4. Effect in the HexA activity of different concentrations of the thioureas DGJNAc thioureas 1–4 and the isothiourea derivatives 5–8 in wild type (A) and G269S TSD fibroblasts (B) relative to controls in the absence of any compound. Bars represent average values ± SD (n = 3).

Figure 5. Effect of compound 6 on the traffic of HexA to lysosomes in WT and in TSD-GS fibroblast. Fibroblasts were treated with 20 µM complexed chaperone. Lysosomal marker (lysosome associated membrane protein 2, LAMP-2). HexA is visualised in green; in the merged images, yellow denotes colocalization in lysosomes. The shown pictures are representative of more than 100 cells in 10 randomly obtained images.