Pharmacological Chaperoning: A Potential Treatment for PMM2-CDG

Supp. Fig. S1. Human PMM2 protein structure showing the location of the residues affected by the selected missense mutations detected in PMM2-CDG patients. Val44, Asp65, Pro113, Arg123, Arg141, Arg162, Thr237, Cys241 have been located in the human homodimeric PMM2 protein structure (PDB ID 2AMY). Changes affecting the represented residues are desestabilizing mutations. However, residues highlighted in blue, Arg123 and Arg141, also affect the catalitic propierties of the protein and the residue depicted in orange, Pro113, is implicated in protein dimerization. Changes in the residues highlighted in red are pure folding mutations (Yuste-Checa, et al., 2015). The initiation codon was termed codon 1

Supp. Fig. S2. Stabilizing effect of the four selected potential PCs (compounds IV, VI, VII and VIII) on WT-PMM2 and PMM2 mutants determined by DSF (a, b) and by degradation time course analysis (c). (a) Tm values of WT-PMM2 and PMM2 mutants (p.Val44Ala, p.Asp65Tyr, p.Arg123Gln, p.Arg141His, p.Arg162Trp, p.Thr237Met and p.Cys241Ser) in the absence (-; light grey bars) or presence of 2% DMSO (intermediate grey bars) or the four selected potential PCs at 40 μM (dark grey bars) or 80 μM (black bars). (b) Representative thermal denaturation profiles of WT-PMM2 and an unstable mutant (caused by the mutation p.Asp65Tyr) incubated with DMSO (grey) and with compounds IV, VI, VII and VIII at 40 μM or 80 μM (black). The midpoint melting temperature (Tm) was calculated as the temperature for which half of the protein (at relative fluorescence 0.5) was in the unfolded state (dashed lines). ∆Tm was calculated as the difference between the Tm in the presence of the compound and Tm in the presence of DMSO. T (°C): Temperature. (c) Relative half-life (in minutes) of WT-PMM2 and mutant proteins (p.Asp65Tyr, p.Pro113Leu, p.Arg162Trp, p.Thr237Met) in a prokaryotic transcription-translation-coupled system in the absence (light grey bars) or presence of 2% DMSO (intermediate grey bars) or the selected potential PCs at 40 μM (dark grey bars) or 80 μM (black bars). Data represent the mean±SD of at least three independent experiments. ***p<0.001, **p<0.01, *p<0.05 (differences compared to DMSO controls).

Supp. Fig. S2. Effect of the four selected potential PCs (compounds IV, VI, VII and VIII) on the enzyme activity of WT-PMM2 and PMM2 mutants in the cellular disease model. Healthy and patient-derived fibroblasts stably transduced with the WT or their own folding mutation (WT, p.Asp65Tyr, p.Pro113Leu, p.Arg162Trp and p.Thr237Met, respectively) were incubated with different concentrations of compound IV, VI, VII or VIII for 48 h. PMM2 activity in the soluble fraction of the cellular extracts. Baseline enzyme activity was considered as ‘1’ in each cell line (-). The results are the mean±SD of at least three independent experiments. p<0.001, **p<0.01, *p<0.05 (differences in compound activity with respect to the corresponding baseline)

Supp. Table S1. Physicochemical properties of the hits compounds.

Supp. Table S2. Analysis of the pharmaco-chemical properties of the four compounds (IV, VI, VII and VIII) using the SmartsFilter website.