Improvement of Mutant Galactose-1-Phosphate Uridylyltransferase (GALT) Activity by FDA-Approved Pharmacochaperones: A Preliminary Study

Abstract

Classic galactosemia is a rare disease with long-term consequences that seriously affect the quality of life of patients. To date, various therapeutic approaches are being developed, but treatments that target the molecular defects in the mutant galactose-1-phosphate uridylyltransferase (GALT) gene are lacking. We conducted a computational search for putative pharmacochaperones by applying a drug repurposing strategy, and we found that one compound, already active as a pharmacochaperone in another pathology, doubled the enzymatic activity of the purified mutant enzyme in an in vitro test. Furthermore, an extensive computational search in a database of known active molecules found another compound able in its turn to improve in vitro enzymatic activity. Both compounds are predicted to interact with a cavity at the enzyme interface previously supposed to be an allosteric site for the GALT enzyme. In vitro tests confirmed also the reduced accumulation of galactose-1-phosphate (G1P) in fibroblasts of patients. Although these results must be considered preliminary, our findings pave the way for future research lines focused on the search for promising pharmacochaperones that can directly rescue the activity of the enzyme.

Keywords: classic galactosemia; drug repurposing; pharmacochaperones; protein mutations; rare diseases; virtual screening.

Figure 1

The Leloir pathway for galactose metabolism. This biochemical pathway is composed of four steps, each involving a different enzyme. In the first step, β-D-galactose is converted to α-D-galactose by the enzyme galactose mutarotase (GALM). Then, α-D-galactose is converted to galactose-1-phosphate (G1P) by galactokinase 1 (GALK1). Galactose-1-phosphate uridyltransferase (GALT) transfers a uridine monophosphate group from uridine diphosphate (UDP)-glucose to galactose-1-phosphate to form UDP-galactose. Finally, UDP-galactose-4′-epimerase (GALE) interconverts UDP-galactose and UDP-glucose. The figures of the sugars have been downloaded from PubChem (https://pubchem.ncbi.nlm.nih.gov/) (accessed on 12 January 2025), whereas the figures of the enzymes have been downloaded from the Galactosemia Proteins Database (http://protein-variants.eu/galactosemia/) (accessed on 12 January 2025) and rendered in cartoon mode with ChimeraX (https://www.rbvi.ucsf.edu/chimerax/) (accessed on 12 January 2025).

Figure 2

Chemical structures of the drugs tested in this work. Panel (a): ambroxol; panel (b): pyrimethamine; panel (c): ciclopirox; panel (d): ombrabulin. The first three molecules (panels (a–c)) were selected from the literature, as described in Section 2.1.1, whereas ombrabulin was identified by a virtual screening procedure based on the pharmacophore derived from ciclopirox, as described in Section 2.1.2. These structures have been downloaded from PubChem (https://pubchem.ncbi.nlm.nih.gov/) (accessed on 12 January 2025).

Figure 3

Interactions between wtGALT (left panels) with selected drugs, and between p.Gln188Arg enzyme (right panels) with selected drugs, identified by docking focused on the putative allosteric site (site X). All the docking simulations were made in the presence of both substrates in the active sites. Panels (a,b) show the docking of ambroxol in the box focused on site Xa (cyan) or Xb (blue); panels (c,d) show the docking of pyrimethamine in the box focused on site Xa (yellow) or Xb (orange); panels (e,f) show the docking of ciclopirox in the box focused on site Xa (pink) or Xb (magenta). The three drugs are represented in CPK mode. The substrates are represented in stick mode and colored black, the Zn ion as a sphere and colored grey. In contrast to ambroxol (panels (a,b)) and pyrimethamine (panels (c,d)), ciclopirox (panels (e,f)) identified only one conformation within the X site, thus showing a higher specificity of interaction with that site.

Figure 4

Detailed interactions between ciclopirox and wtGALT or p.Gln188Arg enzymes identified by docking focused on the putative allosteric site (site X). In all cases, the pose with best energy is also representative of the most populated cluster. Panel (a): result of docking on site Xa on wtGALT. Panel (b): result of docking on site Xa on p.Gln188Arg. Panel (c): result of docking on site Xb on wtGALT; Panel (d): result of docking on site Xb on p.Gln188Arg. In all cases, the interactions of ciclopirox with the enzyme were the same, confirming that the molecule binds specifically to the enzyme pocket.

Figure 5

Further identification of pharmacological hits specific to site X of wtGALT and p.Gln188Arg enzymes starting from ciclopirox. Pharmacophoric models (PM) were obtained for each best pose of the docking of ciclopirox with site Xa and Xb. For each of these starting poses, the PM with the highest selectivity was used to make the corresponding pharmacophore hit search in DrugBank. Only hits with a fitness score > 3 (as recommended) were selected. Then, selection criteria were applied as described in the scheme, to identify the final 25 best hits.

Figure 6

Detailed interactions between DB12882 (ombrabulin) and wtGALT or p.Gln188Arg enzymes identified by docking focused on the putative allosteric site (site X). Panel (a): result of docking on site Xa on wtGALT, pose with best energy. Panel (b): result of docking on site Xa on wtGALT, pose representative of the most populated cluster. Panel (c): result of docking on site Xb on wtGALT, pose with best energy. Panel (d): result of docking on site Xb on wtGALT, pose representing the most populated cluster. Panel (e): result of docking on site Xa on p.Gln188Arg, pose with best energy. Panel (f): result of docking on site Xa on p.Gln188Arg, pose representative of the most populated cluster. Panel (g): result of docking on site Xb on p.Gln188Arg, pose with best energy. Panel (h): result of docking on site Xb on p.Gln188Arg, pose representing the most populated cluster. The interactions between DB12882 (ombrabulin) and the putative allosteric site X involve most of the residues interacting with ciclopirox, thereby confirming the predicted ability of ombrabulin to interact with site X similarly to the parent compound ciclopirox, especially in the poses with the best energy.

Figure 7

Effect of putative PCs on purified mutant GALT enzyme activity and galactose-1-phosphate (G1P) accumulation in patient fibroblasts. Panel (a): Purified mutant GALT enzyme activity was determined in the presence of different compounds. Enzyme activity is expressed as units/mg enzyme/minute. Error bars indicate mean activity ± standard deviation of the mean (n = 3 independent experiments). Panel (b): G1P levels detected in untreated wt, untreated patient, and patient fibroblasts treated with various compounds. Error bars represent mean G1P/protein concentrations ± standard deviation (n = 3 independent experiments. Statistical significance: * p < 0.05, compared to untreated patient fibroblasts (Student’s t-test). ns: not significant.