Drug screening identifies tazarotene and bexarotene as therapeutic agents in Multiple Sulfatase Deficiency (2023)
Figure 1

Fig. 1. Drug screen and evaluation of positive hit drugs using immortalized MSD patient derived cells.

(A) Indicative plot of one 96-well screening plate of the MSD-ARSA high-throughput screening assay with negative and positive controls included. Individual OD values were given for each well (circles) and indicate ARSA activity (grey area: baseline activity). Three hit drugs exceeded the upper baseline OD range. N = 1 experiment per well and drug treatment, final concentration of each drug 10 µM, treatment time 48 hours.

(B) ARSA activity quantification (nmol/h/mg) after treatment of MSDi cells with a selection of four positive hit drugs at a final concentration of 10 µM on MSDi cells in 25cm2 cell culture flasks for three days. Data represent mean ±SD of 7 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. Difference against negative control: ** p<0.01, **** p<0.0001. See details on p-values in appendix table S3.

(C) ARSA activity quantification (nmol/h/mg) after treatment of MSDi cells with increasing concentrations of tazarotene for 3 days. Data represent mean ±SD of 3-9 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. Displayed are significance levels for the next significant difference between adjacent concentrations. # p<0.05, ## p<0.01. Difference against 0 µM control: ** p<0.01, **** p<0.0001. See details on p-values in appendix table S4.

(D) GALNS activity quantification after treatment of MSDi cells with increasing concentrations of tazarotene for 3 days. Data represent mean ±SD of 4 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. Difference against 0 µM control: ** p<0.01, *** p<0.001, **** p<0.0001. See details on p-values in appendix table S5.

(E) Analysis of different retinoids' potential to restore ARSA activity in MSDi cells in comparison to tazarotene after treatment for 3 days at a final concentration 10 µM of each drug (Adapalene 1 µM). Data represent mean ±SD of 3 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. Difference against DMSO control: ** p<0.01, *** p<0.001, **** p<0.0001. See details on p-values in appendix table S6.

(F) ARSA activity quantification after treatment of MSDi cells with increasing concentrations of bexarotene for 3 days. Data represent mean ±SD of 3-10 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. Displayed are significance levels for the next significant difference between adjacent concentrations. # p<0.05. Difference against 0 µM control: * p<0.05, *** p<0.001, **** p<0.0001. See details on p-values in appendix table S7.

(G) ARSA activity quantification after simultaneous treatment of MSDi cells with increasing concentrations of tazarotene and bexarotene in a fixed combination of 1:2 for 3 days. Data represent mean ±SD of 3-6 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. Displayed are significance levels for the next significant difference between adjacent concentrations. #### p<0.0001. Difference against 0/0 µM control: **** p<0.0001. See details on p-values in appendix table S8.

(H) Analysis and quantification of a time dependent increase of ARSA activity in MSDi cells simultaneously treated with 10 and 20 µM tazarotene and bexarotene, respectively. Data represent mean ±SD of 3-10 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. Displayed are significance levels for the next significant difference between treatment times. # p<0.05, ### p<0.001, #### p<0.0001. Difference against 0 days control: * p<0.05, **** p<0.0001. See details on p-values in appendix table S9.