Fig 2: Validation of SCCPDH as an off-target of VX-445. (A) Representative immunoblot showing enrichment of PAL-modified SCCPDH by desthiobiotin–streptavidin pulldown. HEK293T cells transiently expressing SCCPDH-Myc construct were treated with probe, UV cross-linked, and clicked with TMR. SCCPDH-Myc was enriched dose-dependently. Competition with VX-445 at 5- or 10-fold excess led to dose-dependent loss of enrichment. (B) Quantification of labeled SCCPDH normalized to 3 μM VU439 treatment reiterates the specificity of VU439 binding (n = 3). Statistical differences were computed via one-sample t test comparing against 3 μM VU439 treatment. Statistical differences were computed via one-way ANOVA with Geisser–Greenhouse correction and post hoc Dunnett’s multiple comparisons testing against SCCPDH-Myc expressing cells treated with 3 μM VU439. p-values as shown. VX-445 outcompeted VU439 in binding to SCCPDH.

Fig 3: Inhibition of SCCPDH by VX-445 may result in amino acid metabolism dysregulation resulting in side effects. Amino acids such as glutamate and glutamine were decreased upon overexpression of SCCPDH and VX-445 mitigated this decrease, which may be the underlying cause of psychological symptoms experienced by patients on VX-445. Additionally, metabolites reported to be dysregulated in CF were observed to change in identical directions upon SCCPDH overexpression. VX-445 may inhibit SCCPDH to counteract these dysregulations resulting in unforeseen outcomes such as side effects.

Fig 4: Pathways change with SCCPDH overexpression and VX-445 drives these changes in the opposite direction. (A) Heatmap shows metabolites significantly altered across all conditions (FDR < 5%, L1 and L2 annotation only). Metabolites were hierarchically clustered using Pearson distance and average clustering. Among the metabolites that drastically change with SCCPDH overexpression, VX-445 treatment further enhanced the observed changes. See also Figure S3. (B) Pathway analysis performed with the 19 metabolites from (A). Cysteine and methionine metabolism (1), and arginine biosynthesis (2) were the most significant at p < 0.01. Alanine, aspartate, and glutamate metabolism (3), glutathione metabolism (4), glycine, serine, and threonine metabolism (5), and arginine and proline metabolism (6) were observed at p < 0.05. Other pathways at p > 0.05 are listed in Table S4 in order of decreasing significance. Node color and size represent the p value and pathway impact score, respectively. (C) Cysteine and methionine metabolic pathway (1) from (B). Overexpression of SCCPDH resulted in serine accumulation and a decrease of downstream metabolite 2-aminoacrylic acid. Addition of VX-445 in GFP control further highlighted this finding. Metabolites detected in our data set were filled into the pathway as annotated in the key. See also Figure S4. (D) Arginine biosynthesis pathway (2) from (B). Overexpression of SCCPDH resulted in the loss of glutamic acid and glutamine with a small increase in ornithine and arginine. Addition of VX-445 in GFP control further highlighted this finding. Metabolites detected in our data set were filled into the pathway as annotated in the key. See also Figure S5.

Fig 5: SCCPDH was identified as an off-target of VX-445 via affinity purification mass spectrometry. (A) Off-target identification workflow. Cells are treated with VU439, UV cross-linked, and click-modified with TAMRA desthiobiotin azide (TMR). Streptavidin-enriched proteins are digested and analyzed by LC-MS/MS. Competition with the parent compound results in loss of specific targets. (B) Representative SDS-PAGE gel image showing streptavidin-enriched proteins in doxycycline-inducible CFBE F508del cells treated with vehicle (DMSO) or PAL probe (VU439 at 1 μM) or competition (VU439 at 1 μM and VX-445 at 10 μM). Addition of probe allows enrichment of cross-linked proteins modified by TMR. (C) Workflow A was followed and analyzed by data-independent acquisition (DIA) mass spectrometry (n = 8). Volcano plot shows log2 fold change of enriched protein abundance of probe condition compared to that of vehicle. Enriched proteins with a standard deviation of at least one from the normal distribution of all identified proteins were selected (red dots) for comparison against the competition condition. (D) log2 fold changes of filtered proteins were compared to competition conditions to identify probe-specific targets (red). SCCPDH showed clear enrichment effectively competed by the parent compound as a specific off-target of VX-445 (blue).