Discovery of a Novel Small Molecule Inhibitor Targeting the Frataxin/Ubiquitin Interaction via Structure-Based Virtual Screening and Bioassays

Friedreich’s ataxia (FRDA) is an autosomal recessive neuro- and cardiodegenerative disorder for which there are no proven effective treatments. FRDA is caused by decreased expression and/or function of the mitochondrial protein frataxin. Here, we report findings that frataxin is degraded via the ubiquitin–proteasomal pathway and that it is ubiquitinated at residue K¹⁴⁷ in Calu-6 cells. A theoretical model of the frataxin-K¹⁴⁷/Ub complex, constructed by combining bioinformatics interface predictions with information-driven docking, revealed a hitherto unnoticed, potential ubiquitin-binding domain in frataxin. Through structure-based virtual screening and cell-based assays, we discovered a novel small molecule (compound (+)-<b>11</b>) able to prevent frataxin ubiquitination and degradation. (+)-<b>11</b> was synthesized and tested for specific binding to frataxin by an UF-LC/MS based ligand-binding assay. Follow-up scaffold-based searches resulted in the identification of a lead series with micromolar activity in disrupting the frataxin/Ub interaction. This study also suggests that frataxin could be a potential target for FRDA drug development

Discovery of a Novel Small Molecule Inhibitor Targeting the Frataxin/Ubiquitin Interaction via Structure-Based Virtual Screening and Bioassays

Friedreich’s ataxia (FRDA) is an autosomal recessive neuro- and cardiodegenerative disorder for which there are no proven effective treatments. FRDA is caused by decreased expression and/or function of the mitochondrial protein frataxin. Here, we report findings that frataxin is degraded via the ubiquitin–proteasomal pathway and that it is ubiquitinated at residue K¹⁴⁷ in Calu-6 cells. A theoretical model of the frataxin-K¹⁴⁷/Ub complex, constructed by combining bioinformatics interface predictions with information-driven docking, revealed a hitherto unnoticed, potential ubiquitin-binding domain in frataxin. Through structure-based virtual screening and cell-based assays, we discovered a novel small molecule (compound (+)-<b>11</b>) able to prevent frataxin ubiquitination and degradation. (+)-<b>11</b> was synthesized and tested for specific binding to frataxin by an UF-LC/MS based ligand-binding assay. Follow-up scaffold-based searches resulted in the identification of a lead series with micromolar activity in disrupting the frataxin/Ub interaction. This study also suggests that frataxin could be a potential target for FRDA drug development

Mutation of Ser 227 impairs PKG-mediated increase of H₂S production on T24 cells.

<p>(A) Western blot analysis of proteins from CBSΔT24 cells transiently transfected with plasmids expressing HA-CBS WT and HA-CBS mutants (HA-CBS S227A, HA-CBS S525A), incubated with 8-Br-cGMP or vehicle for 15 min. Loading in the gel lanes was controlled by detection of GAPDH protein. (B) Production of H₂S from the same samples. Mutation of Ser227 significantly reduces H₂S production in basal condition and following incubation with 8-Br-cGMP compared with the paired WT (^#p<0.05 and ^###p<0.001). The levels of H₂S are not affected by the mutation of Ser 525. Indeed, H₂S levels are similar to those obtained from HA-CBS WT either in basal condition or upon cyclic nucleotide stimulation. 8-Br-cGMP significantly increases H₂S production in HA-CBS WT and in HA-CBS S525A compared to vehicle (*p<0.05, ^§§§p<0.001). Data are calculated as nanomoles per milligram of protein per min and expressed as mean±SE of three different experiments.</p

CBS and CSE expression and activity in human urothelium.

<p>(A) Both CBS and CSE are expressed in human urothelium as demonstrated by the western blot analysis. Loading in the gel lanes was controlled by detection of GAPDH protein. (B) Human urothelium expresses CBS and CSE mRNA as determined by quantitative RT-PCR analysis. Data were calculated as Δct and expressed as mean±SE of four separate specimens. (C) Human urothelium homogenate incubation with the vehicle (basal) or with L-cysteine (the substrate) generates detectable amount of H₂S (**p<0.01). (D) The incubation with 8-Br-cGMP significantly increases H₂S production compared to vehicle (*p<0.05). This effect is abrogated by KT5823, a selective PKG inhibitor (°p<0.05). The stimulation with d<b>-</b>cAMP does not affect H₂S production either in the presence or in the absence of KT5720, a selective PKA inhibitor. H₂S levels are calculated as nanomoles per milligram of protein/min and expressed as mean±SE of five different specimen.</p

The pCBS^Ser227 antibody selectively recognizes the CBS phosphorylated form in human urothelium and T24 cells.

<p>(A) Expression of CBS phosphorylated form in human urothelium tissue treated or untreated with 8-Br-cGMP for 15 min. Protein extracts are analyzed by immunoblotting using the anti-pCBS^Ser227 and anti-CBS. Loading in the gel lanes was controlled by detection of GAPDH protein. (B) T24 cells treated or untreated with 8-Br-cGMP for 15 min. Protein extracts are analyzed by immunoblotting using the anti-pCBS^Ser227 and anti-CBS. Loading in the gel lanes was controlled by detection of GAPDH protein. A more robust pCBS specific signal is detected by anti-pCBS^Ser227 in lysates from T24 cells treated with 8<b>-</b>Br-cGMP. No difference in signal intensity is appreciable in the same samples incubated with the anti<b>-</b>CBS. (C) Protein levels are evaluated by densitometric analysis expressed as arbitrary units. (D) Proteins from T24 cells transiently transfected with constructs expressing HA<b>-</b>CBS WT or HA<b>-</b>CBS S227A and treated with 8-Br-cGMP are specifically immunoprecipitated with antibody against the HA epitope. Immunoprecipitates (Ip) are separated by SDS–PAGE and immunoblotted with anti-pCBS^Ser227. Note the absence of signal in cells transfected with HA<b>-</b>CBS S227A mutant construct.</p

PKG phosphorylates CBS in T24 cells.

<p>(A) CBS is isolated by immunoprecipitation (Ip) from starved T24 cells treated with 8-Br-cGMP or vehicle in the presence or the absence of PKG inhibitor. CBS phosphorylation is evaluated by western blotting assay using anti<b>-</b>pS/T. 8<b>-</b>Br<b>-</b>cGMP treatment causes a time-dependent increase of CBS phosphorylation (*p<0.05, ***p<0.001 vs vehicle; ^### p<0.001 vs 5 and 30 min.). PKG inhibition (KT5823) abolishes CBS phosphorylation induced by the 8-Br-cGMP (°°p<0.01 and °°°p<0.001 vs matched time point). (B) CBS is isolated by Ip from starved T24 cells treated with d<b>-</b>cAMP or vehicle in the presence or absence of PKA inhibitor. CBS phosphorylation is evaluated by western blotting assay using anti<b>-</b>pS/T. The stimulation with d<b>-</b>cAMP in the presence or in the absence of PKA inhibitor, KT5720, does not cause changes in phosphorylated CBS level. Data are calculated as % of CBS phosphorylation and expressed as mean ± SE of three separate experiments. (C) Representative protein expression of PKG and PKA in human T24 urothelial cells, urothelium, detrusor muscle and full thickness bladder tissue homogenates. Loading in the gel lanes was controlled by detection of GAPDH protein. PKG is expressed in all samples. PKA is highly expressed in human detrusor and whole bladder but not in T24 cell lines or human urothelium. The blot is representative of three separate experiments.</p

PKG phosphorylates Ser227 and Ser525 of CBS protein.

<p>(A) Schematic representation of CBS protein domains and predicted PKG phosphorylation sites by computational analysis. (B) Proteins from T24 cells transiently transfected with plasmids expressing HA-CBS WT or HA-CBS mutants (HA-CBS S32A, HA-CBS S227A, HA-CBS S525A), are specifically immunoprecipitated with antibodies against the HA epitope. Immunoprecipitates (Ip) are used in a cell free kinase assay and analyzed for incorporation of ³²P by autoradiography. The same samples are immunoblotted with antibodies versus HA epitope. A significant reduction of labeling with ³²P in the HA-CBS S227A and HA-CBS S525A proteins compared to HA-CBS WT protein is reported (***p<0.001). Data are calculated as % of CBS phosphorylation and expressed as mean ± SE of three separate experiments. (C) Proteins from T24 cells transiently transfected with plasmids expressing HA-CBS WT or HA-CBS mutants (HA-CBS S32A, HA-CBS S227A HA-CBS S525A), untreated and treated with 8-Br-cGMP are specifically immunoprecipitated with antibodies against the HA epitope. Immunoprecipitates are separated by SDS–PAGE and immunoblotted with anti-pS/T and anti-HA. The activation of PKG by 8-Br-cGMP leads to a highly significant increase of pCBS produced by cells transfected with HA-CBS WT and HA-CBS S32A mutant (***p<0.001 and °°°p<0.001). Levels of pCBS in cells tranfected with HA-CBS S227A and HA CBS-S525A are increased as compared to untreated control (^###p<0.001 and ^§§§p<0.001). Levels of pCBS in HA-CBS S227A and HA-CBS S525A are significantly reduced when compared with HA-CBS WT (^ççp<0.01). Data are expressed as mean ± SE of three different experiments.</p

Effect of CBS silencing on H₂S production in human T24 cells.

<p>(A) Generation of CBSΔT24 cells. Five clones resistant to puromycin were screened for CBS expression using western blotting analysis. Clone F was selected for the further experiments. GAPDH was used as a protein loading control. (B) The H₂S production in CBSΔT24 is markedly lower than in control T24 cells (*p<0.05). Data are calculated as nanomoles per milligram of protein per min and expressed as mean±SE of four different experiments.</p