recent studies have indicated that members 
 of the 2-aminobenzamide class of histone deacetylase inhibitors show 
 promise as therapeutics for the neurodegenerative diseases friedreich s 
 ataxia ( frda ) and huntington s disease . in the case 
 of frda , this disorder is caused by transcriptional repression of 
 the nuclear fxn gene encoding the essential mitochondrial 
 protein frataxin . expansion of gaattc 
 triplet repeats in pathogenic fxn alleles cause gene 
 silencing and a loss of frataxin protein in affected individuals . currently there is no effective therapy for frda that addresses the 
 cause of the disease . unlike many triplet - repeat diseases ( e.g. , the 
 polyglutamine expansion diseases ) , expanded gaattc triplets 
 in fxn are in an intron and do not alter the amino 
 acid sequence of the frataxin protein ; thus , gene activation would 
 be of therapeutic benefit . on the basis of the hypothesis that the acetylation state of the 
 histone proteins is responsible for gene silencing in frda , the gottesfeld 
 lab identified one commercially available hdac inhibitor ( bml-210 ) 
 that partially relieves repression of the fxn gene 
 in lymphoid cells derived from frda patients . a library of derivatives of this lead compound has been synthesized , 
 and potent activators of fxn transcription have been 
 identified in cell - based assays . importantly , 
 these compounds consistently increase the level of frataxin mrna in 
 lymphocytes from frda patients to at least the levels found in lymphocytes 
 from unaffected carrier siblings or parents . we find that the hdac 
 inhibitors act directly on the histones associated with the fxn gene , increasing acetylation at particular lysine residues 
 on histones h3 and h4 . biochemical studies , 
 including enzyme inhibition and target identification with affinity - capture 
 probes , provided evidence that hdac3 is a main preferred enzyme target 
 of the inhibitors . importantly , upregulation of the 
 frataxin gene has been observed in two frda mouse models when treated 
 with these compounds , and one member of this drug class has been undergoing 
 preclinical evaluation and has completed a phase ib clinical trial 
 in frda patients , who show increases in fxn mrna 
 in circulating lymphocytes . in 
 the case of huntington s disease ( hd ) , a large body of evidence 
 points to transcriptional dysregulation as one of the key features 
 of this disease , and hdac inhibitors have been the subject of intense 
 investigation to counteract the transcription deficits in hd . we find that members of the 2-aminobenzamide 
 class of hdac inhibitors are beneficial in restoring normal transcriptional 
 activity in both cellular and mouse models for hd and these molecules 
 have beneficial effects on neuromotor function in the r6/2 mouse model . in our previous studies , we surprisingly found that common hdac inhibitors , valproic acid , 
 trichostatin a ( tsa ) , and suberoylanilide hydroxamic acid ( saha ) , 
 some of which are more potent hdac inhibitors than bml-210 and our 
 derivatives , do not have a positive effect on activation of the fxn gene in frda cells . while 
 it is clear that hdac3 is a cellular target of the 2-aminobenzamide 
 class of hdac inhibitors and is inhibited 
 through a slow , tight - binding mechanism in contrast to the rapid - on / rapid - off 
 inhibition mechanism observed for the hydroxamates tsa and saha , inhibition of other class i hdacs ( hdacs 1 and 2 ) may also be involved 
 in the beneficial effects of these compounds in frda and hd , and other 
 hdac interacting proteins may be important . to identify the 
 targets of the 106 compound , we synthesized an activity - based profiling 
 probe ( abpp ) version of one of our hdac inhibitors ( 106 ) and a control 
 probe , which is a derivative of 106 lacking a 2-amino group in the 
 hdac inhibitor portion of the molecule . the control 
 probe is far less active as an hdac inhibitor as shown in a previous 
 study . while our primary interest is identification 
 of targets of 106 that might be involved in regulation of the fxn gene in frda , an unbiased proteomic approach should 
 also identify the broader targets of 106 and their interacting proteins . 
 in the present study , we used a dimethyl stable isotope - labeling approach 
 coupled with multidimensional protein identification technology ( mudpit ) to quantitatively identify the proteins specifically 
 captured by the abpp 106 probe under nondenaturing conditions compared 
 with the control probe . the abpp approach allows us to purify the 
 106 probe - specific targets with vigorous washing to reduce contaminating 
 proteins . dimethyl labeling and mudpit provide powerful tools for 
 defining the targets of the hdac inhibitor 106 probe based on rigorous 
 quantification to the control probe . in total , 4933 proteins were 
 quantified and 1556 proteins were bound to the abpp 106 probe with 
 statistical significance compared with the control probe . many of 
 the specific abpp 106 binders are involved in regulation of gene transcription 
 and posttranscriptional processes , giving insights into frda mechanism 
 and clinical therapy . human 
 friedreich s ataxia ipsc - derived neurospheres were grown in 
 neurobasal - a medium with 2% b-27 supplement , 1% its - a supplement , 
 1% n-2 supplement , 2 mm glutamine , 1% antibiotic / antimycotic , 10 mm 
 hepes , 20 ng / ml basic fgf , and 20 ng / ml egf ( r&d systems ) according 
 to a previous procedure . neurospheres 
 were dissociated to single cells with accutase and plated on matrigel 
 ( bdbiosciences ) at 50,000 cells / cm and passaged every 
 45 days for expansion . cells were centrifuged , and cell pellets 
 were collected and washed with pbs buffer . the new control 
 probe ( structure shown in figure 5a ) was made by reaction of n-(4-(4-aminobenzoyl)phenyl)hex-5-ynamide 
 with acetic anhydride , and probe 2 ( structure is shown in figure 5a ) is obtained by amide 
 reaction of n-(4-(4-aminobenzoyl)phenyl)hex-5-ynamide 
 with 7-((2-((tert - butoxycarbonyl)amino)phenyl)amino)-7-oxoheptanoic 
 acid , followed by boc deprotection . nuclear extracts were prepared by first adding cold 10 mm hepes 
 ( ph 7.9 ) , 10 mm kcl , 1.5 mm mgcl2 , 0.5 mm dtt , and 0.2 
 mm pmsf to washed cell pellets ( 100 l / million cells ) ; after 
 incubation on ice for 10 min , the lysed cells were centrifuged at 
 3000  g for 15 min , and the soluble fractions were removed . the pellet was resuspended in a 1:1 mixture of low salt buffer ( 20 
 mm hepes [ ph 7.9 ] , 25% glycerol , 20 mm kcl , 1.5 mm mgcl2 , 0.2 mm edta , 0.5 mm dtt , and 0.2 mm pmsf ) and high salt buffer 
 ( 20 mm hepes [ ph 7.9 ] , 25% glycerol , 1.2 m kcl , 1.5 mm mgcl2 , 0.2 mm edta , 0.5 mm dtt , and 0.2 mm pmsf ) and was subjected to 
 homogenization , followed by stirring at 4 c for 30 min . the 
 lysed nuclear pellet solution was centrifuged at 14,000  g for 
 30 min at 4 c to provide the nuclear fractions ( supernatant ) 
 and a membrane pellet . western blotting with histone antibodies showed enrichment 
 in the nuclear fraction ( data not shown ) . three 
 hundred l of nuclear extract ( 3.8 mg / ml protein ) in 2100 l 
 pbs was added to different wells in a 6-well plate . two hundred and 
 forty l of trifunctional probe was added to give a final concentration 
 of 4 mm , and incubation was continued on ice for 5 min . samples were 
 then cross - linked with uv at 365 nm for 1 h on ice ; 360 l of 
 click reagent ( a mixture of cuso4 , biotin azide , tcep , 
 and ligand as with previous procedures ) was added to the wells , and the resulting solutions were rotated 
 at ambient temperature for 1 h. one ml of pbs was added to each well , 
 and the solution was kept at 20 c overnight . the next 
 day , the solutions from each well were transferred to separate eppendorf 
 tubes and centrifuged to precipitate proteins , which were then washed 
 with cold methanol ( 1 ml , twice ) , dried , resuspended in 1 ml of 0.2% 
 sds in pbs , and then incubated with 0.8 ml of magnetic streptavidin 
 beads ( invitrogen ) for 2 h. the supernatant was removed from the original 
 bead solution , and the beads were washed with pbs ( 1 ml , twice , prior 
 to use ) . the supernatant was removed , and the beads were washed with 
 0.2% sds in pbs ( 1 ml , twice ) , 6 m urea ( 1 ml , twice ) , and pbs ( 1 
 ml , three times ) ; the resulting beads were eluted with 60 l 
 sds loading buffer at 90 c ; 20 l aliquots were loaded 
 onto three separate sds polyacrylamide gels , and subjected to western 
 blotting . each membrane was immunostained with antibodies to hdac1 , 
 hdac2 , and hdac3 ( all from abcam ) , respectively , followed by antirabbit 
 igg  horseradish peroxidase - conjugated secondary antibody ( cell 
 signaling , ma ) . the proteins bound to abpp 106 probe were enriched using 
 streptavidin beads as described above and then were reduced on beads 
 in 5 mm tcep/100 mm teab . the supernatant containing tryptic peptides were 
 mixed with 4 l of 4% ch2o or cd2o to be labeled with light and heavy formaldehyde , respectively . four l of 0.6 m nabh3cn or nabd3cn were 
 added to the samples to be light or heavy labeled . after incubation 
 for 1 h at room temperature , the reaction was quenched by adding 16 
 l of a 1% ammonia solution . eight l of formic acid was 
 added to each sample to acidify the sample for lc the light and heavy labeled peptides 
 were equally mixed ( w / w ) and were analyzed by a modified 10-step multidimensional 
 protein identification technology ( mudpit ) as described previously . briefly , the peptide mixtures were preloaded onto a 250 m 
 internal diameter ( i.d . ) silica - fused capillary column packed with 
 strong cation exchange ( scx , whatman , clifton , nj ) and reversed phase 
 ( aqua c18 , phenomenex , torrance , ca ) . analytical 
 column packed with reversed phase ( aqua c18 ) was attached with the 
 scx end via a union , and the entire column setting ( biphasic column  union analytical 
 column ) was placed in line with an agilent 1200 quaternary hplc pump 
 ( palo alto , ca ) . eluted peptides were electrosprayed directly into 
 a hybrid ltq - orbitrap mass spectrometer ( thermofisher , san jose , ca ) 
 with the application of a distal 2.4 kv spray voltage . a cycle of 
 one full - scan mass spectrum ( 4001600 m / z ) followed by seven data - dependent ms / ms spectra at a 35% 
 normalized collision energy was repeated continuously throughout each 
 step of the multidimensional separation . the raw data were extracted from the xcalibur data system format 
 into ms1 and ms2 formats using in - house software . the peptides and 
 proteins were identified by the integrated proteomics pipeline - ip2 
 ( integrated proteomics applications , inc . , san diego , ca . the  light  
 and  heavy  dimethylation of n - term and k were searched . proteins with an average stable isotope ratio ( abpp 106 
 versus control probe ) greater than 2 or greater than 1.5 with p < 0.05 were subjected to functional analysis in david as well as ingenuity . hdaci 106 has been shown to increase fxn mrna levels 
 in lymphoblast cell lines and in primary lymphocytes from friedreich s 
 ataxia patients , and a related 2-aminobenzamide 
 has shown similar efficacy in neuronal cells derived from frda patient 
 ipscs . the structures of the 106- and 
 control probes are shown in figure 1a , and 
 the strategy applied in the present study is shown in figure 1b . nuclear proteins were extracted from neural stem 
 cells differentiated from friedreich s ataxia patient - derived 
 ips cells . we use neural stem cells as these cells are easily propagated 
 and can give the required number of cells for the experiments . differentiated 
 neurons , the authentic cells that are affected in frda , generally 
 can not be obtained as a pure population of cells and can not be propagated 
 to give rise to the required numbers of cells . the abpp 106 probe 
 and control probe were incubated with nuclear extracted proteins . 
 afterward , the bound probe was cross - linked to the protein using uv 
 light , conjugated with biotin by using the captured proteins 
 were subjected to extensive washing using harsh denaturing conditions 
 prior to trypsin digestion and labeling of peptides from different 
 samples with  heavy  or  light two of four experimental replicates were forward 
 labeled , and the other two were reverse labeled ( e.g. , label swap ) . 
 the  heavy  and  light  labeled samples 
 were mixed and analyzed by lc  ms / ms . searching tandem mass 
 spectra through the sequence database identified peptides . identified 
 peptides were quantified by calculating the ratio of peptide abundances 
 in the differentially labeled samples , and those changes were then 
 extrapolated to the protein level . this method will identify both 
 direct targets of the 106 probe and proteins that interact with target 
 proteins . for example , in our previous study we identified both hdac3 and its partner protein ncor1 by western 
 blot analysis . structures of the 106- and control probes ( a ) and the 
 experimental strategy in the present study ( b ) . the synthesis procedures 
 of 106- and control probes are shown in the previous study . a total of 
 2096 nonredundant proteins at a protein false discovery rate of 1% 
 were identified in all four experimental replicates and a total of 
 4933 proteins were quantified overall ( table s1 in the supporting information [ si ] ) . a total of 2571 
 proteins were quantified in at least one forward- and reverse - labeled 
 experiment , and the reproducibility of the measurements was determined 
 by plotting this experiment against another . the log base 2 of average 
 ratios of two forward - labeling and two reverse - labeling experiments 
 are shown in figure 2 . the slope of the calculated 
 best fit to the data is 0.9449 ( with an r of 0.7617 ) , indicating that the ratio for each protein in the forward- 
 and reverse - labeled measurements were largely similar ( figure 2 ) . about 77% of the proteins ( 1987 ) have ratios 
 ( abpp 106 versus control probe ) greater than 1 . the protein ids , which are identified 
 in at least one forward- or reverse - labeled replicate were selected 
 for reproducibility evaluation . the log base 2 of average ratios of 
 two forward labeling and two reverse labeling was plotted against 
 each other . the slope of the calculated best fit to the data is 0.9449 
 ( with an r of 0.7617 ) . 3003 
 proteins were quantified in at least two of the replicates , and this 
 set was used for further analysis . one thousand two hundred and thirty - one 
 proteins have an average ratio ( abpp 106 versus control probe ) greater 
 than 1.5 with a p - value < 0.05 , and among those 
 proteins 883 had an average ratio greater than 2 ( figure 3 ) . hdac1 and 2 were identified as 106-probe specific 
 binders and were verified by western blot analysis ( figure 4 ) . hdac1 and 2 were found to be significantly enriched 
 in the abpp 106 incubated samples . log2 ( fold changes ) 
 were plotted against log10 ( p - values ) . photoaffinity labeling of proteins in a nuclear 
 extract from frda - ipsc derived neural stem cells with 106 probe followed 
 by addition of a biotin - azide by click  chemistry , 
 streptavidin capture , and western blotting with antibody to the indicated 
 hdacs . lane 1 , nuclear extract input ( 2% of total , relative to lanes 
 23 ) . for hdacs 1 and 3 , lane 2 , 106-bound proteins ; lane 3 , 
 control ( ctrl ) probe - bound proteins . for hdac2 western , lane 2 , control 
 probe - bound proteins ; lane 3 , 106 probe bound proteins . a total of 1556 proteins ( 10 keratin - contaminating proteins were 
 discarded ) , which have average ratios ( abpp 106 versus control probe ) 
 greater than 2 or greater than 1.5 with p - value < 0.05 
 ( we define as abpp 106 binders ) , were subjected to functional analysis 
 in david as well as in ingenuity . gene ontology ( go ) analysis 
 for cellular components showed that the abpp 106 binders are significantly 
 enriched in broad go fat categories , including ribonucleoprotein complex 
 ( p = 1.68  10 ) , spliceosome 
 ( p = 1.84  10 ) , chromatin 
 remodeling complex ( p = 2.30  10 ) , transcriptional repressor complex ( p = 9.78  
 10 ) , nurd complex ( p = 9.14  
 10 ) , swi / snf chromatin remodeling complex ( 3.22 
  10 ) , histone deacetylase complex ( p = 7.62  10 ) , and sin3 complex 
 ( p = 0.002 ) . go analysis for molecular functions 
 showed that abpp 106 binders are mostly enriched in the go fat category 
 of rna binding ( p = 7.93  10 ) . the go fat molecular function categories ( p < 
 0.001 ) in which abpp 106 binders are significantly enriched are shown 
 in figure s1a in the si . go analysis for 
 biological processes showed that abpp 106 binders are mostly enriched 
 in the go fat category of translation elongation ( p = 7.31  10 ) . the top ranking categories 
 ( p < 1  10 ) are shown 
 in figure s1b in the si . the sp - pir 
 keywords mostly enriched ( p < 1  10 ) in our abpp 106 binder set are shown in figure s2 
 in the si ; 66.09% of the abpp 106 binders 
 belong to the category of acetylation , which makes the enrichment 
 most significant ( p = 1.25  10 ) . the kegg pathway analysis shows 16 significantly enriched 
 categories ( p < 0.05 ) for abpp 106 binders ( figure 
 s3a in the si ) , including ribosome , proteasome , 
 spliceosome , etc . the biocarta pathway analysis found 13 significant 
 enrichment categories ( figure s3b in the si ) with the top category of control of gene expression by vitamin 
 d receptor . the role of histone deacetylases in vitamin d - regulated 
 gene expression is well established . the finding 
 of ribosome - associated pathways is at first surprising since we used 
 a nuclear extract in these experiments ; however , it is well documented 
 that ribosomes are assembled in nucleoli and many translation factors 
 localize in the nucleus and participate in nuclear functional annotation of 
 proteins binding abpp 106 , which combines the gene - term enrichment 
 analysis done by go , sp - pir keywords , up_seq_feature , kegg and biocarta 
 pathways , interpro and smart protein domains , is shown in table s1 
 in the si ( top ranking terms which have p < 1  10 ) . we find that the 
 most significant biological term associated with abpp 106 binders 
 across those analysis tools is acetylation , as would be expected . other significant enrichment categories ranking on the top include 
 ribonucleoprotein complex , rna binding , rna recognition motif , mrna 
 metabolic process , rna splicing , mrna processing , etc . the role of 
 protein acetylation in these processes is beginning to be appreciated . by clustering 
 functional annotation groups with similar annotations together according 
 to shared gene members , the top 10 clusters out of 
 56 clusters ( high classification stringency ) , which have significant 
 group enrichment scores ( < 0.05 , equivalent to 1.3 in minus log 
 scale ) , are shown in table s2 in the si . the most enriched annotation cluster is rna recognition motif ( representative 
 annotation term ) . a gene functional classification analysis 
 distributes the abpp 106 binders into 10 functional related gene clusters 
 ( highest classification stringency ) , which have significant functional 
 enrichment scores ( < 0.05 , equivalent to 1.3 in minus log ) . the 
 top gene group includes several ribosomal proteins associated with 
 the major biology term of translation / ribosome / rna binding ( table 
 s3 in the si , the associated biology terms 
 are manually summarized on the basis of gene terms enriched for each 
 functional group ) . the pathway analysis shows that abpp 106 binders 
 are mostly enriched in the eif2 signaling pathway ( p = 1.26  10 ) . the molecules ( shown in 
 red ) , which are enriched in the eif2 signaling pathway , are illustrated 
 in figure s4a in the si . the top ranking 
 categories ( p < 1  10 ) are shown in figure s4b in the si . interestingly , pathogenesis of cardiomyopathy 
 in a mouse model for frda correlates with the early and persistent 
 eif2 phosphorylation , which precedes activation of autophagy 
 and apoptosis . the disease analysis shows 
 that the  neurological disease  ranks at the top among 
 the enriched disease categories ( table s4 in the si ) . one of the identified targets of the 106 probe is the transcription 
 elongation factor tceb2 . interestingly , the gene encoding tceb2 was 
 found to be up - regulated by hdaci 106 in primary lymphocytes from 
 friedreich s ataxia patients . tceb2 
 is of interest since down regulation of fxn mrna synthesis is the 
 primary cause of frda , and transcription elongation as well as initiation 
 has been shown to be affected by the gaa repeats . to validate tceb2 as a bonafide target or target - interacting 
 protein of the 106-probe , we used western blotting of the affinity - captured 
 proteins with antibody to tceb2 ( figure 5b ) . for this experiment , we validated capture of 
 tceb2 with a second version of the activity - based probe and a second 
 control probe , whose structures are shown in figure 5a . figure 5b clearly shows that tceb2 
 is captured by both specific probes , but not by the control probe , 
 providing validation of the proteomic analysis for tceb2 . validation 
 experiments for other identified targets is beyond the scope of the 
 present study . ( a ) structures of a second control probe and a second 106 
 probe ( probe 2 ) . ( b ) photoaffinity labeling , followed by addition 
 of a biotin - azide by  click  chemistry , streptavidin 
 capture , and western blotting with antibody to tceb2 followed the 
 protocol outline in ref ( 7 ) . lane 1 , nuclear extract input ( 2% of total , relative to lanes 24 ) . lane 2 , 106-probe - bound protein ; lane 3 , second control probe - bound 
 proteins ; lane 4 , probe 2-bound proteins . in the present study , 
 the targets of hdac inhibitor abpp 106 probe and interacting proteins 
 have been identified in frda patient - derived neural stem cells by 
 dimethyl labeling quantitative mass spectrometry combined with mudpit . 
 the abpp approach , which allows the use of harsh protein denaturing 
 conditions after the probes are cross - linked to the protein to remove 
 noncovalently bound proteins , allowed us to purify either direct targets 
 of 106 probe or interactors in close proximity to the direct targets 
 of the native activity . the inactive 
 analogue of the 106 probe , which differs from 106 by a simple amino 
 group , provides a control for specificity . 
 in our previous study , we found that only 
 the 106 probe , but not the control probe , was able to identify hdacs 
 in nuclear lysates . a competition step with excess free compound 106 can also be employed in the experimental design to further confirm 
 the selectivity of the 106 probe . to differentiate the specific targets 
 from nonspecific binding proteins of the 106 probe dimethyl labeling provided a fast 
 and straightforward quantification method to exclude the nonspecific binding proteins . bantscheff and 
 colleagues revealed hdac complexes selectivity for 16 hdac inhibitors 
 by combining affinity capture and quantitative mass spectrometry . however , hdac3 was not identified in our data 
 set although control western blotting experiments reproducibly detected 
 hdac3 in the 106-probe pull - downs . while detectable by western blotting 
 ( figure 4 ) , hdac3 may have been too low in 
 abundance in the proteome of neural stem cells differentiated from 
 frda patient ips cells for detection by mass spectrometry , or we were 
 unable to digest the protein effectively off the streptavidin bead . recombinant hdac1 and 2 show less affinity for the 106 probe compared 
 to hdac3 , and it is less active in nuclear extracts of lymphoid cell 
 line derived from an frda patient . in 
 contrast , we found hdac1 and 2 were selectively bound to the 106 probe , 
 indicating an interaction of hdac1 and 2 with 106 probe in neural 
 stem cells . we compared the proteins bound to abpp 106 with the interactome 
 of hdac111 identified by cristea and colleagues . the venn diagram ( figure 6 ) shows that 18 proteins are shared among abpp 106 binders and hdac13 
 interactome and 27 proteins are shared among abpp 106 binders and 
 hdac411 interactome . the comparison showed that 106 probe 
 binds a broad range of hdac111 interactors rather than binding 
 to only the interactors of class i hdacs , indicating that the restoration 
 of frataxin gene transcription by 106 probe may be due to the coordination 
 of multiple hdacs . the overlap in the venn diagram ( figure 6 ) is quite low as the overlap between the two data 
 sets may be more representative of the interactors of hdac13 
 rather than hdac411 . eighteen overlapping proteins between abpp 106 binders 
 and hdac13 interactome are listed in the box . on the basis of the functional analyses from david 
 and ingenuity , the proteins specifically binding the abpp 106 probe 
 were found to be mainly enriched in the regulation of transcription 
 and post - transcription events , such as rna splicing and translation . it has been shown that frataxin deficiency in frda is caused by transcriptional 
 silencing . one mechanism for frataxin 
 gene silencing is the epigenetic gene silencing through heterochromatin 
 formation . it has been shown that histones 
 h3 and h4 are hypoacetylated in the first intron of the inactivated 
 frataxin gene , accompanied by trimethylation of lysine 9 of histone 
 h3 , which is a hallmark of heterochromatin . we 
 found abpp 106 probe specific proteins were mostly enriched in the 
 category of acetylation in sp - pir keywords across all the selected 
 gene term enrichment analyses done in david , indicating compound 106 
 may up - regulate frataxin gene transcription by selectively targeting 
 proteins affecting acetylation . the transcription repression complex , 
 the nurd and sin3 complexes which contain hdac1 and hdac2 , were enriched 
 in the abpp 106 specific protein fraction , suggesting that inhibition 
 of hdac1 and 2 may play a role in frataxin gene expression restoration . the wierzbicki lab proposed 
 that rna polymerase v - produced long noncoding rnas guide the swi / snf 
 complex and establish positioned nucleosomes on specific genomic loci 
 to mediate transcriptional silencing , which supports the hypothesis that compound 106 may reverse frataxin 
 gene silencing by targeting the swi / snf complex . we found targets 
 of abpp 106 probe are also involved in rna processing and translation . one study has shown that drosophila small nuclear ribonucleoprotein smd1 , involved in splicing , is required 
 for assembly and function of the small interfering risc , suggesting 
 the role of drosophila smd1 in rnai - mediated 
 gene silencing besides its pre - mrna splicing activity in posttranscriptional 
 gene regulation . proteins involved in 
 the ribonucleoprotein complex and splicesome are enriched in the abpp 
 106 probe specific proteins . surprisingly , we found that the eif2 
 signaling pathway and ribosome are also enriched , suggesting that 
 the compound 106 may affect mrna translation . there exists ample evidence 
 in the literature for localization of many translation factors in 
 the nuclear compartment and their role in mrna metabolism and transport 
 ( refs above ) . moreover , the finding of ribosomal proteins in the nucleus 
 is not surprising since ribosomes are assembled in nucleoli . it has 
 been shown that abnormal control of eif2 and eif2b leads to cach ( childhood 
 ataxia with central nervous system hypomyelination)/vwm ( leukoencephalopathy 
 with vanishing white matter ) syndrome in young children , which is 
 a severe autosomal recessive neurodegenerative disease . the ribosome binding and translation initiation 
 as well as translation elongation and termination strongly influence 
 mrna stability in bacteria . in eukaryotes , 
 translation is also linked to mrna stability , suggesting a general 
 model for cotranslational mrna decay . it is possible that 
 compound 106 could have a positive effect on translation of frataxin 
 mrna in addition to its documented effect on transcription of the fxn gene . additionally , hdac 
 inhibition could have a positive effect on fxn mrna splicing or stability , 
 and this in turn could also result in the observed increases in frataxin 
 protein on treatment of frda cells with 2-aminobenzamide hdac inhibitors . the 
 beneficial effects of hdac inhibition in huntington s disease 
 have been reviewed . in particular , hdac 
 inhibition can have positive effects in restoring global gene expression 
 profiles , in ameliorating cytoskeletal defects and clearance of mutant htt protein by the ubiquitin our current 
 findings of diverse targets of the 2-aminobenzamides suggest that 
 there are other potentially beneficial mechanisms of action , such 
 as increased processing or translation of mrnas that are down - regulated 
 by mutant htt at the transcriptional level , among other possibilities 
 suggested by the wide range of pathways identified as influenced by 
 the 2-aminobenzamides . on a final note , the finding of a large 
 number of targets of the 106 probe or interacting proteins could potentially 
 raise concern for the use of 2-aminobenzamides as human therapeutics 
 due to potential undesirable side effects . similarly , the 2-aminobenzamides 
 induce changes in global gene expression patterns in human lymphocytes 
 treated ex vivo , again raising concern 
 for off - target effects . in spite of these findings , a related 2-aminobenzamide , 
 hdaci 109 , has been subjected to a phase 
 i dose - escalation clinical study in human frda patients , with no reported 
 adverse effects , even on exposure to 240 mg drug / day , suggesting that potential off - target effects are not of 
 serious concern . the 2-aminobenzamide 
 class of histone deacetylase inhibitors is of great value for the 
 neurodegenerative diseases , friedreich s ataxia and huntington s 
 disease . the present study applied dimethyl labeling quantitative 
 mass spectrometry combined with mudpit to identify the targets of 
 compound 106 , and performed functional analyses of the targets . the 
 findings show that the targets of compound 106 are involved not only 
 in transcriptional regulation but also in mrna translation , indicating 
 the complexity of the mechanisms by which compound 106 may exert its 
 effect in up - regulation of frataxin expression .
members 
 of the 2-aminobenzamide class of histone deacetylase ( hdac ) inhibitors 
 show promise as therapeutics for the neurodegenerative diseases friedreich s 
 ataxia ( frda ) and huntington s disease ( hd ) . 
 while it is clear 
 that hdac3 is one of the important targets of the 2-aminobenzamide 
 hdac inhibitors , inhibition of other class i hdacs ( hdacs 1 and 2 ) 
 may also be involved in the beneficial effects of these compounds 
 in frda and hd , and other hdac interacting proteins may be impacted 
 by the compound . to this end , we synthesized activity - based profiling 
 probe ( abpp ) versions of one of our hdac inhibitors ( compound 106 ) , 
 and in the present study we used a quantitative proteomic method coupled 
 with multidimensional protein identification technology ( mudpit ) to 
 identify the proteins captured by the abpp 106 probe . 
 nuclear proteins 
 were extracted from frda patient ipsc - derived neural stem cells , and 
 then were reacted with control and abpp 106 probe . 
 after reaction , 
 the bound proteins were digested on the beads , and the peptides were 
 modified using stable isotope - labeled formaldehyde to form dimethyl 
 amine . 
 the selectively bound proteins determined by mass spectrometry 
 were subjected to functional and pathway analysis . 
 our findings suggest 
 that the targets of compound 106 are involved not only in transcriptional 
 regulation but also in posttranscriptional processing of mrna .