aldehyde dehydrogenases 
 ( aldh ) comprise a superfamily of enzymes 
 that catalyze the nad(p)-dependent oxidation of aldehydes 
 to their corresponding carboxylic acids . enzymes in this superfamily exhibit diversity in their specificity 
 for substrates .
detrimental changes in their contributions to specific 
 metabolic pathways lead to several disease states , including sjgren 
larsson 
 syndrome , type ii hyperprolinemia , hyperammonemia , and alcohol flushing 
 disease as well as cancer . using
known structural and catalytic attributes for several of these 
 family members has led to the discovery and characterization of some 
 selective chemical modulators for aldh2 and aldh1/3 as well as broad - spectrum modulators .
our 
 prior work with a broad - spectrum inhibitor demonstrated that 
 the enzyme catalyzed production of a vinyl - ketone intermediate that 
 inhibited aldh1a1 , aldh2 , and aldh3a1 through the formation of a covalent 
 adduct with their catalytic cysteine residue .
however , to achieve selective inhibition of particular isoenzymes , 
 molecules that do not rely solely on common mechanistic features may 
 be more desirable . therefore , this study looks to further that work 
 by characterizing a class of inhibitors that utilize a common mechanistic 
 feature but that can achieve selectivity through elaboration of the 
 common functional group , indole-2,3-dione .
we report here the kinetic 
 and structural characterization of a diverse group of substituted 
 indole-2,3-diones , from which selective inhibitors for aldh1a1 , aldh2 , 
 and aldh3a1 may be derived .
recently , we reported a 
 class of compounds identified during a 
 high - throughput screen for modulators of aldh2 that showed nonselective 
 covalent inhibition of aldh isoenzymes . to achieve a more selective inhibition of aldh isoenzymes , we reasoned 
 that reliance on mechanistic features common to aldh family members 
 was not desirable .
consequently , we re - evaluated the original high - throughput 
 screening results for compounds that might demonstrate 
 better isoenzyme selectivity .
re - examination of these screens led 
 to the identification of four aldh2 inhibitors with structural similarity 
 to five aldh3a1 inhibitors , some of which showed excellent selectivity 
 toward aldh3a1 . to characterize this 
 group of compounds
further , we obtained an additional 33 structurally 
 similar analogues from chemdiv and chembridge and evaluated their 
 ability to inhibit aldh1a1 , aldh2 , and aldh3a1 using nad(p)-dependent aldehyde oxidation to measure activity .
the compounds 
 in this study are all derived from the indole-2,3-dione 
 parent compound , but three distinct structural groupings can be created 
 on the basis of the nature of the substitutions to the indole-2,3-dione 
 ring system and their ability to inhibit selected aldh isoenzymes . 

these were the least selective between aldh isoenzymes and 
 exhibited low micromolar ic50 values for aldh2 and middle - to - high 
 nanomolar ic50 values for aldh1a1 and aldh3a1 ( table 1 ) .
compounds in group 2 are characterized by 
 the addition of a benzyl 
 moiety via an alkyl chain linker attached to the indole ring nitrogen 
 atom with and without halogen substitutions at the 5-position of the 
 indole ring .
however , the nature of the substitutions can shift the 
 potency 380-fold in favor of aldh1a1 or 40-fold in favor of aldh2 
 ( 1-pentyl-2,3-dihydro-1h - indole-2,3-dione ( compound 3 ) vs 5-bromo-1-(2-phenylethyl)-1h - indole-2,3-dione 
 ( compound 8) , table 1 ) . in general , 
 longer alkyl - chain linkers favor aldh1a1 and aldh3a1 inhibition .
halogens 
 at the 5-position improve potency toward aldh2 , but 5-bromo - substitutions 
 on the indole ring reduce the potency toward aldh1a1 .
substitution 
 of either a 5-chlorine or 5-bromine on the indole ring severely reduces 
 potency toward aldh3a1 ( 1-(2-phenylethyl)-1h - indole-2,3-dione 
 ( compound 6 ) vs 8 , table 1 ) .
the addition of a double bond to the linker between the 
 indole and benzyl rings almost eliminates potency toward aldh2 ( 1-(3-phenyl-2-propen-1-yl)-1h - indole-2,3-dione ( compound 10 ) ) , but introduction 
 of the 5-chloro group to the same molecule restores potency ( 5-chloro-1-[(2e)-3-phenylprop-2-en-1-yl]-2,3-dihydro-1h - indole-2,3-dione ( compound 11 ) ) .
group 3 compounds 
 possess either a piperazine , morpholine , or imidazolidine 
 nonaromatic ring linked to the indole nitrogen ( table 1 ) .
these compounds tend to be the most selective for haldh3a1 
 and show little if any inhibition of aldh2 .
only the compound with 
 a 5-bromo substitution on the indole ring ( 1-{[4-(1,3-benzodioxol-5-ylmethyl)-1-piperazinyl]methyl}-5-bromo-1h - indole-2,3-dione ( compound 21 ) ) was a poor 
 inhibitor of haldh3a1 ( table 1 ) . to understand 
 the mechanism of inhibition for these compounds better , 
 compounds
these inhibitors 
 exhibited noncompetitive mixed - type inhibition with respect to varied 
 coenzyme , and they exhibited competitive inhibition with respect to 
 varied aldehyde substrate for all three aldh isoenzymes ( tables 2 and 3 ) .
consistent with the kinetic 
 data , the crystal structure of aldh2 
 with 3 and 1 bound shows that the compounds 
 bound within the aldehyde substrate binding site with the 3-keto group 
 sandwiched between the active - site cysteine residues 301 and 303 ( figures 1 and 2 ) .
the distance of 
 interaction in these models suggest that both cysteine residues are 
 interacting equivalently on either face of the carbonyl carbon but 
 do not appear to have formed formal adducts .
interestingly , the 
 orientation of their indole-2,3-dione rings are flipped such that 
 the opposing faces are interacting with cys301 and cys303 in the 1 versus 3 structures . the 5-methyl substituent 
 of 1 forms hydrophobic interactions with the side chains 
 of trp177 , leu173 , and met174 , whereas the 7-bromo substituent is 
 oriented toward the solvent - exposed exit of the substrate - binding 
 site .
in contrast , the 5-position of 3 is oriented toward 
 the solvent , and the 1-benzyl substituent is tucked tightly into the 
 substrate binding side adjacent to the side chain of phe465 , which 
 is displaced from the position found in all other aldh2 crystal structures . 

there is an inverse correlation between ordered positioning of the 
 1-n - benzyl group and that of phe465 , suggesting that one or the other 
 is mobile in this complex .
the close contacts between these aromatic 
 rings is likely responsible for the relatively high ic50 exhibited by aldh2 for 3 . despite the flipped orientations 
 of their common ring system ,
the indole-2,3-diones of 1 and 3 maintain the same aromatic -stacking interactions 
 with the side chains of phe170 and phe459 .
( a ) side view of the 
 protein surface containing compound 1 ( left ) and ( b ) 
 top view with the original unbiased figure of merit , a - weighted , 
 2fo  fc ( blue ; contoured at one standard deviation of the map ) and fo  fc electron 
 density map ( green ; contoured at 2.5 standard deviations of the map ) 
 for 1 prior to its inclusion in the model superimposed 
 on the final refined model .
( a ) side view of the 
 protein surface containing compound 3 ( left ) and ( b ) 
 top view with the original unbiased figure of merit , a - weighted , 
 2fo  fc ( blue ; contoured at one standard deviation of the map ) and fo  fc electron 
 density map ( green ; contoured at 2.5 standard deviations of the map ) 
 for 3 prior to its inclusion in the model superimposed 
 on the final refined model .
in contrast , the crystal structure of aldh3a1 in a complex 
 with 
 1-{[4-(1,3-benzodioxol-5-ylmethyl)-1-piperazinyl]methyl}-1h - indole-2,3-dione ( compound 20 ) ( figure 3 ) shows the 3-keto group of the indole-2,3-dione 
 ring bound within the substrate - binding site and forming an adduct 
 with the active - site nucleophile ( cys243 in aldh3a1 ) .
the distance 
 between cys243 and the carbonyl - carbon as well as the out - of - plane 
 displacement of the carbonyl oxygen is consistent with the formation 
 of an adduct between these two reactive groups . that this covalent 
 bond is reversible is supported by the fact that addition of dithiothreitol 
 to the reaction solution after preincubation restores the enzymatic 
 activity .
there is sufficient electron density to model the indole-2,3-dione 
 and the n - piperazine moiety , but insufficient electron density is 
 present to model the terminal benzyl - dioxol moiety .
computational 
 placement of the benzyl - dioxol moiety onto the crystallographically 
 observed partial structure suggests that the benzyl - dioxol lies at 
 the interface between the exit of the substrate - binding site and bulk 
 solvent , where it can apparently adopt multiple conformations .
( a ) side view of 
 the protein surface containing the crystallgraphically observed portion 
 of compound 20 .
( b ) top view with the original unbiased 
 figure of merit , a - weighted , 2fo  fc ( blue ; contoured at one 
 standard deviation of the map ) and fo  fc electron density map ( green ; contoured at 
 2.5 standard deviations of the map ) prior to its inclusion in the 
 model superimposed on the final refined model .
there is insufficient 
 electron density to fit and refine properly the terminal benzyldioxol 
 group in the structure .
consequently , we have placed and refined only 
 that portion that can be accounted for by the available electron density .
there 
 are 19 members of the aldh superfamily in the human genome , 
 with many of the family members participating in defined metabolic 
 pathways , such as proline , valine , retinal , folate , and gaba metabolism .
in contrast , several members have less well - defined 
 substrate preferences and constitute a cellular defense system against 
 endogenously and exogenously generated aldehydes .
in particular , aldh1a1 , 
 aldh2 , and aldh3a1 demonstrate broad and overlapping substrate specificities 
 and are frequently expressed in the same tissues or cell types .
for instance , aldh1a1 and aldh3a1 have both 
 been shown to be biomarkers for cancer as well as cancer stem cells .
aldh1a1 overexpression is indicative of high - grade ductal carcinoma , 
 multiple myeloma , and acute myeloid leukemia .
aldh3a1 is generally found in stratified squamous epithelium , and both aldh3a1 and aldh1a1 confer resistance to the cell - killing 
 effects of cyclophosphamide . however , aldh2 has 
 primary contributions to the elimination of acetaldehyde and to the 
 bioactivation of nitroglycerin .
thus , selective inhibitors of these general aldehyde - oxidizing enzymes 
 could provide important research tools for the assessment of their 
 individual contributions to their common substrates or cellular functions . 
 toward this end
, we have examined derivatives of indole-2,3-diones 
 for their ability to inhibit differentially these three forms of aldh .
members of this class of compounds were identified in separate 
 inhibitory screens on aldh2 and aldh3a1 , although the diversity of 
 the substitutions on the indole rings of these initial hits suggested 
 that selectivity could be achieved through optimization of those substitutions . 
 that this level of selectivity could be achieved is further supported 
 by the differences in the chemical and surface topologies of their 
 respective substrate - binding sites .
aldh2 and aldh3a1 share only 30% 
 sequence identity , and despite sharing the common catalytic residues 
 they have distinct substrate - binding - site characteristics ( figure 4 ) .
similarly , aldh2 and aldh1a1 share 68% sequence 
 identity , and , although their substrate - binding sites are both largely 
 hydrophobic in nature , the middle and exterior regions of the site 
 in aldh1a1 are considerably wider because of smaller amino acid side 
 chains at positions 124 and 459 .
it is in this 
 context that the structure  activity relationships for these 
 substituted indole-2,3-diones should be interpreted .
comparison of aldib interactions 
 with active - site cysteines in 
 aldh2 and aldh3a1 .
( a ) crystal structure of aldh2 with 1 ( gray ) has been overlaid with that of 3 ( magenta ) to 
 show how the steric hindrance of the surrounding hydrophobic residues 
 determines the mode of binding .
( b ) surface map of 3 ( magenta ) 
 bound to aldh2 ( cyan ) aligned to the crystallographically observed 
 partial structure of 20 ( orange ) bound to aldh3a1 ( yellow ) .
the kinetic data is consistent 
 with these inhibitors binding in 
 a manner that is competitive with respect to aldehyde binding .
although 
 the details of the interactions between the aldib inhibitors and the 
 enzymes differ in the precise details , all compounds occupy the substrate - binding 
 site in a manner that requires their displacement prior to productive 
 binding of substrate aldehydes .
their mixed - type noncompetitive inhibition 
 patterns with respect to varied coenzyme is consistent with the largely 
 ordered bi 
bi mechanism followed by aldh family members that 
 have been characterized in this manner .
the 
 distinct differences in the nature of the interactions between 
 the indole-2,3-dione rings and aldh3a1 and aldh2 highlight the structural 
 and functional differences of these distantly related isoenzymes . 

consistent with this level of sequence identity , a structural alignment 
 yields a rmsd of 2.1  for 395 similarly positioned c 
 atoms .
the amino acids lining their respective substrate - binding sites 
 are both largely hydrophobic , but the differences in their positioning 
 and identity create unique topographical features ( figures 3 and 4 ) .
the manner 
 in which 20 is bound within the aldh3a1 
 substrate site also provides key information with respect to the manner 
 in which aldh3a1 selectivity is achieved when compared to the substrate - binding 
 sites of aldh1a1 and aldh2 ( figure 3 ) .
it is 
 these structural and functional differences that underlie the distinct 
 way these substituted indole-2,3-dione molecules bind to these aldh 
 active sites .
the 3-keto group in these inhibitory molecules is a 
 well - known electrophile , so it was not surprising that this group 
 would seek a reactive nucleophile within the aldh active site . however , 
 it was surprising that only in aldh3a1 did these compounds form an 
 adduct with the catalytic nucleophile , as the active site of aldh2 
 is generally thought to contain the stronger cysteine nucleophile . 

aldh2 generally exhibits low to submicromolar km values for aldehydes , whereas aldh3a1 exhibits km values at least 2 orders of magnitude higher . why then 
 does 20 bind to the catalytic nucleophile in aldh3a1 
 but neither 1 nor 3 does so in aldh2 ?
an 
 alignment of the respective active sites demonstrates that in order 
 for either 1 or 3 to bind to cys302 the 
 side chain of trp177 has to move , but its position is restricted by 
 the side chains of leu173 , met174 , and leu477 ( figure 4 ) .
this explains why the indole-2,3-dione ring can not bind 
 productively to cys302 in aldh2 and why the sar for aldh2 was so difficult 
 to understand before the structures of 1 and 3 were available . the most difficult features of the aldh2 sar 
 to understand were 
 the favorability of larger halogens at the 5-position and how the 
 changes in inhibition strength varied as the alkyl linker between 
 the indole ring and a benzene substituent on the n1 position was lengthened . 

if the indole ring bound similarly to that in aldh3a1 , then we would 
 expect the sar on halogens at the 5-position to follow that of aldh3a1 ; 
 namely , that larger halogens are detrimental to inhibitory potency 
 ( table 1 , 8 vs 5-chloro-1-(2-phenylethyl)-1h - indole-2,3-dione ( compound 7 ) vs 6 , 20 vs 21 ) .
however , this is not the case 
 for aldh2 , as increasing the size of the halogen at the 5-position 
 either has little effect ( 1-benzyl-5-bromo-1h - indole-2,3-dione 
 ( compound 5 ) , 1-benzyl-5-chloro-2,3-dihydro-1h - indole-2,3-dione ( compound 4 ) , and 3 , table 1 ) or increases potency ( 8 vs 7 vs 6 , 10 vs 11 , table 1 ) .
in addition , lengthening 
 the alkyl linker to the benzene ring had no real impact on potency , 
 which is opposite that seen with aldehyde substrates where the longer 
 and more hydrophobic the alkyl chain , the better the substrate .
however , 
 the presence of a double bond in this linker virtually abolishes inhibitory 
 potency ( 10 vs 1-(3-phenylpropyl)-1h - indole-2,3-dione ( compound 9 ) , table 1 ) , whereas addition of a 5-chloro group to 10 ( 11 ) restores potency .
this data suggests that the 
 binding modes for the halogen versus non - halogen - substituted compounds 
 differ .
we suggest that the binding mode observed for 3 in aldh2 is maintained for group 2 compounds lacking halogens at 
 the 5-position ( table 1 ) , as the ability of 
 the longer alkyl chains to adopt new conformations necessary to exit 
 the active site past the position of cys302 requires a flexible linker . 

however , this binding mode is inconsistent with the increased potency 
 of halogen substitution at the 5-position because the 5-position of 3 is within van der waals contact to val120 , met124 , and phe296 . 

consequently , the sar would suggest that 5-halogen - substituted group 
 2 compounds adopt the position observed for 1 , where 
 the 5-methyl substituent will approximate the position of the 5-chloro 
 or 5-bromo substituents and the indole nitrogen is now pointing toward 
 the solvent - exposed exit of the substrate - binding site .
in addition , 
 the aldh2 crystal structures presented here show that 
 cys302 is pointing in the direction of the cofactor pocket and away 
 from the substrate pocket , which has been referred to as the resting 
 conformation .
lang et al . suggested that 
 this resting conformation contributes to cys301/303 having the primary 
 role in stimulation of the cys302 thio - carbonyl adduct formation , 
 lowering the acidity of cys302 .
furthermore , 
 comparison of aldh2 with other enzyme family members suggests that 
 cys303 interacts with substrates via hydrogen bonding or by an ion 
the crystal structures of 1 and 3 presented here suggest that cys301/303 
 may contribute to substrate binding through a trapping mechanism and 
 possibly by serving to dehydrate the hydrated aldehydes that form 
 in solution .
weiner and colleagues proposed that elements of the enzyme s 
 proton - relay system performed this function , but this proposal preceded 
 structure determination by 12 years .
the proton - relay 
 system is identical in aldh1a1 and aldh3a1 , but the two residues immediately 
 surrounding the catalytic nucleophile in the active - site loop are 
 not . in this
regard , aldh1a1 has ile at 301 and a single additional 
 cys residue at 303 , whereas aldh3a1 lacks cys residues at either equivalent 
 position ( thr and val , respectively ) , which correlates with their 
 decreased catalytic efficiency for small aliphatic aldehydes with 
 the greatest level of hydration .
in contrast to the complex sar for aldh2 , selectivity for aldh3a1 
 is more simply achieved through the addition of a nonaromatic ring 
 system linked by a single carbon atom to the indole nitrogen ( table 1 ) .
this addition to the indole ring alone abrogates 
 any potency toward aldh2 and severely diminishes potency toward aldh1a1 . 

addition of hydrophobic substituents at the 4-position of the piperazine 
 ring further improves potency up to 4-fold ( 1-(4-morpholinylmethyl)-1h - indole-2,3-dione ( compound 12 ) versus 20 ) while still maintaining a 40-fold selectivity over aldh1a1 . 

although the benzyl - dioxol substituent is disordered in our crystal 
 structure , presumably because of the multiple binding modes near the 
 exit of the substrate - binding site , these additional hydrophobic substituents 
 likely interact with the side chains of trp233 and met237 ( figure 3 ) .
substitution of the central nonaromatic ring 
 with an aromatic ring reduces potency by about 6-fold ( 3 versus 12 ) .
this is likely due to the generation of 
 an unfavorable steric contact between the aromatic ring and met237 
 ( figure 3 ) .
addition of one or more additional 
 linking carbon atoms between the indole and aromatic rings improves 
 potency by more than 15-fold , presumably by bypassing the restrictive 
 space occupied by met237 ( 3 vs 6 or 9 , table 1 ) .
however , these changes 
 come at the cost of selectivity for aldh3a1 as neither 6 nor 9 are selective inhibitors for aldh3a1 .
another 
 unique characteristic of aldh3a1 is the negative impact that substitution 
 of large halogen atoms at the 5-position of the indole ring has on 
 potency .
this effect can be explained by the close proximity of the 
 side chains of phe401 , tyr412 , and his413 ( figure 3 ) .
substitution of anything larger than a hydrogen atom would 
 crowd this location . with the exception of the compounds in 
 group 3 , most aliphatic 
 or aromatic n - substituted indole-2,3-diones are potent inhibitors 
 of aldh1a1 ( table 1 ) .
only the substitution 
 of a nonaromatic ring linked to the indole nitrogen or the presence 
 of a 5-bromo group is detrimental to aldh1a1 potency .
consistent with 
 its ability to oxidize retinaldehyde with high efficiency , aldh1a1 
 has the largest and most hydrophobic substrate - binding site , and substituents on the indole nitrogen that 
 enhance hydrophobic interactions improve the potency of these compounds 
 toward this isoenzyme .
the structure  activity data for aldh1a1 
 is consistent with a binding mode for the substituted indole-2,3-diones 
 that is similar to that for 20 in aldh3a1 .
this data 
 is supported by presence of gly124 in aldh1a1 , rather than met124 
 in aldh2 ; this substitution provides some additional room for a 5-chloro 
 group but is still insufficient for a 5-bromo substitution , which 
 would still clash with trp177 .
the aim 
 of this work was to develop further a new class of small 
 molecules that can be adapted to selectively inhibit aldh enzymes . 

as such , several compounds were identified that show reasonable selectivity 
 ( > 40-fold ) toward aldh1a1 ( 3 ) , aldh2 ( 8) , 
 or aldh3a1 ( 20 or 1-{[4-(4-fluorobenzyl)-1-piperazinyl]methyl}-1h - indole-2,3-dione ( compound 16 ) ) based on 
 the size and chemical characteristics of each isoenzyme s substrate - binding 
 site .
the competition assays show that these inhibitors are noncompetitive 
 with respect to the coenzyme - binding site and are competitive toward 
 the substrate - binding site .
x - ray crystallography data confirms inhibition 
 occurs through direct interactions between the 3-keto group of the 
 indole ring and conserved cysteine residues within the active site . 

selective inhibition can be achieved through substitutions at the 
 c5 and n1 positions of the indole ring system .
these compounds may 
 act as useful tools in elucidating the contributions of individual 
 aldh isoenzymes toward specific , as well as overlapping , metabolic 
 pathways .
compounds used for the inhibitor screen were 
 found using a 80% structure similarity search against 5-methyl-1h - indole-2,3-dione using the pubchem project .
the following compounds were purchased from 
 chembridge for further in vitro assay screening : 5301889 , 1-benzyl-5-bromo-1h - indole-2,3-dione ( 5 ) ; 5192630 , 1-(2-phenylethyl)-1h - indole-2,3-dione ( compound 6 ) ; 7196590 , 5-chloro-1-(2-phenylethyl)-1h - indole-2,3-dione ( 7 ) ; 6047303 , 5-bromo-1-(2-phenylethyl)-1h - indole-2,3-dione ( 8) ; 6378722 , 7-bromo-5-methyl-1h - indole-2,3-dione ( 1 ) ; 6433626 , 1-(3-phenylpropyl)-1h - indole-2,3-dione ( 9 ) ; 6997087 , 1-(3-phenyl-2-propen-1-yl)-1h - indole-2,3-dione ( compound 10 ) ; 6505720 , 
 1-{[4-(1,3-benzodioxol-5-ylmethyl)-1-piperazinyl]methyl}-1h - indole-2,3-dione ( 20 ) ; 8918814 , 1-{[4-(4-fluorobenzyl)-1-piperazinyl]methyl}-1h - indole-2,3-dione ( 16 ) ; 8918815 , 1-{[4-(3-chlorobenzyl)-1-piperazinyl]methyl}-1h - indole-2,3-dione ( compound 18 ) ; 8918818 , 
 1-{[4-(2-fluorobenzyl)-1-piperazinyl]methyl}-1h - indole-2,3-dione 
 ( compound 15 ) ; 8918819 , 1-{[4-(3-fluorobenzyl)-1-piperazinyl]methyl}-1h - indole-2,3-dione ( compound 17 ) ; 8919812 , 
 1-{[4-(3-methoxybenzyl)-1-piperazinyl]methyl}-1h - indole-2,3-dione 
 ( compound 19 ) ; 6989645 , 1-{[4-(1,3-benzodioxol-5-ylmethyl)-1-piperazinyl]methyl}-5-bromo-1h - indole-2,3-dione ( 21 ) ; 5250099 , 1,1-[1,3-imidazolidinediylbis(methylene)]bis(1h - indole-2,3-dione ) ( compound 22 ) ; 5115436 , 
 1-(4-morpholinylmethyl)-1h - indole-2,3-dione ( 12 ) ; 5260280 , 1-[(4-methyl-1-piperazinyl)methyl]-1h - indole-2,3-dione ( compound 13 ) ; and 5115499 , 
 1-[(4-benzyl-1-piperazinyl)methyl]-1h - indole-2,3-dione 
 ( compound 14 ) .
the following compounds were purchased 
 from chemdiv for further screening : 0764 - 0625 , 1-pentyl-2,3-dihydro-1h - indole-2,3-dione ( compound 2 ) ; 0764 - 0628 , 
 1-benzyl-2,3-dihydro-1h - indole-2,3-dione ( 3 ) ; and 5353 - 0854 , 1-benzyl-5-chloro-2,3-dihydro-1h - indole-2,3-dione ( 4 ) .
on the basis of the sar data 
 collected , compound bbv-138984 , 5-chloro-1-[(2e)-3-phenylprop-2-en-1-yl]-2,3-dihydro-1h - indole-2,3-dione ( 11 ) , was purchased from 
 molport as proof of the selective - inhibition scheme .
the nmr spectra 
 confirming > 95% compound purity provided by the vendors is included 
 in the supporting information .
human aldh1a1 ( haldh1a1 ) , aldh2 
 ( haldh2 ) , and aldh3a1 ( haldh3a1 ) were expressed and purified as described 
 elsewhere .
crystals of aldh3a1 at 3 mg / ml in 10 mm hepes , ph 7.5 , were grown 
 from solutions containing 0.2 m potassium acetate and 18% peg3350 
 at 25 c .
enzyme complexes with inhibitors were generated through 
 direct - soaking experiments by first equilibrating the crystals overnight 
 in a solution containing 2% dmso , which was then supplemented with 
 100 m compound 20 .
crystals 
 of aldh2 were grown from protein solutions containing 8 mg / ml of aldh2 
 in 100 mm aces , ph 6.26.8 , 100 mm guanidine - hcl , 10 mm mgcl2 , and 1419% peg 6000 .
the aldh2 complexes with inhibitors 
 were generated through direct - soaking experiments by first equilibrating 
 the crystals overnight in solutions containing 2% dmso , which was 
 then supplemented with 100 m of either compound 1 or 3 .
all diffraction data were collected at 
 a wavelength of 0.9869  
 and at 100 k. all diffraction data were indexed , integrated , and scaled 
 using the hkl3000 program suite .
the structure of the aldh3a1 complex with 20 was solved 
 by molecular replacement using the aldh3a1 apo structure ( pdb code 3sza ) .
the presence of 
 inhibitor was determined after inspection of the initial -a 
 weighted fo  fc electron density maps .
the structures of aldh2 complexed 
 with 1 and 3 were solved using the coordinates 
 of the refined aldh2 in the p21 space 
 group after removal of solvent and ligands ( 1cw3 ) .
the aldh2 complex with 1 demonstrates high - occupancy 
 binding in subunits a and h and insufficient electron density for 
 unequivocal assignment in the remaining subunits .
the aldh2 complex 
 with 3 shows high - occupancy binding in subunits a , b , 
 e , and h and insufficient electron density for unequivocal assignment 
 in the remaining subunits .
ic50 inhibition 
 curves for the inhibitors were measured using the activity of haldh2 , 
 haldh1a1 , and haldh3a1 as described elsewhere . in short ,
the enzyme was incubated with the inhibitor and 
 coenzyme for 2 min prior to initiation of the reaction with aldehyde 
 substrate .
the inhibition curves were fit to the four - parameter ec50 equation using sigmaplot ( version 11 , statsys ) .
inhibition of aldh 
 activity was measured using the activity of haldh2 , haldh1a1 , and 
 haldh3a1 to determine the kinetic mode of inhibition versus varied 
 coenzyme or aldehyde substrate .
coenzyme competition was determined 
 by measuring aldh activity for various concentrations of compound 
 while varying the concentration of nad for haldh2 and 
 haldh1a1 and nadp for haldh3a1 .
likewise , substrate competition 
 was determined for different concentrations of each compound while 
 varying propionaldehyde concentration for haldh2 and haldh1a1 and 
 benzaldehyde concentration for haldh3a1 . in each experiment , the concentration 
 of the nonvaried substrate was set to saturating levels , and the varied 
 substrate concentration ranged at least 10-fold spanning the calculated km value .
similarly , the concentration range 
 for the inhibitors was varied a minimum of 5-fold , exclusive of the 
 control ( no inhibitor ) reactions , which spanned the calculated ki values .
the kinetic mode of inhibition was 
 determined by fitting data to the competitive , noncompetitive , and 
 uncompetitive inhibition equations and evaluating the goodness of 
 fit to each equation .
data fitting and 
 analysis was performed using sigmaplot ( version 11.0 ) with the enzyme 
 kinetics module ( version 1.3 ) .
 aldehyde 
 dehydrogenases ( aldh ) participate in multiple metabolic 
 pathways and have been indicated to play a role in several cancerous 
 disease states . 
 our laboratory is interested in developing novel and 
 selective aldh inhibitors . 
 we looked to further work recently published 
 by developing a class of isoenzyme - selective inhibitors using similar 
 indole-2,3-diones that exhibit differential inhibition of aldh1a1 , 
 aldh2 , and aldh3a1 . 
 kinetic and x - ray crystallography data suggest 
 that these inhibitors are competitive against aldehyde binding , forming 
 direct interactions with active - site cysteine residues . 
 the selectivity 
 is precise in that these compounds appear to interact directly with 
 the catalytic nucleophile , cys243 , in aldh3a1 but not in aldh2 . in 
 aldh2 , 
 the 3-keto group is surrounded by the adjacent cys301/303 . 
 
 surprisingly , the orientation of the interaction changes depending 
 on the nature of the substitutions on the basic indole ring structure 
 and correlates well with the observed structure  activity relationships 
 for each aldh isoenzyme . 
Introduction
Results
Discussion and Conclusions
Experimental Procedures