There is insufficient electron density to fit and refine properly the terminal benzyldioxol group in the structure

There is insufficient electron density to fit and refine properly the terminal benzyldioxol group in the structure. is surrounded by the adjacent Cys301/303. Surprisingly, the orientation of the Rabbit polyclonal to PTEN interaction changes depending on the nature of the substitutions on the basic indole ring structure 6b-Hydroxy-21-desacetyl Deflazacort and correlates well with the observed structureCactivity relationships for each ALDH isoenzyme. Introduction Aldehyde dehydrogenases (ALDH) comprise a superfamily of enzymes that catalyze the NAD(P)+-dependent oxidation of aldehydes to their corresponding carboxylic acids.1 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 Sj?grenCLarsson syndrome, type II hyperprolinemia, hyperammonemia, and alcohol flushing disease as well as cancer.2?6 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 ALDH27?9 and ALDH1/310,11 as well as broad-spectrum modulators.12,13 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.12 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 6b-Hydroxy-21-desacetyl Deflazacort ALDH1A1, ALDH2, and ALDH3A1 may be derived. Results 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.12 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 results12,13 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 ALDH3A1.13 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. Group 1 is 6b-Hydroxy-21-desacetyl Deflazacort represented by substitutions 6b-Hydroxy-21-desacetyl Deflazacort that lack additional ring systems. 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). Table 1 Open in a separate window Open in a separate window 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. This group comprises the most potent inhibitors of ALDH1A1 and ALDH2. 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-1 em H /em -indole-2,3-dione (compound 3) vs 5-bromo-1-(2-phenylethyl)-1 em H /em -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)-1 em H /em -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)-1 em H /em -indole-2,3-dione (compound 10)), but introduction of the 5-chloro group to the.