AID	Name	Type	Comment
1030	qHTS Assay for Inhibitors of Aldehyde Dehydrogenase 1 (ALDH1A1)	confirmatory	qHTS Validation Assay for Inhibitors of aldehyde dehydrogenase 1 (ALDH1A1)
2407	Probe Development Summary for Inhibitors of HPGD (15-Hydroxyprostaglandin Dehydrogenase)	summary
493210	Inhibitors of Aldehyde Dehydrogenase 1 (ALDH1A1): Followup Confirmation and Counterscreen	confirmatory

Aldehyde dehydrogenase 1 (ALDH1A1) catalyzes the NAD+ dependent oxidation of a variety of endogenous and exogenous aldehydes to the corresponding carboxylic acids. The enzyme is the critical step in the metabolic activation of retinoic acid, which plays essential roles as nuclear receptor ligand. Furthermore, the precursor, retinaldehyde has recently been shown to play a fundamental role in adipogenesis and obesity, which makes inhibitor development a possible target in metabolic diseases. See [1] through [4] for more information on ALDH1A1.

Inhibition of ALDH1A1 activity was screened by utilizing propionaldehyde as an electron donor and NAD+ as an electron acceptor/cofactor. An increase in the fluorescence intensity due to conversion of NAD+ to NADH was used to measure the enzyme activity. See [5] for screening methodology used.

References:
[1] Manzer R, Qamar L, Estey T, Pappa A, Petersen DR, Vasiliou V. Molecular cloning and baculovirus expression of the rabbit corneal aldehyde dehydrogenase (ALDH1A1) cDNA. DNA Cell Biol. 2003 May;22(5):329-38.

[2] Collard F, Vertommen D, Fortpied J, Duester G, Van Schaftingen E. Identification of 3-deoxyglucosone dehydrogenase as aldehyde dehydrogenase 1A1 (retinaldehyde dehydrogenase 1). Biochimie. 2007 Mar;89(3):369-73.

[3] Ziouzenkova O, Orasanu G, Sharlach M, Akiyama TE, Berger JP, Viereck J, Hamilton JA, Tang G, Dolnikowski GG, Vogel S, Duester G, Plutzky J. Retinaldehyde represses adipogenesis and diet-induced obesity. Nat Med. 2007 Jun;13(6):695-702.

[4] Molotkov A, Duester G. Genetic evidence that retinaldehyde dehydrogenase Raldh1 (Aldh1a1) functions downstream of alcohol dehydrogenase Adh1 in metabolism of retinol to retinoic acid. J Biol Chem. 2003 Sep 19;278(38):36085-90.

[5] Inglese J, Auld DS, Jadhav A, Johnson RL, Simeonov A, Yasgar A, Zheng W, Austin CP. Quantitative high-throughput screening: a titration-based approach that efficiently identifies biological activities in large chemical libraries. Proc Natl Acad Sci. 2006 Aug 1;103(31):11473-8.

NIH Chemical Genomics Center [NCGC]
Structural Genomics Consortium [SGC]

Buffer: 100 mM Hepes pH 7.5, 0.01% Tween 20.

Reagents/Controls:

[1] Buffer in columns 3 and 4 as negative control (no enzyme).

[2] Substrate/cofactor solution: 1 mM NAD+ and 80 uM propionaldehyde (Sigma-Aldrich, St. Louis, MO) final concentrations dispensed throughout the plate.

[3] Enzyme: 50 nM ALDH1A1 final concentration in columns 1, 2, 5-48. Column 1 is neutral (100% activity).

[4] Control: Pintool transfer of control inhibitor Bay 11-7085 (Lopac B5681 ) to column 2 of all assay plates. Two-fold, 16 pt dilution in duplicate: 5.75 uM - 0.175 nM.

Assay Steps:
Three uL of enzyme were dispensed to 1536-well Greiner black solid bottom plates. Compounds (23 nL) were transferred via Kalypsys PinTool. The plates were incubated for 15 min at room temperature, and then 1 uL of substrate/cofactor solution was added to start the reaction. The plates were immediately transferred to and read twice on ViewLux High-throughput CCD imager (Perkin-Elmer) 20 minutes apart using 360 nm excitation and 450 nm emission fluorescence protocol. The fluorescence intensity difference between the second and the first time points was used to compute reaction progress.

Keywords: NIH Roadmap, MLSCN, MLPCN, MLI, MLSMR, SGC, qHTS, NCGC, ALDH1A1, aldehyde dehydrogenase 1 family, member A1, ALDH1, 5U54MH084681-02 U54 CDP

Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.

2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.

Grant Number: 5U54MH084681-02