Inhibition of human recombinant N-terminal His6-tagged AKR1C4 expressed in Escherichia coli BL21(DE3) cells using 8-Acetyl-2,3,5,6-tetrahydro-1H,4H-11-oxa-3a-aza-benzo[de]anthracen-10-one as substrate at 3 uM after 1 hr by fluorimetric analysis
Inhibition of human recombinant N-terminal His6-tagged AKR1C4 expressed in Escherichia coli BL21(DE3) cells using 8-Acetyl-2,3,5,6-tetrahydro-1H,4H-11-oxa-3a-aza-benzo[de]anthracen-10-one as substrate after 1 hr by fluorimetric analysis
Inhibition of His-tagged human AKR1C4 expressed in Escherichia coli BL21 (DE3) assessed as inhibition of reduction of non-fluorescent ketone probe to fluorescent alcohol after 1 hr by competitive fluorescence assay
Activity of human recombinant AKR1C4 expressed in HEK293 cells assessed as (1R,2S,5S)-N-((2S,3R)-4-amino-1-cyclobutyl-3-hydroxy-4-oxobutan-2-yl)-3-((S)-2-(3-tert-butylureido)-3,3-dimethylbutanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide formation per mg protein after 120 mins in presence of NADPH by LC-MS/MS/FSA method
Discontinuous Radiometric Assay: Compounds may be evaluated as selective reversible inhibitors of AKR1C3 by screening them against homogeneous recombinant AKR1C1-AKR1C4 expressed in E. coli. In each case, a discontinuous radiometric assay may be used to monitor the inhibition of progesterone reduction (20-ketosteroid reduction) catalyzed by AKR1C1, the inhibition of Δ4-AD reduction (17-ketosteroid reduction) catalyzed by AKR1C3, and the inhibition of 5α-DHT reduction (3-ketosteroid reduction) catalyzed by AKR1C2 and AKR1C4 (by measuring the formation of 20α-hydroxyprogesterone, testosterone or 3α-androstanediol by radiochromatography). Secondary screens of the compounds of interest include: (a) a full-screen against all nine human recombinant AKR enzymes to ensure there are no-intended off-target effects (in this context AKR1B10 (retinal reductase; SEQ ID NO:5) has been shown to be potently inhibited by N-phenylanthranilates) (Endo et al., 2010, Biol. Pharm. Bull. 33:886-90); (b) a screen against COX-1 and COX-2 to reaffirm that compounds do not act as NSAIDs; and (c) an expanded screen against other nuclear receptors (especially other steroid hormone receptors).
Inhibition Assay: Inhibitors were initially screened for an ability to block the NADP+ dependent oxidation of the artificial substrate S-tetralol catalyzed by AKR1C3. S-tetralol was used since it is also a substrate of the highly related AKR1C1 and AKR1C2 enzymes. Inhibition of AKR1C1 and AKR1C2 is undesirable in the context of prostate cancer since they are involved in the elimination of DHT (see FIG. 1B; see also Rizner et al, 2003; Steckelbroeck et al. 2004). All assays were performed at Km so that IC50 values across enzyme forms were comparable.An exemplary screening strategy was against homogeneous recombinant AKR1C3 and its highly related enzyme AKR1C2 to generate full dose-response curves and IC50 values.