20alpha-hydroxysteroid dehydrogenase (AKR1C1): AKR1C1 (Ki = 4 nM); AKR1C2 (Ki = 87 nM); AKR1C3 (Ki = 4.2 μM); AKR1C3 (Ki = 18.2 μM)

In BAEC that overexpress AKR1C1, AKR1C1-IN-1 efficiently inhibits progesterone metabolism with an IC50 of 460 nM [1].

Enzyme and Activity Assays[1]
The recombinant AKR1C1, AKR1C2, AKR1C3, and AKR1C4 were expressed in Escherichia coli JM109 and purified to homogeneity as previously described. Protein concentration was determined by a bicinchoninic acid protein assay reagent kit using bovine serum albumin as the standard. The NADP+-linked S-tetralol dehydrogenase activity of the enzymes was assayed by measuring the rate of change in NADPH fluorescence (at 455 nm with an excitation wavelength of 340 nm) or its absorbance (at 340 nm) at 25 °C, as described previously. In the inhibition assays, the IC50 values for the inhibitors were initially determined with the S-tetralol concentration (0.1 mM for AKR1C1 and 1 mM for other enzymes) using a software ED50 and IC50 for graded Response version 1.2. The inhibition patterns were determined by fitting the initial velocities using five substrate concentrations (0.2−2 × Km for AKR1C3 and 0.5−5 × Km for other enzymes) in the presence of the inhibitor concentrations (0−0.5 × IC50) to Lineweaver−Burk and Dixon plots. The Ki values were calculated by using the appropriate programs of ENZFITTER and are expressed as the mean ± standard error of at least three determinations.

Evaluation of Inhibitors in the Cells[1]
BAECs were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37 °C in a 5% CO2 incubator. In all experiments, the cells were used at passages 4−8, and the endothelial cobblestone morphology was confirmed microscopically before use. The expression vector with the cDNA for AKR1C1 was constructed according to the method previously reported. The cDNA was initially amplified from the bacterial expression vector pGEX/AKR1C1 by PCR using the primer pairs consisting of a forward primer (5′-GAGTCGACgccaccATGGATTCGAAATATCAGTGT-3′) and a reverse primer (5′-AGGTCGACTTAATATTCATCAGAAAATGGA-3′), in which SalI site, a Kozak sequence and a start codon are expressed in italic letters, small letters, and underlined letters, respectively. The PCR product was verified by automated DNA sequencing and subcloned at the SalI site of the eukaryotic expression vector pGW1. The expression vector with the insert was then transfected into subconfluent BAECs using Lipofectamine 2000. The transfected cells were maintained in the medium containing 2% fetal bovine serum for 24 h and then used to evaluate the inhibitory effects of 3,5-dibromosalicylic acid and compounds 4 and 9 on the metabolism of progesterone in the cells. The cells were pretreated for 2 h with various concentrations of inhibitors in serum-free growth medium prior to incubating for 6 h with 30 μM progesterone. The culture media were collected by centrifugation, and the lipidic fraction of the media was extracted twice by ethyl acetate. The metabolite, 20α-hydroxyprogesterone, was quantified on a LC-MS using a Chiralcel OJ-H 5 μm column as described previously.

[1]. El-Kabbani O, et al. Structure-guided design, synthesis, and evaluation of salicylic acid-based inhibitors targeting a selectivity pocket in the active site of human 20alpha-hydroxysteroid dehydrogenase (AKR1C1). J Med Chem. 2009 May 28;52(10):3259-64.

The first design, synthesis, and evaluation of human 20alpha-hydroxysteroid dehydrogenase (AKR1C1) inhibitors based on the recently published crystal structure of its ternary complex with inhibitor are reported. While the enzyme-inhibitor interactions observed in the crystal structure remain conserved with the newly designed inhibitors, the additional phenyl group of the most potent compound, 3-bromo-5-phenylsalicylic acid, targets a nonconserved hydrophobic pocket in the active site of AKR1C1 resulting in 21-fold improved potency (K(i) = 4 nM) over the structurally similar 3alpha-hydroxysteroid dehydrogenase isoform (AKR1C2). The compound is hydrogen bonded to Tyr55, His117, and His222, and the phenyl ring forms additional van der Waals interactions with residues Leu308, Phe311, and the nonconserved Leu54 (Val in AKR1C2). Additionally, the metabolism of progesterone in AKR1C1-overexpressed cells was potently inhibited by 3-bromo-5-phenylsalicylic acid, which was effective from 10 nM with an IC(50) value equal to 460 nM.[1]
In summary, the use of the recently determined crystal structure of AKR1C1 complexed with an inhibitor in conjunction with a GRID analysis of the inhibitor-binding site has allowed the design of a new salicylic acid-based inhibitor (compound 4) with improved potency (Ki = 4 nM) and selectivity (21-fold) over that of AKR1C2. Moreover, compound 4 significantly decreased the metabolism of progesterone in the cells with an IC50 value of 460 nM, which is comparable or superior to the IC50 values of the previously known two most potent inhibitors of AKR1C1, benzbromarone and 3′,3′′,5′,5′′-tetrabromophenolphthalein. Compound 4 was designed to target a selectivity pocket in the active site of AKR1C1 lined by the three apolar residues Leu54, Leu308, and Phe311. Leu308 is one of two nonconserved C-terminal residues (the other residue is Leu306) responsible for the greater than 4000-fold difference in inhibitor potency between AKR1C1 and the two isoforms AKR1C3 and AKR1C4. Since the active sites of AKR1C1 and AKR1C2 differ only by one amino acid residue, which is Leu54 in AKR1C1 and is Val54 in AKR1C2, and the current inhibitors show similar potency for the two enzymes, newly designed inhibitors that capture the maximum interactions with Leu54 in AKR1C1 are needed in order to improve their selectivity over AKR1C2. Thus, future developments of new derivatives of compound 4 are likely to improve on the selectivity of the currently known AKR1C1 inhibitors. We have also illustrated that while large chemical database searches are useful in discovering new enzyme inhibitors, the use of the high resolution crystal structure of an enzyme−inhibitor complex is an effective tool in optimizing the enzyme−inhibitor interaction by exploiting the small structural differences between the different enzyme isoforms.[1]

C13H9BRO3

293.12

291.974

C, 53.27; H, 3.10; Br, 27.26; O, 16.37

4906-68-7

268734

Typically exists as White to pink solids at room temperature

1.6±0.0 g/cm3

439.2±0.0 °C at 760 mmHg

219.4±0.0 °C

0.0±0.0 mmHg at 25°C

1.663

4.43

2

3

2

17

276

0

C1=CC=C(C=C1)C2=CC(=C(C(=C2)Br)O)C(=O)O

XVZSXNULHSIRCQ-UHFFFAOYSA-N

InChI=1S/C13H9BrO3/c14-11-7-9(8-4-2-1-3-5-8)6-10(12(11)15)13(16)17/h1-7,15H,(H,16,17)

3-bromo-2-hydroxy-5-phenylbenzoic acid

NSC-109116; NSC 109116; NSC109116; 5-Bromo-4-hydroxy-[1,1'-biphenyl]-3-carboxylic acid; 3-Bromo-5-phenyl salicylic acid; AKR1C1-IN-1; 3-bromo-5-phenylsalicylic acid; 3-bromo-2-hydroxy-5-phenylbenzoic acid; NSC-109116; CHEMBL387536;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~341.17 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.53 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (8.53 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (8.53 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)