20alpha-hydroxysteroid dehydrogenase (AKR1C1): AKR1C1 (Ki = 4 nM); AKR1C2 (Ki = 87 nM); AKR1C3 (Ki = 4.2 μM); AKR1C3 (Ki = 18.2 μM)
The primary target of NSC-109116 is 20α-hydroxysteroid dehydrogenase (AKR1C1), a member of the aldo-keto reductase superfamily. It acts as a highly potent and selective inhibitor of AKR1C1 with a Ki of 4 nM. The compound also inhibits AKR1C2 with a Ki of 87 nM and AKR1C3 with Kis of 4.2 μM and 18.2 μM. The target of the compound is a selective pocket in the AKR1C1 active site composed of three nonpolar residues, Leu54, Leu308, and Phe311. By binding to this pocket, NSC-109116 selectively inhibits AKR1C1 activity, preventing the reduction of steroid hormones.

In BAEC that overexpress AKR1C1, AKR1C1-IN-1 efficiently inhibits progesterone metabolism with an IC50 of 460 nM [1].
In vitro, NSC-109116 is a highly potent and selective inhibitor of AKR1C1 with a Ki of 4 nM. It also inhibits AKR1C2 with a Ki of 87 nM and AKR1C3 with Kis of 4.2 μM and 18.2 μM. The compound's activity is typically measured using enzyme assays that monitor the reduction of a substrate (e.g., 20α-hydroxysteroid) in the presence of NADPH. IC50 and Ki values are determined from dose-response curves. These assays confirm that NSC-109116 is a potent and selective AKR1C1 inhibitor.

In vivo, NSC-109116 has been studied for its potential therapeutic applications in hormone-dependent cancers, such as prostate and breast cancer. By selectively inhibiting AKR1C1, the compound prevents the reduction of steroid hormones, which may slow the growth of hormone-dependent tumors. While specific in vivo efficacy data for NSC-109116 are not extensively detailed in the available literature, its mechanism of action and in vitro activity suggest that it would be effective in reducing tumor growth in hormone-dependent cancers.

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.
In vitro enzyme/receptor binding assays for NSC-109116 involve AKR1C1 enzyme assays using purified enzyme. The compound's inhibitory activity is measured by monitoring the reduction of a substrate (e.g., 20α-hydroxysteroid or a fluorescent substrate) in the presence of NADPH. IC50 and Ki values are determined from dose-response curves. Selectivity profiling against other AKR enzymes (AKR1C2, AKR1C3) and other dehydrogenases is performed to assess the compound's specificity. X-ray crystallography or molecular docking studies can be employed to confirm the compound's binding to the selective pocket in the AKR1C1 active site.

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.
In vitro cellular assays for NSC-109116 are conducted in hormone-dependent cancer cell lines, such as prostate cancer (e.g., LNCaP) or breast cancer (e.g., MCF-7) cells. Cells are treated with varying concentrations of NSC-109116, and cell proliferation is measured using MTT, CellTiter-Glo, or colony formation assays. Steroid hormone metabolism is assessed by measuring the levels of reduced steroid hormones using LC-MS or other analytical techniques. These assays confirm that NSC-109116 engages its target in a cellular context and produces the expected inhibition of AKR1C1 activity and cancer cell growth.

In vivo animal studies for NSC-109116 would typically be conducted in mouse xenograft models using hormone-dependent cancer cell lines. Animals would be administered the compound, and tumor growth inhibition would be monitored. Steroid hormone levels in tumors and plasma could be measured to assess the compound's effects on hormone metabolism. Pharmacokinetic studies would be performed to determine the compound's bioavailability, half-life, and tissue distribution. These studies would confirm that NSC-109116 is effective in vivo and provide information about its therapeutic potential.

Pharmacokinetic properties of NSC-109116 indicate that it has a molecular weight of 293.12 and a molecular formula of C13H9BrO3. The compound is soluble in DMSO, facilitating its use in in vitro assays and formulation for in vivo administration. For storage, the powder should be kept under appropriate conditions to maintain stability. The compound's purity is typically high, ensuring quality and reproducibility in experimental studies.

The toxicological profile of NSC-109116 is primarily derived from its use as a research compound in preclinical studies. As an AKR1C1 inhibitor, potential on-target effects could include changes in steroid hormone metabolism, which could affect reproductive and endocrine function. Comprehensive toxicology studies would be required for therapeutic development, including assessments of endocrine, reproductive, and hepatic function.

[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.

This article reports the first design, synthesis, and evaluation of human 20α-hydroxysteroid dehydrogenase (AKR1C1) inhibitors based on the recently published crystal structure of a ternary complex of AKR1C1 and its inhibitor. While the newly designed inhibitors retain the enzyme-inhibitor interactions observed in the crystal structure, the additional phenyl group of the most active compound, 3-bromo-5-phenylsalicylic acid, targets a non-conserved hydrophobic pocket in the AKR1C1 active site, resulting in a 21-fold increase in inhibitory activity (K_i = 4 nM) compared to the structurally similar 3α-hydroxysteroid dehydrogenase isoenzyme (AKR1C2). This compound forms hydrogen bonds with Tyr55, His117, and His222, and its benzene ring also forms van der Waals interactions with Leu308, Phe311, and the non-conserved Leu54 (Val in AKR1C2) residues. In addition, 3-bromo-5-phenylsalicylic acid effectively inhibited progesterone metabolism in AKR1C1-overexpressing cells at an effective concentration of 10 nM and an IC(50) value of 460 nM. [1] In summary, using the recently determined crystal structure of the AKR1C1-inhibitor complex and GRID analysis of the inhibitor binding site, a new salicylic acid-based inhibitor (compound 4) was designed, which showed superior potency (Ki = 4 nM) and selectivity (21-fold) compared to AKR1C2. Furthermore, compound 4 significantly reduced intracellular progesterone metabolism with an IC50 value of 460 nM, which is comparable to or better than the IC50 values of the two previously known most potent AKR1C1 inhibitors—benzbromarone and 3′,3′′,5′,5′′-tetrabromophenolphthalein. The target of compound 4 was the selective pocket in the AKR1C1 active site composed of three nonpolar residues, Leu54, Leu308, and Phe311. Leu308 is one of two non-conserved C-terminal residues (the other being Leu306), and it is these two residues that cause the difference in inhibitory potency between AKR1C1 and its two isoforms, AKR1C3 and AKR1C4, exceeding 4000-fold. Since the active sites of AKR1C1 and AKR1C2 differ by only one amino acid residue (Leu54 in AKR1C1 and Val54 in AKR1C2), and existing inhibitors have similar inhibitory potency against these two enzymes, it is necessary to design novel inhibitors that can interact with Leu54 in AKR1C1 to the greatest extent possible in order to improve their selectivity against AKR1C2. Therefore, the development of new derivatives of compound 4 is expected to improve the selectivity of currently known AKR1C1 inhibitors. We also demonstrate that while searching large chemical databases is helpful in discovering new enzyme inhibitors, using the high-resolution crystal structure of enzyme-inhibitor complexes to analyze the small structural differences between different enzyme isozymes is an effective tool for optimizing enzyme-inhibitor interactions. [1]
NSC-109116 (AKR1C1-IN-1) is a highly potent and selective inhibitor of 20α-hydroxysteroid dehydrogenase (AKR1C1). It has a Ki of 4 nM for AKR1C1, 87 nM for AKR1C2, and 4.2 μM and 18.2 μM for AKR1C3. The compound targets a selective pocket in the AKR1C1 active site. NSC-109116 is not approved for clinical use and is available from research chemical suppliers for preclinical studies.

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.)