2.1.1. Cell lines and culture conditions

The A549 cell line was obtained from ATCC. LNCaP cell line was generously donated by the lab of William Farrar at Frederick National Laboratories. Cells were cultured in RPMI 1640 medium supplanted with 10% dialyzed fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin. For the LNCaP cell line, arachidonic acid was added at 10 μM. The cultures were maintained in a 37 °C incubator with 5% CO₂ and under a humidified atmosphere.

2.1.2. HPGD qHTS Assay

Inhibition of 15-PGDH (HPGD) activity was screened by utilizing prostaglandin 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. All compounds were screened using a qHTS approach, where compounds are assayed using at least seven concentrations to generate concentration-response curves for every compound. Briefly, qHTS uses an inter-plate dilution method where the first plate contains the highest concentration of a set of compounds in DMSO, while subsequent plates contain the same compounds in the same well locations, but at successive lower concentrations.

Three μL of enzyme (20 nM final concentration) 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 μL of substrate/cofactor solution (Substrate/cofactor solution: 1 mM NAD⁺ and 30 μM prostaglandin (Sigma, prostaglandin E2, #P5640)) were added to start the reaction. The plates were immediately transferred to and read 4 times every 30 seconds on a ViewLux High-throughput CCD imager (Perkin-Elmer) using 340 nm excitation and 450 nm emission fluorescence protocol. The fluorescence intensity difference between the last and the first time points, normalized against controls, was used to compute reaction progress. [PubChem AID 743171]

2.1.3. Prostaglandin E₂ (PGE₂) HTRF Cell-based 384-well Assay: A549 or LNCaP Cell Line

A549 cells, LNCaP cells or media were dispensed in 384-well plates (ProxiPlate-384 Plus, PerkinElmer, Shelton, CT) at 5,000 cells/9.9 μL/well using a CappAero384 multichannel pipette (Odense, Denmark). Separately on the plate, 10 μL of PGE₂ calibration standards at a starting assay concentration of 5,000 pg/mL (1:2 dilution, 12-points, n = 1, prepared in media, Cisbio, Bedford, MA) were dispensed. Compounds (100 nL, final concentration range from 41.9 nM to 100 μM) were transferred using an ATS Acoustic Liquid Dispenser (EDC Biosystems, Fremont, CA). After being mixed using an automated liquid handler (CyBio, Jena, Germany), samples were incubated (37 °C, 5% CO_2, under a humidified atmosphere) for 16 hours. PGE₂ labeled conjugates were added to each well, first 5 μL of PGE₂-d₂, followed by 5 μL of anti-PGE₂-cryptate, centrifuged (1,500 rpm, 15 sec) and incubated (RT, protected from light) for 5 hours. Time-resolved fluorescence intensity (320 nm excitation, 615 and 665 nm emission) was measured using an Envision plate reader (PerkinElmer). Data was expressed as the ratio of 665 nm/615 nm emissions. Concentration (pg/mL) of samples were calculated from a 4-parameter logistic calibration curves of the PGE₂ standards. [PubChem AID 743172]

2.1.4. Prostaglandin E₂ (PGE₂) HTRF Cell-based 1,536-well Assay: A549 Cell Line

A549 cells or media were dispensed onto a 1,536-well solid-bottom T/C white plate (Greiner Bio One, Monroe, NC) at 2,500 cells/5 μL/well using a Thermo Scientific Multidrop Combi (Thermo Fisher Scientific Inc., Waltham, MA). Separately on the plate, 5 μL of PGE₂ calibration standards at a starting assay concentration of 5,000 pg/mL (1:2 dilution, 12-points, n = 2, prepared in media, Cisbio, Bedford, MA) were manually dispensed, followed by compounds (23 nL, final concentration range from 775 pM to 45.8 μM) and positive control titration (CID- 3243760, final concentration range 1.4 nM to 45.8 μM) transferred using a Kalypsys Pin-tool dispenser (Wako Automation, San Diego, CA). Plates were incubated (37 °C, 5% CO₂, under a humidified atmosphere) at 4, 8, 12, 16, and 20 hours. PGE₂ labeled conjugates were added to each well, first 2.5 μL of PGE₂-d₂ (or buffer), followed by 2.5 μL of anti-PGE₂-cryptate, centrifuged (1,000 rpm, 15 sec) and incubated (RT, protected from light) for 5 hours. Time-resolved fluorescence intensity (320 nm excitation, 615 and 665 nm emission) was measured using an Envision plate reader (PerkinElmer). Data was expressed as the ratio of 665 nm/615 nm emissions. Concentration (pg/mL) of samples were calculated from a 4-parameter logistic calibration curves of the PGE₂ standards. [PubChem AID 743167]

2.1.5. HSD17β4 Counterscreen

The mitochondrial enzyme 17β-hydroxysteroid dehydrogenase 1 (HSD17β4 catalyzes the NAD⁺-dependent oxidation of a variety of substrates including acyl-CoAs, androgens and neurosteroids, as well as bile acids. HSD17β4 activity was screened by utilizing an electron donor (estradiol) 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.

Briefly, 3 μL of enzyme (final concentration 100 nM) or buffer were dispensed into a 1,536-well solid-bottom black plate (Greiner Bio One, Monroe, NC) followed by pin-tool transfer (23 nL) of compound (final concentration range 324 pM to 57.2 μM) and control (CID-24867619, final concentration range 5.23 nM to 11.4 μM). Samples were incubated (RT, protected from light) for 15 minutes followed by a 1 μL substrate addition of NAD⁺ and Estradiol (final concentrations of 1 mM and 50 μM, respectively, in 15% DMSO). Plates were centrifuged at 1,000 rpm for 15 seconds, then read immediately on a ViewLux High-throughput CCD imager (Perkin-Elmer) equipped with standard UV fluorescence optics (340 nm excitation, 450 nm emission), incubated for 1 hour (RT, protected from light) followed by a second read. Data were normalized (Read 2_{1 hour} – Read 1_{0 hour}) against no-enzyme (positive) and enzyme-containing (neutral) wells. A stepwise description of the 1536-well assay is shown in Table 4.

Table 4

Stepwise protocol of the HSD17β4 counterscreen in 1536-well format.

2.1.6. ALDH1A1 Counterscreen

Aldehyde dehydrogenase 1 family, member A1 (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 an essential role as a 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. ALDH1A1 shares some homology with HPGD, and it served as a suitable anti-target of HPGD to determine chemical probe selectivity. The assay system is similar to the HPGD primary screening protocol. 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.

Briefly, 3 μL of enzyme (final concentration 50 nM) or buffer were dispensed into a 1,536-well solid-bottom black plate (Greiner Bio One, Monroe, NC) followed by pin-tool transfer (23 nL) of compound (final concentration range 324 pM to 57.2 μM) and control (Bay 11-7085 (CID-5353432), final concentration range 1.31 nM to 2.86 μM). Samples were incubated (RT, protected from light) for 15 minutes followed by a 1 μL substrate addition of NAD⁺ and Propionaldehyde (final concentrations of 1 mM and 80 μM, respectively). Plates were centrifuged at 1,000 rpm for 15 seconds, then read in kinetic mode on a ViewLux High-throughput CCD imager (Perkin-Elmer) equipped with standard UV fluorescence optics (340 nm excitation, 450 nm emission) for 10 minutes at 5 minute intervals. Data were normalized (Read 2_{10 min} – Read 1_{0 min}) against no-enzyme (positive) and enzyme-containing (neutral) wells. A stepwise description of the 1536-well assay is shown in Table 5.

Table 5

Stepwise protocol of the LDHA counterscreen in 1536-well format.

2.1.7. LDHA Counterscreen

LDHA converts pyruvate to lactate while regenerating NAD⁺ from NADH. LDHA enzymatic activity is then coupled to the activity of diaphorase, which reversibly catalyzes the reaction of NAD⁺ to NADH while simultaneously reducing resazurin to the fluorescent resorufin. This change in fluorescence signal (excitation 544 nm; emission, 590 nm) was monitored using the ViewLux CCD imager (Perkin Elmer). Data were normalized against ‘no enzyme’ control wells and ‘no compound’ control wells.

The enzymatic reaction was performed in buffer consisting of 200 mM Tris-HCl pH 7.4, containing 0.01% Tween 20, and the assay was run in black solid-bottom Greiner plates. A stepwise description of the 1536-well assay is shown in Table 6.

Table 6

Stepwise protocol of the LDHA counterscreen in 1536-well format.

2.1.8. LDHB Counterscreen

LDHB converts pyruvate to lactate while regenerating NAD⁺ from NADH. LDHB enzymatic activity is then coupled to the activity of diaphorase, which reversibly catalyzes the reaction of NAD⁺ to NADH while simultaneously reducing resazurin to the fluorescent resorufin. This change in fluorescence signal (excitation 544 nm; emission, 590 nm) was monitored using the ViewLux CCD imager (Perkin Elmer). Data were normalized against ‘no enzyme’ control wells and ‘no compound’ control wells.

The enzymatic reaction was performed in buffer consisting of 200 mM Tris-HCl pH 7.4, containing 0.01% Tween 20, and the assay was run in black solid-bottom Greiner plates. A stepwise description of the 1536-well assay is shown in Table 7.

Table 7

Stepwise protocol of the LDHA counterscreen in 1536-well format.

2.1.9. Rabbit GAPDH Counterscreen

The counterscreen for non-specific dehydrogenase activity was conducted using a rabbit glyceraldehyde-3-phosphate dehydrogenase (rGAPDH; Sigma Aldrich, St. Louis, MO) coupled assay system. In this assay, the rGAPDH-catalyzed conversion of NAD⁺ to NADH is coupled downstream to an additional enzyme, diaphorase (Sigma Aldrich, St. Louis, MO), which reduces the non-fluorescent molecule, resazurin, to a red fluorescent product, resorufin, in an NADH-dependent fashion. Thus, rGAPDH activity leads to an increase in fluorescence at 598 nm, and inhibitors of the reaction lead to a decrease in resorufin production and a subsequent decrease in fluorescence. The change in fluorescence between 0 and 30 minutes was measured, and data were normalized to control columns representing maximum signal (all components) and background (no rGAPDH). The buffer consisted of 100mM triethanolamine, pH 7.6, 5mM MgCl₂ and 0.01% Tween-20, and the assay was run in black, solid-bottom, non-tissue culture-treated Greiner plates. A stepwise description of the 1536-well assay is shown in Table 8.

Table 8

Stepwise protocol of the rGAPDH counterscreen in 1536-well format.

2.1.10. NF-κB-bla ME180 Cell Line and Culture Conditions

The CellSensor™ NF-κB-bla ME180 cell line (NF-κB-bla cells) were obtained from Invitrogen (Carlsbad, CA). Cells were cultured in High Glucose GlutaMAX™ DMEM medium supplemented with 10% dialyzed fetal bovine serum, 0.1 mM non-essential amino acids, 25 mM HEPES (pH 7.3), 100 U/mL penicillin and 100 μg/mL streptomycin, and 5 μg/mL of blasticidin. The cultures were maintained in a 37 °C incubator with 5% CO₂ and under a humidified atmosphere.

2.1.11. NF-κB β-Lactamase Reporter Gene Assay (NF-κB bla assay) and Cell Viability Assay

The NF-κB-bla cells were suspended in OPTI-MEM medium supplemented with 0.5% dialyzed fetal bovine serum, 0.1 mM nonessential amino acids, 25 mM sodium pyruvate, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were dispensed at 2,000 cells/5 μL/well using a Thermo Scientific Multidrop Combi (Thermo Fisher Scientific Inc., Waltham, MA) and incubated (37 °C, 5% CO_2, under a humidified atmosphere) overnight. Compounds (23 nL, final concentration range from 647 pM to 38.2 μM) and positive control titration (MG132, final concentration range 17.5 nM to 38.2 μM) were transferred to a 1,536-well clear-bottom T/C black plate (Greiner Bio One) using a Kalypsys Pin-tool dispenser (Wako Automation, San Diego, CA). Cells were incubated (37 °C) 15 minutes, followed by addition of either 1 μL assay buffer with or without tumor necrosis factor alpha (TNF-α, final concentration 1 ng/mL). The plates were incubated for 4, 8, 12, 18, and 24 hours at 37 °C, then 1 μL of LiveBLAzer™ B/G FRET substrate (Invitrogen, CA) detection mix was added and the plates incubated at room temperature for 2 hours. Fluorescence intensity (405 nm excitation, 460 and 530 nm emission) was measured using an Envision plate reader (Perkin Elmer, Shelton, CT). Data was expressed as the ratio of 460nm/530 nm emissions. At the completion of the read, each plate received a 2.5 μL dispense of CellTiter-Glo (Promega, Madison, WI), then after a 10 minute incubation (RT) samples were analyzed for luminescence intensity using a ViewLux High-throughput CCD imager (Perkin-Elmer) equipped with clear filters.

2.2.1. ML387 (Probe 1)

(1-(4-methoxyphenyl)-1H-benzo[d]imidazol-5-yl)(piperidin-1-yl)methanone, TFA (ML387)

LC-MS rt: 4.15 min; ¹H NMR (400 MHz, DMSO-d₆) δ 8.81 – 8.70 (m, 1H), 7.77 (s, 1H), 7.66 – 7.60 (m, 2H), 7.58 (d, J = 8.5 Hz, 1H), 7.37 (d, J = 8.6 Hz, 1H), 7.23 – 7.15 (m, 2H), 3.86 (s, 3H), 3.65 – 3.35 (m, 4H), 1.68 – 1.58 (m, 2H), 1.58 – 1.44 (m, 4H); ¹³C NMR (100 MHz, DMSO-d₆) δ 168.9, 159.2, 144.4, 133.6, 131.5, 128.0, 125.8, 123.0, 117.4, 115.2, 111.0, 55.6, and 24.1; HRMS (ESI) m/z 336.1703 (M + H)⁺ (C₂₀H₂₂N₃O₂ requires 336.1707).

Figure 1¹H and¹³C NMR spectra of ML387

Figure 2LC-MS of ML387

Probe analogs

Figure 3Structures of the five analogs of ML387 that have been submitted to the MLSMR

View in own window

Internal ID	MLS ID	SID	CID	ML #	Type	Source
NCGC00262228	MLS005565827	162211043	3474778	ML387	Probe	NCGC
NCGC00262223	MLS004857446	162211012	71307827		Analog	NCGC
NCGC00262277	MLS004857445	162210983	71307865		Analog	NCGC
NCGC00262222	MLS004857448	162210969	71307836		Analog	NCGC
NCGC00262238	MLS004857449	162211008	71307876		Analog	NCGC
NCGC00262242	MLS004857450	162211025	71307812		Analog	NCGC

Figure 4Stability of ML387 over time in aqueous solution at room temperature, as measured by the area under the curve (AUC) of the probe HPLC signal observed at 254 nm

PBS: phosphate buffer saline, pH 7.4; HPGD: assay buffer; GSH: 10 mM GSH in PBS buffer.

2.2.2. ML388 (Probe 2)

3-(2,5-dimethyl-1-(p-tolyl)-1H-pyrrol-3-yl)-6,7,8,9-tetrahydro-5H-[1,2,4]triazolo[4,3-a]azepine, TFA (ML388)

LC-MS rt: 4.75 min; ¹H NMR (400 MHz, DMSO-d₆) δ 7.39 (d, J = 8.0 Hz, 2H), 7.26 (d, J = 8.2 Hz, 2H), 6.19 (s, 1H), 4.30 – 4.06 (m, 2H), 3.16 – 3.01 (m, 2H), 2.41 (s, 3H), 2.05 (s, 3H), 2.02 (s, 3H), 1.90 – 1.78 (m, 4H), 1.75 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 157.0, 150.0, 138.4, 134.2, 131.4, 130.2, 130.1, 127.6, 106.5, 69.8, 46.5, 29.3, 26.7, 25.4, 23.9, 20.7, 12.5, and 11.8; HRMS (ESI) m/z 321.2076 (M + H)⁺ (C₂₀H₂₅N₄ requires 321.2074).

Figure 5¹H and¹³C spectra of ML388

Figure 6LC-MS of ML388

Probe analogs

Figure 7Structures of the five analogs of ML388 that have been submitted to the MLSMR

View in own window

Internal ID	MLS ID	SID	CID	ML #	Type	Source
NCGC00249661	MLS005565826	162211017	71307851	ML388	Probe	NCGC
NCGC00249658	MLS004857440	162211019	71307849		Analog	NCGC
NCGC00249646	MLS004857441	162211016	71307856		Analog	NCGC
NCGC00249647	MLS004857442	162210974	71307853		Analog	NCGC
NCGC00249642	MLS004857443	162210963	71307824		Analog	NCGC
NCGC00249643	MLS004857444	162210962	71307846		Analog	NCGC

Figure 8Stability of ML388 over time in aqueous solution at room temperature, as measured by the area under the curve (AUC) of the probe HPLC signal observed at 254 nm

PBS: phosphate buffer saline, pH 7.4; HPGD: assay buffer; GSH: 10 mM GSH in PBS buffer.

2.3.1. ML387 (Probe 1)

(4-fluoro-3-nitrophenyl)(piperidin-1-yl)methanone

A mixture containing 4-fluoro-3-nitrobenzoic acid (2.00 g, 10.80 mmol), and oxalyl chloride (4.75 mL, 54.0 mmol) in DCM (54mL) was carefully treated with DMF (0.084 mL, 1.080 mmol). The reaction mixture was stirred at 23 °C for 3 hr. The reaction mixture was concentrated under diminished pressure. The obtained residue was taken up in 10 mL of toluene, and the mixture was concentrated under diminished pressure. This process was repeated to yield crude 4-fluoro-3-nitrobenzoyl chloride as a clear, dark yellow syrup.

A solution containing 4-fluoro-3-nitrobenzoyl chloride (2.20 g, 10.8 mmol) in DCM (27 mL) was treated with piperidine hydrochloride (1.71 g, 14.0 mmol), and with diisopropylethylamine (2.10 mL, 11.9 mmol) at 23 °C for 69 hr. The reaction mixture was diluted with 25 mL of DCM. The organic layer was washed with two 25 mL portions of 0.50 M aqueous HCl. The product was extracted with two 25 mL portions of DCM. The combined organic layer was dried (MgSO₄), and was then concentrated under diminished pressure. The obtained clear pale yellow oil was applied to a silica gel column (50 g); eluting with 80:20 → 20:80 Hex-EtOAc afforded (4-fluoro-3-nitrophenyl)(piperidin-1-yl)methanone as a clear pale yellow syrup: yield 1.72 g (63 % over two steps); ¹H NMR (400 MHz, CDCl₃) δ 8.11 (dd, J = 7.0, 2.2 Hz, 1H), 7.69 (ddd, J = 8.5, 4.2, 2.2 Hz, 1H), 7.34 (dd, J = 10.4, 8.5 Hz, 1H), 3.70 (brs, 2H), 3.36 (brs, 2H), 1.63 (m, 6H).

2.3.2. ML388 (Probe 2)

2,5-dimethyl-1H-pyrrole-3-carbohydrazide

A solution containing 2,5-dimethyl-1H-pyrrole-3-carboxylic acid (2.00 g, 14.37 mmol) in THF (29 mL) was treated with CDI (2.56 g, 15.81 mmol) at 23 °C for 3.5 hr. The resulting brown/orange suspension was then treated with hydrazine monohydrate (2.1 mL, 43.1 mmol) at 23 °C for 18 hr. Within 1 hr, the precipitate had dissolved and a clear orange solution was obtained. The solvent was evaporated under diminished pressure; the resulting mixture was applied to a silica gel column (50 g); eluting with 95:5 → 75:25 DCM-MeOH led to a mixture containing the product and imidazole. The obtained solid was recrystallized from EtOAc to afford 2,5-dimethyl-1H-pyrrole-3-carbohydrazide as a pale yellow crystalline solid: yield 1.43 g (65.0 %); ¹H NMR (400 MHz, DMSO-d₆) δ 10.62 (s, 1H), 8.65 (s, 1H), 6.08 – 5.97 (m, 1H), 4.13 (s, 2H), 2.35 (s, 3H), 2.07 (s, 3H).

3-(2,5-dimethyl-1-(p-tolyl)-1H-pyrrol-3-yl)-6,7,8,9-tetrahydro-5H-[1,2,4]triazolo[4,3-a]azepine, TFA (ML388)

In a sealed vial, a mixture containing 3-(2,5-dimethyl-1H-pyrrol-3-yl)-6,7,8,9-tetrahydro-5H-[1,2,4]triazolo[4,3-a]azepine (150 mg, 0.651 mmol), p-toluyl iodide (170 mg, 0.782 mmol), cesium carbonate (318 mg, 0.977 mmol), copper(I) oxide (9.32 mg, 0.065 mmol), 4,7-dimethoxy-1,10-phenanthroline (L) (31.3 mg, 0.130 mmol), and poly(ethylene glycol) (150 mg, approx. 100 mg/mmol of pyrrole) in NMP (1.30 mL) was stirred at 100 °C for 18 hr. The reaction mixture was diluted with 3 mL of acetonitrile, and was then filtered. The collected clear brown solution was concentrated under diminished pressure and the resulting residue was purified by reversed phase HPLC to afford 3-(2,5-dimethyl-1-(p-tolyl)-1H-pyrrol-3-yl)-6,7,8,9-tetrahydro-5H-[1,2,4]triazolo[4,3-a]azepine, TFA; yield: 38.3 mg (14%).

3.3.1. In-vitro ADME profiles of ML387 and ML388

3.3.1. Selectivity profiles of ML387 and ML388