Placental Alkaline Phosphatase (PLAP) Luminescent HTS assay - Probe 1

a. The original goal for probe characteristics

This MLSCN carry-forward project was an early Cycle 2 assay proposal and a formal CPDP was not filed. It was derived on work with tissue non-specific alkaline phoshatase inhibitor and later activator work, that suggested that PLAP specific inhibitors would also useful tools for this class of isozyme with yet to be elucidated biological function.

b. Assay implementation and screening

i. PubChem Bioassay Name(s), AID(s), Assay-Type (Primary, DR, Counterscreen, Secondary)

Table

Primary assay details are described below.

PLAP screening was developed and performed at the Burnham Center for Chemical Genomics (BCCG) within the Molecular Library Screening Center Network (MLSCN) as a selectivity screen for tissue nonspecific alkaline phosphatase (TNAP, AID-518). XO1 MH077602-01, Pharmacological inhibitors of tissue-nonspecific alkaline phosphatase (TNAP), Assay Provider Dr. José Luis Millán, Burnham Institute for Medical Research, La Jolla, CA.

Protocol

PLAP assay materials

1. PLAP protein was provided by Dr. José Luis Millán (Burnham Institute for Medical Research, San Diego, CA). The CDP-star was obtained from New England Biolabs.
2. Assay Buffer: 250 mM DEA, pH 9.8, 2.5 mM MgCl₂, and 0.05 mM ZnCl₂.
3. PLAP working solution contained a 1/6400 dilution in assay buffer. The solution was prepared fresh prior to use.
4. CDP-star working solution contained 212.5 µM CDP-star in MQ water.
5. TCEP working solution −5 mM in 10% DMSO.

PLAP HTS protocol

1. 4 uL of 100 µM compounds in 10% DMSO were dispensed in columns 3–24 of Greiner 384-well white small volume plates (784075).
2. Using a Thermo wellmate dispenser 4 uL of the following solutions were added:
   1. TCEP working solution - column 1 (positive control).
   2. 10% DMSO - column 2 (negative control).
3. 8 uL of PLAP working solution was added to the whole plate using a WellMate bulk dispenser (Matrix).
4. 8 uL of CDP-star working solution was added to the whole plate using WellMate bulk reagent dispenser (Matrix).
5. Final concentrations of the components in the assay were as follows:
   1. 100 mM DEA, pH 9.8, 1.0 mM MgCl₂, 0.02 mM ZnCl₂ (columns 1–24)
   2. 1/16000 dilution PLAP (columns 1–24)
   3. 85 µM CDP-star (columns 1–24)
   4. 1 mM TCEP (columns 1)
   5. 2 % DMSO (columns 1–24)
   6. 20 µM compounds (columns 3–24)
6. Plates were incubated for 30 minutes at room temperature.
7. Luminescence was measured on an Envision plate reader (Perkin Elmer).
8. Data analysis was performed using CBIS software (ChemInnovations, Inc).

PLAP dose-response confirmation screening protocol

1. Dose-response curves contained 10 concentrations of compounds obtained using 2-fold serial dilution. Compounds were serially diluted in 100% DMSO, and then diluted with water to 10% final DMSO concentration. 4 uL compounds in 10% DMSO were transferred into columns 3–22 of Greiner 384-well white small-volume plates (784075). Columns 1–2 and 23–24 contained 4 uL of TCEP working solution and 10% DMSO, respectively.
2. 8 uL of PLAP working solution was added to the whole plate using a WellMate bulk reagent dispenser (Matrix).
3. 8 uL of CDP-star working solution was added to the whole plate using a WellMate reagent bulk dispenser (Matrix).
4. Plates were incubated for 30 mins at room temperature.
5. Luminescence was measured on an Envision plate reader (Perkin Elmer).
6. Data analysis was performed using CBIS software (ChemInnovations, Inc) using a sigmoidal dose-response equation through non-linear regression

iii. Summary of Results

Primary hits from the PLAP HTS assay were confirmed and tested in parallel against TNAP for selectivity. A few selective hits were identified. Those hits were purchased along with 56 analogues (Table 1). There are many commercial analogues available and so Analogue-By-Catalogue (ABC) was performed in an iterative fashion. From the purchased compounds we were able to conclude that the dihydroxyl groups of the catechol are essential for PLAP activity. The SAR was completed and a probe molecule CID-665093 (MLS-0014097) was identified (see Table 1 below).

Table 1

SAR results generated using analogue-by-catalogue (ABC).

Medicinal chemistry focused on the two final probe candidates, CID-2102207 (MLS-0107074) and CID-665093 (MLS-0014097) (Figure 1) that were identified as low-micromolar inhibitors of PLAP through primary screening.

Figure 1

Re-synthesis of these targets was undertaken and these compounds were subjected to the BCCG target independent ADME assay panel (see also Table 1 below)

c. Probe Optimization

Description of SAR & chemistry strategy (including structure and data) that led to the probe

SAR was developed primarily by the ABC approach for this MLSCN project due to the availability of numerous commercial analogues. Analysis of the SAR identified some key structural features, including: (1) Reposition or derivatization of the hydroxyl groups leads to inactive compounds (SID-56373705, SID-56373700 and SID-56373696). (2) Selectivity for PLAP apparently resides in the N-substituted triazole group (Figure 1). Unsubstituted triazoles also potently inhibit PLAP but do not display selectivity vs. TNAP and IAP. Though 58 analogues were purchased to explore R1 and R2 (see Figure 2, and attached triazole list at end), no significant improvement in either selectivity or potency was achieved. For example, analogue SID-57287606 is in the submicromolar range for PLAP, but it is not as selective as the proposed probe molecule. The objective of chemistry was to develop a tractable route for the synthesis of the probe candidates, prepare authentic samples, confirm structure and purity, and provide enough material for submission to MLSMR.

Figure 2

Probe analogs R1 & R2 variations.

a. Chemical name

1-(3,4-dihydroxyphenyl)-2-[[5-(2-methoxyphenyl)-4-prop-2-enyl-1,2,4-triazol-3-yl]sulfanyl]ethanone [ML085]

b. Probe chemical structure

Figure 3Probe compound structure

c. Structural Verification Information of probe SID

SID-56373725

Spectral data supporting proposed structure: ¹H NMR (400 MHz, Acetone-D6) δ 7.54 (ddd, J = 8.4, 7.5, 1.8, 1H), 7.46 – 7.33 (m, 3H), 7.17 (d, J = 8.1, 1H), 7.07 (dt, J = 7.5, 0.9, 1H), 6.72 (d, J = 8.2, 1H), 5.89 – 5.60 (m, 1H), 5.10 (dd, J = 10.4, 1.1, 1H), 4.91 (dd, J = 17.2, 1.1, 1H), 4.78 (s, 2H), 4.49 (dd, J = 3.7, 1.6, 2H), 3.83 (s, 3H). ¹³C NMR (101 MHz, Acetone-D6) δ 191.32, 159.49, 158.26, 154.41, 151.21, 148.89, 133.16, 132.97, 132.87, 124.45, 123.57, 121.53, 117.97, 117.73, 115.86, 113.80, 112.18, 55.92, 47.44, 42.01. MS (ESI) calculated C₂₀H₁₉N₃O₄S m/z = 397.11, found m/z = 398.01 [M+H].

Figure 4NMR or Probe compound

Figure 5LC-MS traces for probe purity

d. PubChem CID (corresponding to the SID)

CID-665093

e. Availability from a vendor

The probe is commercially available from InterBioScreen (ID # STOCK4S-90308).

f. MLS# that verifies the submission of probe molecule and five related samples that were submitted to the SMR collection

Table 2

Submission information on Probe and analogs.

g. Mode of action for biological activity of probe

The probe is a biochemical inhibitor of PLAP. The mode of action for the biological activity of this probe has not yet been elucidated.

h. Detailed synthetic pathway for making probe

Solid 2-methoxybenzoic acid (5.0g, 32.9mmol) was transferred to a 250ml round-bottom flask and dissolved in 50 mL dry MeOH. 8 drops of c.H₂SO₄ were added to the reaction mixture. The reaction mixture was heated at reflux for 12h. After 12 h the reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure. The crude reaction mixture was dissolved in 50 mL diethylether and washed with saturated NaHCO₃ (2 × 50mL) and brine solution (2 × 50mL). The organic layer was collected and the solvent was removed under reduced pressure to yield the methyl ester as a white solid (5.4 g, 99%). ¹H NMR (400 MHz, CDCl₃) δ 7.82 (dd, J = 7.9, 1.8, 1H), 7.49 (ddd, J = 8.5, 7.4, 1.8, 1H), 7.00 (dtd, J = 4.7, 3.4, 1.0, 2H), 3.93 (s, 3H), 3.91 (s, 3H).

Methyl anisate (50.0mg, 0.3 mmol) was transferred to a glass vial and dissolved in 5 mL dry MeOH. 4-Allyl thio-semicarbazide (59.2 mg, 0.45 mmol) was added to the reaction mixture. This was followed by addition of 0.2 mL of 25%(wt) NaOMe solution. The resultant reaction mixture was heated overnight at 85^oC in a sealed vial. After the reaction was complete, the solvents were evaporated under reduced pressure. The reaction mixture was cooled to 0^oC and 20 mL deionized water was added to it. The pH of the reaction mixture was lowered to 5 using 10% AcOH (dropwise addition). The resultant white precipitate (4-allyl-3-(2-methoxyphenyl)-1H-1,2,4-triazole-5(4H)-thione) was filtered, dried and weighed (55mg, 74.3%). ¹H NMR (400 MHz, CDCl₃) δ 7.65 – 7.47 (m, 1H), 7.35 (dd, J = 7.5, 1.6, 1H), 7.08 (ddd, J = 19.5, 13.1, 4.6, 2H), 5.75 (ddd, J = 16.0, 11.4, 5.7, 1H), 5.10 (d, J = 10.3, 1H), 4.91 (d, J = 17.1, 1H), 4.60 (d, J = 5.8, 2H), 3.86 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 167.79, 157.60, 150.56, 133.03, 131.79, 130.63, 120.98, 118.47, 114.67, 111.15, 55.58, 46.99.

Solid 4-allyl-3-(2-methoxyphenyl)-1H-1,2,4-triazole-5(4H)-thione (25.0 mg, 0.1mmol) was dissolved in 5 mL dry acetonitrile. 2-Chloro-3′,4′-dihydroxyacetophenone (18.8mg, 0.1mmol) was added to the reaction mixture followed by addition of Cs₂CO₃( 33mg, 0.1mmol). the resultant reaction mixture was stirred at room temperature for 8 hrs. The solid was filtered from the reaction mixture and the filtrate was concentrated to yield the final product 2-(4-allyl-5-(2-methoxyphenyl)-4H-1,2,4-triazol-3-ylthio)-1-(3,4-dihydroxyphenyl)ethanone (25mg, 62.5%). The product was further purified by crystallization using acetone. ¹H NMR (400 MHz, Acetone-D6) δ 7.54 (ddd, J = 8.4, 7.5, 1.8, 1H), 7.46 – 7.33 (m, 3H), 7.17 (d, J = 8.1, 1H), 7.07 (dt, J = 7.5, 0.9, 1H), 6.72 (d, J = 8.2, 1H), 5.89 – 5.60 (m, 1H), 5.10 (dd, J = 10.4, 1.1, 1H), 4.91 (dd, J = 17.2, 1.1, 1H), 4.78 (s, 2H), 4.49 (dd, J = 3.7, 1.6, 2H), 3.83 (s, 3H). ¹³C NMR (101 MHz, Acetone-D6) δ 191.32, 159.49, 158.26, 154.41, 151.21, 148.89, 133.16, 132.97, 132.87, 124.45, 123.57, 121.53, 117.97, 117.73, 115.86, 113.80, 112.18, 55.92, 47.44, 42.01. MS (ESI) calculated C₂₀H₁₉N₃O₄S m/z = 397.11, found m/z = 398.01 [M+H].

i. Summary of probe properties (solubility, absorbance/fluorescence, reactivity, toxicity, etc.)

Our internal counter screen efforts has showed CID-665093 to be inactive in two assays TNAP inhibition (AID-518) and G6DPH (AID-1020), and 10-fold less active vs. IAP (AID-1017), a very close family member of PLAP.

In Vitro Pharmacology

Compounds CID-665093 (MLS-0014097) and CID-2102207 (MLS-0107074) were subjected to a battery of in vitro pharmacology assays to assess aqueous solubility, cellular permeability, plasma protein binding (PPB), plasma stability, and microsome stability. The results of these assays are presented in Table 2.

Table 3

Pharmacologic Properties of MLS-01014097, MLS-0107074.

CID-665093 (MLS-0014097) exhibits modest solubility in aqueous buffer and poor cell permeability. On the other hand, since PLAP is an extracellular enzyme, poor permeability could provide an added advantage to the probe, helping to ensure selectivity for its biological function. The compound is most soluble in aqueous buffer at pH 7.4, indicating that the compound will be sufficiently soluble for use in cellular assays conducted under normal culture conditions. CID-665093 (MLS-0014097) is extensively bound to plasma proteins in human plasma. In mouse plasma, the percent compound bound is slightly less than in human plasma. The amount of free compound available to bind the target (PLAP) is therefore greater in mice suggesting that the efficacy of this compound might be greater in mice than in humans. The compound also exhibits robust metabolic stability when exposed to both mouse and human microsome preparations.

CID-2102207 (MLS-0107074) exhibits poor solubility in aqueous buffer at all pH levels tested. In order to assess the cellular permeability, it was required that a cosolvent (20% ACN) be used in the assay to facilitate solubility. Despite the use of ACN, cellular permeability remained poor. The compound is extensively bound to plasma proteins in human and mouse plasma, and exhibits moderate stability in human and mouse plasma. CID-2102207 (MLS-0107074) was significantly metabolized in human and mouse hepatic microsomes. This suggests that the compound will be subjected to extensive first pass metabolism, limiting the use of the compound in vivo. Taken together these attributes indicate that this probe is best suited for biochemical experiments, but may also be used in cell based experiments when formulated with an appropriate co-solvent.

j. Properties Computed from Structure

Table

a. Comparative data on (1) probe, (2) similar compound structures (establishing SAR) and (3) prior probes

Table 5

All Purchased Analogs.

b. Comparative data showing probe specificity for target

Table 4

SAR and Selectivity.