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Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-.

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

, , , , , , , , , , , , , , and .

Received: ; Last Update: September 2, 2010.

Placental alkaline phosphatase (PLAP) is highly expressed in primate placental tissue. Its biological function and relevance are still unknown, but PLAP-like enzymes are detected in serum of patients with primary testicular tumors, in particular seminoma and other cancers. Consequently, the identification of PLAP-specific inhibitors with selectivity over tissue non-specific alkaline phosphatase (TNAP) and intestinal alkaline phosphatase (IAP) may provide the necessary tools to characterize its biological role. Currently, inhibitors of PLAP lack either potency or selectivity. The small molecule probe ML085 (CID-25067483) is a biochemical inhibitor of PLAP, and will be useful to elucidate the key biological functions and natural substrates of human PLAP.

Assigned Assay Grant #: 1 R03 MH077602-01

Screening Center Name & PI: Conrad Prebys Center for Chemical Genomics (formerly Burnham Center for Chemical Genomics) & Dr. John C. Reed

Chemistry Center Name & PI: Conrad Prebys Center for Chemical Genomics (formerly Burnham Center for Chemical Genomics) & Dr. John C. Reed

Assay Submitter & Institution: Dr José Luis Millán & Sanford-Burnham Medical Research Institute (formerly Burnham Institute for Medical Research)

PubChem Summary Bioassay Identifier (AID): AID-1577

Probe Structure & Characteristics

Image ml085fu1
CID/MLTarget NameIC50/EC50 (nM) [SID, AID]Anti-target Name(s)IC50/EC50 (μM) [SID, AID]SelectivitySecondary Assay(s) Name: IC50/EC50 (nM) [SID, AID]
CID-665093

ML085		PLAP2,600 nM IC50 SID-56373725 AID-1512TNAP>100 µM IC50 SID-56373725 AID-518> 42
IAP>6.25 µM IC50 SID-56373725 AID-1017>2.4

Recommendations for the scientific use of this probe

Placental alkaline phosphatase (PLAP) is highly expressed in primate placental tissue. Its biological function is still unknown (1,2). PLAP-like enzymes could be detected in serum of patients with primary testicular tumors, in particular seminoma (3) and other cancers (4). This PLAP-specific inhibitors with selectivity over tissue non-specific alkaline phosphatase (TNAP) and intestinal alkaline phosphatase (IAP) can be used as a tools to characterize the biological role of PLAP. The small molecule probe CID-665093 will be useful to elucidate the key biological functions and natural substrates of human placental alkaline phosphatase (PLAP)

1. Scientific Rationale for Project

Alkaline phosphatases (EC 3.1.3.1) (APs) catalyze the hydrolysis of phosphomonoesters, releasing phosphate and alcohol. APs are dimeric enzymes found in most organisms. In human, four isozymes of APs have been identified: three isozymes are tissue-specific and the fourth one is tissue-nonspecific. Placental alkaline phosphatase (PLAP) is highly expressed in primate placental tissue. Its biological function is still unknown. However, the identification of PLAP-specific inhibitors with selectivity over tissue non-specific alkaline phosphatase (TNAP) and intestinal alkaline phosphatase (IAP) will provide the necessary tools to characterize its biological role

2. Project Description

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)

PubChem BioAssay NameAIDsProbe TypeAssay TypeAssay FormatAssay Detection & well format
Luminescent assay for HTS discovery of chemical inhibitors of placental alkaline phosphatase690InhibitorPrimaryBiochemicalLuminescence (1536)
Luminescent assay for HTS discovery of chemical inhibitors of placental alkaline phosphatase confirmation1512InhibitorConfirmatoryBiochemicalLuminescence (384)
TNAP luminescent HTS assay [Confirmatory]518InhibitorSecondary Assay for specificitybiochemicalLuminescence (384)
Luminescent assay for identification of inhibitors of human intestinal alkaline phosphatase [Confirmatory]1017InhibitorCounterscreen Assay for specificitybiochemicalLuminescence (1536)
Luminescent assay for identification of inhibitors of bovine intestinal alkaline phosphatase [Primary Screening]1019InhibitorCounterscreen Assay for specificitybiochemicalLuminescence (1536)

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

ii. Assay Rationale & Description

For the assay rationale, description and reagents protocols see section 2a above.

For this screen 95857 compounds were tested. The average Z′ for the assay was 0.69, the average signal to background was 38.6, the average signal to noise was 122.7 and the average signal window was 7.8. Initially 192 compounds were identified as primary positives with >= 50% inhibition of activity in the assay. After performing dose-response experiments with liquid DMSO stocks of these compounds, 82 of the hits generated a dose-response curve.

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).

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.

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.

Figure 2

Probe analogs R1 & R2 variations.

3. Probe

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 3. Probe compound 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 4. NMR or Probe compound.

Figure 4NMR or Probe compound

Figure 5. LC-MS traces for probe purity.

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 2Submission information on Probe and analogs

Probe/AnalogMLS-# (BCCG#)CIDSIDSource (vendor or BCCG syn)Amt (mg)Date ordered/submitted
Probe001409766509356373725InterBioScreen204/15/09
Analog 10390843140975957287589InterBioScreen204/15/09
Analog 20390850140069857287596InterBioScreen204/15/09
Analog 30390851140024657287597InterBioScreen204/15/09
Analog 40390857139834557287604InterBioScreen204/15/09
Analog 50390858139453657287605InterBioScreen204/15/09

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

Image ml085fu28

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 85oC in a sealed vial. After the reaction was complete, the solvents were evaporated under reduced pressure. The reaction mixture was cooled to 0oC 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 3Pharmacologic Properties of MLS-01014097, MLS-0107074

Cpd-IDAqueous Solubility (μg/mL) pH 5.0/6.2/7.4 (AID-1615)Permeability (×10−6 cm/s) pH 5.0/6.3/7.4 (AID-1557)Plasma Protein Binding (% Bound) (AID-1617)Plasma Stability (%Remaining) Human/Mouse (AID-1591/AID-1592)Microsomal Stability (%Remaining) Human/Mouse (AID-1555/pending)
Human (10μM/1μM)Mouse (10μM/1μM)
CID-665093

MLS-0014097		8.4/8.4/18.090/98/5898.6/98.787.9/89.5100.2/83.747.0/41.8
CID-2102207

MLS-0107074		0.3/0.3/0.998/91/75*99.9/99.199.7/99.382.4/77.517.0/1.8

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

PropertyValue
Molecular Weight397.44756 [g/mol]
Molecular FormulaC20H19N3O4S
XLogP3-AA3.3
H-Bond Donor2
H-Bond Acceptor6
Rotatable Bond Count8
Tautomer Count22
Exact Mass397.109627
MonoIsotopic Mass397.109627
Topological Polar Surface Area123
Heavy Atom Count28
Formal Charge0
Complexity538
Isotope Atom Count0
Defined Atom StereoCenter Count0
Undefined Atom StereoCenter Count0
Defined Bond StereoCenter Count0
Undefined Bond StereoCenter Count0
Covalently-Bonded Unit Count1

4. Appendices

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

Table 5All Purchased Analogs

StructureCmpd IDIC50(µM)SIDCIDVendorVendorID
PLAPTNAP
Image ml085fu29.jpg
MLS-00140972.6>10056373725665093InterBio-ScreeningSTOCK4S-90308
Image ml085fu30.jpg
MLS-039085013.6>100572875961400698InterBio-ScreeningSTOCK4S-91062
Image ml085fu31.jpg
MLS-03908433.97>100572875891409759InterBio-ScreeningSTOCK4S-87186
Image ml085fu32.jpg
MLS-03908516.02>100572875971400246InterBio-ScreeningSTOCK4S-92693
Image ml085fu33.jpg
MLS-03908589.4>100572876051394536InterBio-ScreeningSTOCK5S-03688
Image ml085fu34.jpg
MLS-0390855>100>100572876011409691InterBio-ScreeningSTOCK4S-97188
Image ml085fu35.jpg
MLS-03908577.45>100572876041398345InterBio-ScreeningSTOCK4S-99075
Image ml085fu36.jpg
MLS-039084911.8>100572875951400269InterBio-ScreeningSTOCK4S-90841
Image ml085fu37.jpg
MLS-03908412.712.1572875871416608InterBio-ScreeningSTOCK4S-84785
Image ml085fu38.jpg
MLS-02978022.92>100572875841400947InterBio-ScreeningSTOCK4S-15744
Image ml085fu39.jpg
MLS-01088352.53/40.3563737241399636InterBio-ScreeningSTOCK4S-90782
Image ml085fu40.jpg
MLS-010135111.8>100572876023878415InterBio-ScreeningSTOCK4S-97273
Image ml085fu41.jpg
MLS-03908448.76>100572875901398070InterBio-ScreeningSTOCK4S-88147
Image ml085fu42.jpg
MLS-03908428.31>100572875881402438InterBio-ScreeningSTOCK4S-86311
Image ml085fu43.jpg
MLS-01070743.87>100573091712102207BCCGPMK-14-59
Image ml085fu44.jpg
MLS-03156812.7993.456373578838842Chem-Bridge5672011
Image ml085fu45.jpg
MLS-039085611.7>100572876031400150InterBio-ScreeningSTOCK4S-97686
Image ml085fu46.jpg
MLS-02978274.9>100572875833735808InterBio-ScreeningSTOCK3S-98364
Image ml085fu47.jpg
MLS-03156887.292.78563736391314069Chem-Bridge7587885
Image ml085fu48.jpg
MLS-03156846.956.5356373634715398Chem-Bridge6593203
Image ml085fu49.jpg
MLS-031568612.221.756373636715363Chem-Bridge6617586
Image ml085fu50.jpg
MLS-03156851.7810.956373635715330Chem-Bridge6607811
Image ml085fu51.jpg
MLS-0390847>100>100572875931400320InterBio-ScreeningSTOCK4S-90030
Image ml085fu52.jpg
MLS-03908537.22>100572875991400627InterBio-ScreeningSTOCK4S-94912
Image ml085fu53.jpg
MLS-03908528.73>100572875981399934InterBio-ScreeningSTOCK4S-94867
Image ml085fu54.jpg
MLS-03908545.59>100572876001401217InterBio-ScreeningSTOCK4S-95328
Image ml085fu55.jpg
MLS-039084613>100572875921383304InterBio-ScreeningSTOCK4S-89768
Image ml085fu56.jpg
MLS-039083925.0>100572875851973575InterBio-ScreeningSTOCK4S-16633
Image ml085fu57.jpg
MLS-03908406.6340.7572875861984977InterBio-ScreeningSTOCK4S-20993
Image ml085fu58.jpg
MLS-03908484.9419.2572875944283152InterBio-ScreeningSTOCK4S-90562
Image ml085fu59.jpg
MLS-03156901.588.5856373641946537Chem-Bridge7746693
Image ml085fu60.jpg
MLS-02380241.895.9856373642713665Chem-Bridge7764114
Image ml085fu61.jpg
MLS-00081121.425.93563735721515366Chem-Bridge7764254
Image ml085fu62.jpg
MLS-02552121.763.48563736432010274Chem-Bridge7775407
Image ml085fu63.jpg
MLS-0315689>10078.5563736401305489Chem-Bridge7642884
Image ml085fu64.jpg
MLS-03156871.857.1956373637715454Chem-Bridge6622859
Image ml085fu65.jpg
MLS-03156801.566.7156373577720372Chem-Bridge5484218
Image ml085fu66.jpg
MLS-00519312.164.8256373536586278ChemDiv3294-0014
Image ml085fu67.jpg
MLS-03156838.77>100563736335350662Chem-Bridge6588132
Image ml085fu68.jpg
MLS-03158874.795.44563737034206177SpecsAI-204/31718048
Image ml085fu69.jpg
MLS-03158863.064.15563737024450383SpecsAI-204/31685046
Image ml085fu70.jpg
MLS-03158853.423.03563737014066568SpecsAI-204/31680049
Image ml085fu71.jpg
MLS-0315884>100>10056373700996625SpecsAG-690/40754100
Image ml085fu72.jpg
MLS-0315888>100>10056373704698547SpecsAM-807/12427007
Image ml085fu73.jpg
MLS-0315883>100>100563736961223257SpecsAE-848/36959517
Image ml085fu74.jpg
MLS-0315682>1003.71563735792877377Chem-Bridge5933089
Image ml085fu75.jpg
MLS-0315889>100>10056373705715329SpecsAP-048/15613011
Image ml085fu76.jpg
MLS-0080017>100>5056373623881297Chem-Bridge6385545
Image ml085fu77.jpg
MLS-031567839.3>100563739222830496Chem-Bridge5147768
Image ml085fu78.jpg
MLS-031567918.8>100563739232841226Chem-Bridge5317936
Image ml085fu79.jpg
MLS-0390862>100>1005728760916415295InterBio-ScreeningSTOCK6S-42849
Image ml085fu80.jpg
MLS-03908590.3743.14572876065922288InterBio-ScreeningSTOCK1S-39868
Image ml085fu81.jpg
MLS-03908600.930.2557287607932151InterBio-ScreeningSTOCK2S-62329
Image ml085fu82.jpg
MLS-0390861157.385728760816000021InterBio-ScreeningSTOCK4S-15750
Image ml085fu83.jpg
MLS-00775342.9529.2563735742940433Chem-Bridge7471251
Image ml085fu84.jpg
MLS-0068603>100>10056373638805519Chem-Bridge6680720
Image ml085fu85.jpg
MLS-00737298.43>100563739139551841InterBio-ScreeningSTOCK2S-13283

b. Comparative data showing probe specificity for target

Table 4SAR and Selectivity

Compound IDAssay FormatAssay TargetNo of RunsIC50 Value (μM)
MLS-0014097LuminescentTNAP4>100
MLS-0014097LuminescentPLAP~ 62.34
MLS-0014097LuminescentIAP120.7
MLS-0014097ColorimetricGAPDH2>100
MLS-0014097FPBCL-b/TR31>100
MLS-0014097FPBfl-11>100

5. Bibliography

1.
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Wennberg C, Kozlenkov A, Di Mauro S, Fröhlander N, Beckman L, Hoylaerts MF, Millán JL. Structure, genomic DNA typing, and kinetic characterization of the D allozyme of placental alkaline phosphatase (PLAP/ALPP). Human Mutation. 2002;19(3):258–267. [PubMed: 11857742]
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Wahren B 1, Hinkula J, Stigbrand T, Jeppsson A, Andersson L, Esposti PL, Edsmyr F, Millán JL. Phenotypes of placental-type alkaline phosphatase in seminoma sera. Int J of Cancer. 2006;37(4):595–600. [PubMed: 3957465]
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Fishman WH. Clinical and biological significance of an isozyme tumor marker—PLAP. Clin Biochem. 1987;20:387–92. [PubMed: 3325192]

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