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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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Probe Reports from the NIH Molecular Libraries Program [Internet].

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Placental Alkaline Phosphatase (PLAP) Luminescent HTS assay - Probe 2

, , , , , , , , , , , , 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 ML095 (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 (This series of compounds were developed with our collaborators at the Human Bimolecular Research Institute (HBRI) – Dr. Marion Lanier and Dr. John Cashman)

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 ml095fu1
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-25067483

ML095
PLAP4,240 nM IC50
SID-56405584
AID-1512
TNAP>100 µM IC50
SID-56405584
AID-1056
> 27
IAP>100 µM IC50
SID-56405584
AID-1017
> 27

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) but PLAP-like enzymes are detected in serum of patients with primary testicular tumors, in particular seminoma (3) and other cancers (4). PLAP-specific inhibitors with selectivity over other tissue non-specific (5) alkaline phosphatase (TNAP) and intestinal alkaline phosphatase (IAP) can be used as tools to characterize the biological role of PLAP. The small molecule probe CID-25067483 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) catalyze the hydrolysis of phosphomonoesters, releasing phosphate and alcohol. Alkaline phosphatases are dimeric enzymes found in most organisms. In human, four isozymes of alkaline phosphatases 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. Currently, inhibitors of PLAP lack either potency or selectivity. Several amino acids (L-tryptophan, L-phenylalanine and L-leucine) and some other compounds (such as levamisole) inhibit PLAP and other APs with mM IC50 values. L-phenylalanylglycylglycine, the state of the art inhibitor of PLAP, is the only compound that demonstrates >10-fold selectivity to PLAP vs IAP or TNAP, yet its IC50 values in PLAP assays are 100–300 µM (6).

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 based on work with tissue non-specific alkaline phosphatase inhibitors and later activator work, that suggested that PLAP specific inhibitors could also be 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)
In Vitro TNAP Dose Response
Luminescent Assay for SAR Study
1056InhibitorSecondary Assay for specificityBiochemicalLuminescence (384)
Luminescent assay for identification of inhibitors of human intestinal alkaline phosphatase [Confirmatory]1017InhibitorCounterscreen Assay for specificityBiochemicalLuminescence (1536)

Primary assay details are described below.

PLAP screening was developed and done 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-1056). X01MH077602-01, 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 freshly prepared 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 μL 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 μL of the following solutions were added:
    1. TCEP working solution - column 1 (positive control).
    2. 10% DMSO - column 2 (negative control).
  3. 8 μL of PLAP working solution was added to the whole plate using a WellMate bulk dispenser.
  4. 8 μL of CDP-star working solution was added to the whole plate using WellMate bulk reagent dispenser.
  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 done 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 dilutions. Compounds were serially diluted in 100% DMSO, and diluted with water to 10% final DMSO concentration. 4 μL 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 μL of TCEP working solution and 10% DMSO, respectively.
  2. 8 μL of PLAP working solution was added to the whole plate using a WellMate bulk reagent dispenser (Matrix).
  3. 8 μL of CDP-star working solution was added to the whole plate using a WellMate reagent bulk dispenser (Matrix).
  4. Plates were incubated for 30 min 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, reagents and protocols, see section 2a above.

For this screen 95,857 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 dose-response curves.

iii. Summary of Results

Primary hits from the PLAP HTS assay were confirmed and tested in parallel against TNAP for selectivity. A few hits were identified. There are many commercial analogues available and so Analogue-By-Catalogue (ABC) was performed in an iterative fashion. These were described in the initial probe report for PLAP focused on CID-665093. From the purchased compounds we were able to conclude that the dihydroxyl group of the catechol moiety were essential for inhibition of alkaline phosphatase activity. One of the series of molecules identified was compound 1, CID-715454 (see Figure 1) This compound inhibited PLAP and TNAP with similar potency (1.9 and 7.2 µM, respectively) yet possessed significantly lower potency vs. IAP (50 µM). Interestingly, IAP and PLAP have much higher sequence similarity between themselves than with TNAP. Thus the above result suggested that additional compounds that are selective to PLAP are achievable.

Figure 1. Structure of the lead catechol MLS-0315687 (CID-715454) compound 1.

Figure 1

Structure of the lead catechol MLS-0315687 (CID-715454) compound 1.

Our collaborative partners at the Human BioMolecular Research Institute (www.hbri.org) concentrated on developing the SAR of compound 1 (CID-715454) by synthesis of a series of compounds based on this scaffold.

c. Probe Optimization

i. SAR & chemistry strategy that led to the probe

The SAR was developed primarily by the targeted synthesis to improve selectivity against TNAP and IAP while maintaining the potency against PLAP. The objective of the hit-to-lead refinement chemistry was to develop a tractable route for the synthesis of probe candidates, prepare authentic samples, confirm structure and purity, and provide enough material for submission to the MLSMR. Figure 2 below summarizes the points of variation of the series. All three regions of the lead were explored: left hand side (LHS), Linker and right hand side (RHS).

Figure 2. Probe analogs variations: 3 regions were explored by chemical synthesis: the left hand side (LHS), linker and right hand side (RHS).

Figure 2

Probe analogs variations: 3 regions were explored by chemical synthesis: the left hand side (LHS), linker and right hand side (RHS).

Establishing an SAR

A library of 34 compounds was synthesized according to the procedures illustrated in Scheme 2, below. Compounds were compared to CID-715454 (MLS-0315687) in the different bioassays (PLAP, TNAP and IAP in some cases). Three main areas of the lead structure were examined for the SAR (Figure 2).

Scheme 2. Synthesis of analogs of lead structure CID-715454 (MLS-0315687).

Scheme 2

Synthesis of analogs of lead structure CID-715454 (MLS-0315687).

Chemical re-synthesis of CID-715454 (MLS-0315687) was done to compare an authentic sample with the commercially purchased compound in the bioassays used. Exploration of the left hand side of the molecule (Table 5) showed that the 3,4-dihydroxy phenyl was crucial for functional inhibitory activity. Removing one the hydroxyl groups, CID-25067456 (MLS-0315814) and CID-25199542 (MLS-0390951), or capping one or both hydroxyl groups, CID-25067475, CID-25067473 and CID-694596 (MLS-0315844, MLS-0315843, MLS-0315815, respectively) led to inactive compounds. Replacing the 3,4-dihydroxyphenyl by a 2,4-dichloro-phenyl substitution, CID-974963 (MLS-0390949) that is a favored feature for TNAP inhibitory activity in other series 5 afforded an inactive compound.

Table 5. Exploration of the left hand side of the lead structure.

Table 5

Exploration of the left hand side of the lead structure.

A small survey of the linker region was done and we observed that any substitution tried led to significant deterioration of inhibitory activity (Table 6).

Table 6. Exploration of the linker.

Table 6

Exploration of the linker.

Exploration of the RHS of the molecule was done using preliminary data generated from the PLAP HTS assay as well as newly synthesized compounds. Compound CID-715454 (MLS-0315678) and other alkyl-substituted analogs (CID-25067503 (MLS-0315858), not shown) suggested that an aromatic ring was necessary for activity. HTS assay results showed also, that all the catechol compounds active carried various heteroaromatic rings, mostly linked through a sulfur atom and were weakly or non selective for TNAP [Table 7, compounds CID-586278, -1515366, and -2940433 (MLS-0051931, 0008112, 0077534, respectively)]. Exploring the RHS region with heterocycles such as imidazoles, triazoles and pyrazoles directly attached to the core led to compounds active vs. PLAP and selective over TNAP, CID-25067472, -25067487, -747227 (MLS-0315813, 0315850, 0390948, respectively). A more thorough exploration followed using the imidazole ring.

Table 7. Exploration of the right hand side of the lead structure.

Table 7

Exploration of the right hand side of the lead structure.

A small library of substituents to explore the inhibitory potency around the imidazole moiety was prepared and tested against PLAP, TNAP and IAP. The results are summarized in Table 8. Significantly, utilization of imidazole core provided improved selectivity vs. TNAP yet decreased selectivity vs. IAP. The presence of a hydrogen or small alkyl (methyl, ethyl) at the 2-position (R7) had little influence on TNAP inhibitory activity. With the introduction of a bulkier group at the 2-position such as a phenyl, CID-25067481 (MLS-0315847), inhibition activity decreased (IC50=17.2 µM). At the 4 and 5 positions (R8) of the imidazole group, a small group (methyl), CID-25199538 (MLS-0390945), maintained activity compared to the unsubstantiated imidazole. A larger group such as bromine, CID-2506747 (MLS-0315846), led to a decrease in activity by about 7-fold. Adding a 2-methyl substituent to the broom compound had an additive effect and the activity of CID-25067477 (MLS-0315845) decreased to 4.96 µM with concomitant loss of selectivity vs. TNAP. Interestingly, an unsubstituted benzimidazole CID-25067493 (MLS-0315853) was tolerated and more potent than the unsubstituted imidazole (IC50 = 1.53 µM and 3.2 µM respectively) but was also less selective vs. TNAP.

Table 8. Exploration around MLS-0615813.

Table 8

Exploration around MLS-0615813.

Selectivity over TNAP

The most interesting results for this series of imidazoles were the selectivity inhibition of PLAP compared with TNAP and IAP. Results are summarized in Table 8. While substitution at the 2 position of the imidazole (R7) allowed to maintain the selectivity over TNAP, CID-25067472 (MLS-0315813) IC50>100 µM, CID-25067491 (MLS-0315852) IC50>100 µM, CID-25067483 (MLS-0315848) IC50=59.6 µM, CID-25067481 (MLS-0315847) IC50>100 µM, substitution at the 4 position (R8) resulted in significant loss of selectivity over TNAP. The presence of a benzimidazole instead of an imidazole led to a compound with an IC50 below 15 µM for TNAP. In summary, varying the R8 GROUP can control selectivity over TNAP. When R8 is hydrogen, the compound showed no activity for TNAP CID-25067493 (MLS-0315813). R7 had a weak influence on TNAP selectivity.

Selectivity over IAP

The 7 best compounds in the PLAP assay were also tested in the IAP assay. Results are summarized in Table 8. An early trend appears to show that the R7 position was critical for IAP selectivity. When the R7 substituent volume increased from hydrogen to methyl to ethyl, the IC50 in the IAP assay increased and led to inactive compounds (IC50 = 22.4 µM, 64.6 µM, >100 µM respectively). Similarly, in the two benzimidazoles, CID-25067493 (MLS-0315853) and CID-25067495 (MLS-0315854) methyl group at R7 position led to >7-fold increase in selectivity vs. IAP.

In summary, we have been able to develop a series of imidazole and pyrazolo catechols that maintained inhibitory activity of the original screening hit CID-715454 (MLS-0315687) vs. PLAP while generating selectivity over the 2 isozymes, TNAP and IAP, and further differential selectivity between these two latter isozymes.

Image ml095fu35

3. Probe

a. Chemical name

1-(3,4-dihydroxyphenyl)-2-(2-ethylimidazol-1-yl)ethanone hydrochloride [ML095]

b. Probe chemical structure

Figure 3. Structure of probe compound MLS-0315848 (CID-25067483).

Figure 3Structure of probe compound MLS-0315848 (CID-25067483)

c. Structural Verification Information of probe SID

SID-56405584. Spectral data supporting proposed structure:

1H NMR (300 MHz, CD3OD) δ: 1.29 (t, J = 7.7MHz, 3H), 2.76 (q, J = 7.7MHz, 2H), 4.8 (s, 2H), 6.83 (d, J = 8.4MHz, 1H), 6.99 (s, 2H), 7.42 (s, 1H), 7.43 (d, J = 6.6MHz 1H).

Image ml095fu36
Image ml095fu37
Image ml095fu38

d. PubChem CID (corresponding to the SID)

CID-25067483

e. Availability from a vendor

Not a commercial source

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

Table 4Submission information on Probe and analogs

Probe /AnalogMLS-# (BCCG#)CIDSIDSource (vendor or BCCG syn)Amt (mg)Source Lot
Probe03158482506748356405584HBRI syn15HBRI-MLG-I-126D
Analog 1031568771545456405531HBRI syn15HBRI-100206
Analog 203909452519953857309182HBRI syn15HBRI-MLG-I-182A
Analog 303158542506749556405590HBRI syn15HBRI-MLG-I-128B
Analog 403158522506749156405588HBRI syn15HBRI-MLG-I-126
Analog 503158132506747256405578HBRI syn15HBRI-100205

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

Scheme 1. Synthesis of MLS-0315848 (CID-25067483).

Scheme 1Synthesis of MLS-0315848 (CID-25067483)

To 50 mg (0.27 mmol, Aldrich) of 2-chloro-3’,4’-dihydroxyacetophenone in 1 mL dioxane was added 26 mg (0.27 mmol, TCI) of 2-ethylimidazole. The solution was stirred overnight in a sealed vial at room temperature. The reaction was checked by TLC for completion (dichloromethane/methanol, 90:10, v/v). The reaction product precipitated out of solution. The precipitate was filtered, washed with a minimum amount of dioxane then dried under high vacuum to afford 35 mg of the probe as a beige solid.

1H NMR (300 MHz, CD3OD) δ: 1.29 (t, J = 7.7MHz, 3H), 2.76 (q, J = 7.7MHz, 2H), 4.8 (s, 2H), 6.83 (d, J = 8.4MHz, 1H), 6.99 (s, 2H), 7.42 (s, 1H), 7.43 (d, J = 6.6MHz 1H). MS (ESI) C13H14N2O3, m/z = 246.1, found m/z = 247.27 [M+H].

See spectra above in Sec. 3.c. “Structural Verification Information of Probe SID.

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

Probe compounds is soluble in dimethylsulfoxide at 20 mg/ml.

j. Properties Computed from Structure

Molecular Weight282.7228 [g/mol]
Molecular FormulaC13H15ClN2O3
H-Bond Donor3
H-Bond Acceptor4
Rotatable Bond Count4
Tautomer Count44
Exact Mass282.07712
MonoIsotopic Mass282.07712
Topological Polar Surface Area75.4
Heavy Atom Count19
Formal Charge0
Complexity298
Isotope Atom Count0
Defined Atom StereoCenter Count0
Undefined Atom StereoCenter Count0
Defined Bond StereoCenter Count0
Undefined Bond StereoCenter Count0
Covalently-Bonded Unit Count2

4. Bibliography

1.
Hoylaerts MF, Manes T, Millán JL. Molecular mechanism of uncompetitive inhibition of human placental and germ-cell alkaline phosphatase. Biochem J. 1992;286(Pt 1):23–30. [PMC free article: PMC1133013] [PubMed: 1520273]
2.
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]
3.
Wahren B, 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]
4.
Fishman WH. Clinical and biological significance of an isozyme tumor marker—PLAP. Clin Biochem . 1987;20:387–92. [PubMed: 3325192]
5.
Sidique S, Ardecky R, Su Y, Narisawa S, Brown B, Millán JL, Sergienko E, Cosford NDP. Design and synthesis of pyrazole derivatives as potent and selective inhibitors of tissue-nonspecific alkaline phosphatase (TNAP). Bioorg Med Chem Lett. 2009;19:222–225. [PMC free article: PMC2752324] [PubMed: 19038545]
6.
Goldstein DJ, Rogers C, Harris H. Evolution of alkaline phosphatases in primates. Proc Natl Acad Sci USA. 1982;19:879–883. [PMC free article: PMC345856] [PubMed: 6950431]
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