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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 Report for NPY-Y2 Receptor Antagonists

, , , , , , , , and .

Received: ; Last Update: August 6, 2010.

Due to its expression profile and biological action, neuropeptide Y (NPY)-Y2 receptor (Y2R) is an attractive G protein-coupled receptor (GPCR) target for anxiolytic research. Additionally, NPY-Y2R is predicted to be a therapeutic target in alcoholism. Four different compounds, ML075 (CID-2936384), ML074 (CID-2228302), ML073 (CID-3236979) and ML072 (CID-4460128) are claimed as novel antagonist probes to the NPY-Y2R, producing increased cAMP (3',5'-cyclic-AMP phosphodiesterase) levels. These probes demonstrate submicromolar affinity to the Y2 receptor, do not antagonize NPY-Y1R, do not present significant cytotoxicity, and are blood-brain barrier penetrant. Hence, these probes represent an improvement over previously described NPY-Y2R antagonists and offer greater promise to serve as valuable in vivo pharmacological probes for elucidating the Y2R signaling pathway.

Assigned Assay Grant #: 1 R21 NS056950-01

Screening Center Name & PI: Scripps Research Institute Molecular Screening Center (SRIMSC); Hugh Rosen

Chemistry Center Name & PI: SRIMSC; Hugh Rosen

Assay Submitter & Institution: Claes Wahlestedt, The Scripps Research Institute (TSRI)

PubChem Summary Bioassay Identifier (AID): AID-1791

Probe Structure & Characteristics

Four different compounds are claimed as novel antagonist probes to the NPY-Y2 receptor. These four probes demonstrate sub-micromolar affinity to the Y2 receptor, do not antagonize the Y1R receptor, and do not present significant cytotoxicity. Additionally, they are all blood-brain barrier penetrant. In this regard they all represent an improvement over the current NPY Y2 antagonist probe, BIIE 0246. The four probes are illustrated below:

Scaffold 1 (Piperidine carbothioamide) [ML075].

Scaffold 1 (Piperidine carbothioamide) [ML075]

Scaffold 2 (Arysulfamoyl benzamide) [ML074].

Scaffold 2 (Arysulfamoyl benzamide) [ML074]

Scaffold 3 (Aryl-1,2,4-oxadiazole) [ML073].

Scaffold 3 (Aryl-1,2,4-oxadiazole) [ML073]

Scaffold 4 (Dimethylisoxazole) [ML072].

Scaffold 4 (Dimethylisoxazole) [ML072]

Scaffold IDScaffold NameCID/MLSIDTarget (NPY Y2) IC50 (μM)Anti-Target (NPY Y1) IC50 (μM)Secondary Assay CC50 (μM)Brain Penetrance (ng/mL)
1Piperidine carbothioamide2936384/ML075175073050.220 [AID-1272]>35 [AID-1279]741656 ± 749
2Arysulfamoylbenzamide2228302/ML074174133920.428 [AID-1272]>35.4 [AID-1279]20.6395 ± 16
3Aryl-1,2,4-oxadiazole3236979/ML07342420790.733 [AID-1272]>35 [AID-1279]>991860 ± 135
4Dimethylisoxazole4460128/ML072224132493.917 [AID-1272]>35.4 [AID-1279]>99513 ± 57

Recommendations for the scientific use of this probe

These probes are useful for assays aiming to block cellular NPY Y2 receptor (Y2R) signaling, leading to increased cAMP levels, without inhibiting NPY Y1 receptor (Y1R) activity. The representative (“best-in-class”) compounds from scaffold 1 (SID-17507305), scaffold 2 (SID-17413392), scaffold 3 (SID-4242079), and scaffold 4 (SID-22413249) displace agonist from the Y2R with high affinity. These probes bind to fewer off-target receptors than BIIE 0246, currently the most widely used Y2R antagonist. Most importantly, and in contrast to BIIE 0246, these best-in-class probes are brain penetrant and thus able to reach Y2R binding sites in both the periphery and brain. Additional information regarding the compounds and assays employed in this probe development campaign can be found in (1).

1. Scientific Rationale for Project

Due to its expression profile and biological action, NPY Y2 is an attractive GPCR target for anxiolytic research. Additionally, Y2 is predicted to be a therapeutic target in alcoholism. It has been reported, however, that the complex structure and high molecular weight of BIIE 0246 (the current NPY Y2 antagonist) limit its usefulness as an in vivo pharmacological tool (2). It is therefore necessary to produce brain penetrant, high affinity selective ligands for the Y2 receptor.

2. Project Description

a. Information for each Assay Implemented and Screening Run

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

Table 2PubChem BioAssay

AIDAssay NameAssay TypeTargetPowder SampleCompound Concentration
793Primary cell based high-throughput screening assay for antagonists of neuropeptide Y receptor Y2.Primary Assay (1X %INH)NPY-Y2No2.7μM
1256Counterscreen assay for antagonists of neuropeptide Y receptor Y2 (NPY-Y2): Cell-based high throughput assay to measure NPY-Y1 antagonism.Counterscreen Assay (3X %INH)NPY-Y1No3.6 μM
1257Cell-based high throughput confirmation assay for antagonists of neuropeptide Y receptor.Confirmation Assay (3X %INH)NPY-Y2No2.7 μM
1272Dose response cell-based screening assay for antagonists of neuropeptide Y receptor Y2.Dose Response (3X IC50)NPY-Y2No10-point, 1:3 dilution starting at 35 μM
1040Primary cell-based high-throughput screening assay for antagonists of NPY-Y1Primary Assay (1X %INH)NPY-Y1No3.6 μM
1279Dose response counterscreen for neuropeptide Y receptor Y2 (NPY-Y2): Cell-based high throughput assay to measure NPY-Y1 antagonism.Dose Response Counterscreen (3X IC50)NPY-Y1No10-point, 1:3 dilution starting at 35 μM
2142Late stage results from the probe development effort to identify antagonists of neuropeptide Y receptor Y2 (NPY-Y2)Various (Cytotoxicity and Brain Penetrance assays)NPY-Y2YesVarious

ii. Assay Rationale & Description

Table 3Assay Rationale and Description

AIDAssay RationaleAssay DescriptionZ′S:B
793The purpose of this assay is to determine the ability of test compounds to inhibit NPY Y2 activity.In this assay test compounds are screened for their ability to increase agonist-mediated cAMP levels in a cell line transfected with the NPY-Y2 receptor and a cyclic-nucleotide gated channel (CNG). The cells are treated with isoproterenol to activate adenylate cyclase and open the CNG channel, leading to a change in membrane potential that is measured using a fluorescent probe. The addition of the agonist NPY peptide counteracts cAMP accumulation induced by isoproterenol. As designed, test compounds that act as NPY-Y2 receptor antagonists will reverse the reduction in well fluorescence.0.78+/− 0.045.09 +/− 0.33
1256The purpose of this assay is to determine the ability of test compounds to inhibit NPY Y1 activity.Same as AID-793, except that test compounds are tested in triplicate and are incubated with a cell line that is transfected with the NPY-Y1 receptor.0.79 +/− 0.014.28 +/− 0.04
1257The purpose of this assay is to confirm activity of compounds previously identified as active in a previous set of experiments (PubChem AID 793).Same as AID-793, except that compounds are tested in triplicate..0.81+/− 0.023.99 +/− 0.13
1272The purpose of this assay is to determine dose response curves for compounds identified as active in a previous set of experiments entitled, "Primary cell-based high-throughput screening assay for antagonists of neuropeptide Y receptor Y2 (NPY-Y2)," (PubChem AID 793).Same as AID-793, except that test compounds were assayed in triplicate in a 10-point, 1:3 dilution series starting at a nominal test concentration of 35 μM.0.84 +/− 0.024.04 +/− 0.19
1040The purpose of this assay is to determine the ability of test compounds to inhibit NPY Y1 activity.Same as AID-1256, except that test compounds are incubated with a cell line that is transfected with the NPY-Y1 receptor.0.65+/− 0.063.35 +/− 0.28
1279The purpose of this assay is to determine whether compounds identified as active in a previous set of experiments (PubChem AID-793) were nonselective due to inhibition of the NPY Y1 receptor.Same as AID-1272, except that test compounds are incubated with a cell line that is transfected with the NPY-Y1 receptor.0.70+/− 0.103.56+/−0.09
2142The purpose of this assay is to determine whether probe candidates were cytotoxic and brain penetrant.The purpose of this assay is to determine whether compounds of interest reduce cell viability. Y2R HEK293-CNG cells were seeded at 500 cells per well in 1536-well plates in 5 uL growth media. Compounds (in DMSO) were prepared as 10-point, 1:3 serial dilutions starting at 11 µM final concentration were added to cells. Plates were incubated for 24, 48 or 72 h at 37 C. After incubation, 5 uL of CellTiter-Glo (Promega, Madison, WI) were added to each well and the plates were allowed to incubate for 15 min at room temperature. Luminescence was then measured (ViewLux plate reader, PerkinElmer, Turku, Finland). Viability was measured as a percentage relative to control cells treated with DMSO alone (0% cytotoxicity) and cells treated with 100 µM Doxorubicin (100% cytotoxicity).

Plasma and brain levels of the compounds were assessed in C57Bl6 mice 30 minutes after dosing 10 mg/kg intraperitoneally. Samples were formulated at 2 mg/mL in 10/10/80 DMSO/tween/water. Blood was collected into EDTA containing tubes at 30 min and plasma was generated using standard centrifugation techniques. Brain samples were frozen upon collection and all samples were stored at −80 C until analyzed. Brain tissue was not perfused prior to freezing to prevent diffusion of the compound out of the tissue during the process. Plasma samples were analyzed by treating 25 μL of plasma with 125 μL of acetonitrile containing an internal standard (propanolol) and filtering through a Millipore Multiscreen Solvinter 0.45 μm low binding PTFE hydrophilic filter. The filtrate was analyzed by LC- MS/MS using an API Sciex 4000. MRM methods were developed in positive ion mode and concentrations were determined using a standard curve between 2 to 2000 ng/mL. Samples with concentrations outside of the curve were diluted with blank plasma and reanalyzed. Similar conditions were used to determine brain levels except the samples were weighed and acetonitrile was added (10x, weight by volume). The samples were sonicated to extract the compound from the brain matrix and then filtered as described above. A density of 1 g/mL was used to convert compound per mg tissue into molar equivalents.

Table 4Reagents and Source

AIDReagent (Source)
793, 1256, 1257, and 1272 Neuropeptide Y receptor Y2 HEK293-CNG cells (BD Biosciences, part 344870)
10x ACTOne Membrane Potential Assay Kit (BD Biosciences, part BD354663)
Phosphate Buffered Saline (Invitrogen, part 10010-023)
DMEM (Invitrogen, part 11965-092)
Fetal Bovine Serum (Invitrogen, part 16140-071)
Trypsin-EDTA solution (Invitrogen, part 25200-056)
Geneticin (Invitrogen, part 10131-027)
Puromycin (Sigma, part P9620)
Isoproterenol (Sigma, part I6504)
Ro 20-1724 (Sigma, part B8279)
Neuropeptide Y (American Peptide, part 60-1-11B)
BIIE 0246 (Tocris, part 1700)
1536-well plates (Greiner, part 789072)
T-175 tissue culture flasks (Corning, part 431)
1040 and 1279 As above, with the following changes: Neuropeptide Y receptor Y1 HEK293-CNG cells (BD Biosciences, part 344869) (replaces Y2 cells)
2142Y2R HEK293-CNG cells ; CellTiter-Glo (Promega, Madison, WI); DMSO; Doxorubicin DMSO/tween/water; EDTA; acetonitrile containing an internal standard (propanolol)

iii. Summary of Results

Compared to previously described Y2R antagonists, the compounds described herein offer greater promise for elucidating the Y2R signaling pathway.

b. Probe Optimization

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

Since receptor subtype selective antagonists were desired, compounds worth follow-up from the Primary, Confirmation and Counterscreening were required to exhibit an IC50 value of less than 10 μM at Y2R and greater than 35 μM at Y1 in dose response assays. Structures and data for each probe can be found in the SAR tables. The chemistry strategy that lead to each scaffold is described below:

Scaffold 1: Based on the above criteria, one compound belonging to the piperidine-carbothioamide scaffold was identified: SID-17507305. This compound does not share structural similarities with the known NPY-Y2 antagonist BIIE 0246 or JNJ-5207787. Analogs were purchased in powder form or reordered from the MLSMR in liquid form and tested in dose response assays against both receptors. Results for these analogs are summarized in the SAR table below.

Scaffold 2: Based on the above criteria, a compound belonging to the arysulfamoyl benzamide scaffold was identified: SID-17413392. This compound does not share structural similarities with the known NPY-Y2 antagonist BIIE0246 or JNJ-5207787. Analogs were purchased in powder form or re-ordered from the MLSMR in liquid form and tested in dose response assays against both receptors. Results for these analogs are summarized in the SAR table below.

Scaffold 3: Based on the above criteria, a compound belonging to the aryl-1,2,4-oxadiazole scaffold was identified: SID-4242079. This compound does not share structural similarities with the known NPY-Y2 antagonist BIIE0246 or JNJ-5207787. Analogs were purchased in powder form or re-ordered from the MLSMR in liquid form and tested in dose response assays against both receptors. Results for these analogs are summarized in the SAR table below.

Scaffold 4: Based on the above criteria, a compound belonging to the dimethylisoxazole scaffold was identified: SID-22413249. This compound does not share structural similarities with the known NPY-Y2 antagonist BIIE0246 or JNJ-5207787. Analogs were purchased in powder form or re-ordered from the MLSMR in liquid form and tested in dose response assays against both receptors. Results for these analogs are summarized in the SAR table below.

3. Probe

a. Chemical name

Scaffold 1: N-(4-ethoxyphenyl)-4-[hydroxy(diphenyl)methyl]piperidine-1- carbothioamide [ML075]

Scaffold 2: 4-chloro-3-[(2,5-dimethylphenyl)sulfamoyl]-N-(2-phenylphenyl)benzamide [ML074]

Scaffold 3: 2-(2-methoxyphenyl)-N-[4-[5-(3-methoxyphenyl)-1,2, 4-oxadiazol-3-yl]phenyl]acetamide [ML073]

Scaffold 4: 3,5-dimethyl-4-[(4-methylphenyl)sulfonyl-phenylmethyl]-1,2-oxazole [ML072]

b. Probe chemical structure

Scaffold 1 (Piperidine carbothioamide) [ML075].

Scaffold 1 (Piperidine carbothioamide) [ML075]

Scaffold 2 (Arysulfamoyl benzamide) [ML074].

Scaffold 2 (Arysulfamoyl benzamide) [ML074]

Scaffold 3 (Aryl-1,2,4-oxadiazole) [ML073].

Scaffold 3 (Aryl-1,2,4-oxadiazole) [ML073]

Scaffold 4 (Dimethylisoxazole) [ML072].

Scaffold 4 (Dimethylisoxazole) [ML072]

c. Structural Verification Information of probe SID

LCMS

d. PubChem CID (corresponding to the SID)

Scaffold 1: CID-2936384

Scaffold 2: CID-2228302

Scaffold 3: CID-3236979

Scaffold 4: CID-4460128

e. Availabilty from vendor

Scaffold 1: Asinex, catalog number BAS 01915526

Scaffold 2: ChemBridge, catalog number 7955494

Scaffold 3: ChemDiv, catalog number K907-0250

Scaffold 4: Specs, catalog number AM970/41069539

f. Mode of action for biological activity of probe

Scaffold 1: Using cell-based 125I-PYY binding assays, the Assay Provider has determined that SID-17507305 competes with agonist for binding to the NPY Y2 receptor. In cells expressing NPY Y2, this compound exhibited a Ki value of 1.55 nM (1). No significant blockade of 125I-PYY binding in cells expressing NPY Y1 was detected, even at concentrations up to 50 μM. These results suggest that this probe binds to the NPY Y2 receptor and does not bind to the Y1 receptor subtype.

Scaffold 2: Using cell-based 125I-PYY binding assays, the Assay Provider has determined that SID-17413392 competes with agonist for binding to the NPY Y2 receptor. In cells expressing NPY Y2, this compound exhibited a Ki value of 1.93 nM (1). No significant blockade of 125I-PYY binding in cells expressing NPY Y1 was detected, even at concentrations up to 50 μM. These results suggest that this probe binds to the NPY Y2 receptor and does not bind to the Y1 receptor subtype.

Scaffold 3: Using cell-based 125I-PYY binding assays, the Assay Provider has determined that SID-4242079 competes with agonist for binding to the NPY Y2 receptor. In cells expressing NPY Y2, this compound exhibited a Ki value of 6.0 nM (1). No significant blockade of 125I-PYY binding in cells expressing NPY Y1 was detected, even at concentrations up to 50 μM. These results suggest that this probe binds to the NPY Y2 receptor and does not bind to the Y1 receptor subtype.

Scaffold 4: Using cell-based 125I-PYY binding assays, the Assay Provider has determined that SID-22413249 competes with agonist for binding to the NPY Y2 receptor. In cells expressing NPY Y2, this compound exhibited a Ki value of 60.3 nM (1). No significant blockade of 125I-PYY binding in cells expressing NPY Y1 was detected, even at concentrations up to 50 μM. These results suggest that this probe binds to the NPY Y2 receptor and does not bind to the Y1 receptor subtype.

g. Detailed synthetic pathway for making probe

Not applicable

h. Probe properties

Scaffold 1: Aqueous solubility, −8.47; ADMET BBB, 0.803; ADMET BBB level, 0; ADMET absorption level, 0; ADMET solubility, −5.463; ADMET solubility level, 2

Scaffold 2: Aqueous solubility, −9.18; ADMET BBB, ND; ADMET BBB level, 4; ADMET absorption level, 2; ADMET solubility, −7.195; ADMET solubility level, 1

Scaffold 3: Aqueous solubility, −6.86; ADMET BBB, −.253; ADMET BBB level, 2; ADMET absorption level, 0; ADMET solubility, −4.849; ADMET solubility level, 2

Scaffold 4: Aqueous solubility, −6.11; ADMET BBB, .133; ADMET BBB level, 1; ADMET absorption level, 0; ADMET solubility, −5.237; ADMET solubility level, 2

i. A tabular presentation summarizing known probe properties

Please see Tables of Probe Properties and Probe Names.

Table 5. Probe Properties.

Table 5

Probe Properties.

Table 6. Probe Names.

Table 6

Probe Names.

4. Appendices

  1. Comparative data on (1) probes, (2) similar compound structures (establishing SAR) and (3) prior probes
  2. Comparative data showing probe specificity for target

See Tables 7a-d

Table 7(a). SAR Scaffold 1: Piperidine-carbothioamide.

Table 7(a)

SAR Scaffold 1: Piperidine-carbothioamide.

Table 7(b). SAR Scaffold 2: Arylsulfamoylbenzamide.

Table 7(b)

SAR Scaffold 2: Arylsulfamoylbenzamide.

Table 7(c). SAR Scaffold 3: Aryl-1,2,4-oxadiazole.

Table 7(c)

SAR Scaffold 3: Aryl-1,2,4-oxadiazole.

Table 7(d). SAR Scaffold 4: Dimethylisoxazole.

Table 7(d)

SAR Scaffold 4: Dimethylisoxazole.

5. Bibliography

1.
Saldanha SA, Brothers SP, Spicer T, Cameron M, Mercer BA, Chase P, McDonald P, Wahlestedt C, Hodder PS. Selective and Brain Penetrant Neuropeptide Y Y2 Receptor Antagonists Discovered through Whole-Cell High Throughput Screening. Submitted. [PMC free article: PMC2802430] [PubMed: 19837904]
2.
Bonaventure P, Nepomuceno D, Mazur C, Lord B, Rudolph DA, Jablonowski JA, Carruthers NI, Lovenberg TW. Characterization of N-(1-Acetyl-2,3-dihydro-1H-indol-6-yl)-3-(3-cyanophenyl)-N-[1-(2-cyclopentyl-ethyl)-piperidin-4yl]acrylamide (JNJ-5207787), a small molecule antagonist of the neuropeptide Y Y 2 receptor. J Pharmacol Exp Ther. 2004 Mar;308(3:):1130–7. [PubMed: 14617685]
3.
Tetko IV, Tanchuk VY, Kasheva TN, Villa AE. Estimation of aqueous solubility of chemical compounds using E-state indices. J Chem Inf Comput Sci. 2001;41:1488–1493. [PubMed: 11749573]
4.
Egan WJ, Merz KM Jr, Baldwin JJ. Prediction of drug absorption using multivariate statistics. J Med Chem. 2000;43:3867–3877. [PubMed: 11052792]
5.
Egan WJ, Lauri G. Prediction of intestinal permeability. Adv Drug Deliv Rev. 2002;54:273–289. [PubMed: 11922948]
6.
Cheng A, Merz KM Jr. Prediction of aqueous solubility of a diverse set of compounds using quantitative structure-property relationships. J Med Chem. 2003;46:3572–3580. [PubMed: 12904062]

Brain Penetrance Data

In order to determine whether the identified Y2R antagonists were brain-penetrant, selected compounds were injected intraperitoneally into adult mice at 10 mg/kg and levels in the brain tissue and plasma were measured after thirty minutes. BIIE 0246, the current NPY Y2 antagonist probe, exhibited poor penetration (only 2% of plasma levels). All probes demonstrated a higher brain penetrance than BIIE 0246.

IDBrain Penetration Studies
Plasma Concentration (μM)Brain Concentration (μM)Ratio [B]/[P] (%)
SID-17507305 8.5 ± 3.33.7 ± 1.744
SID-17413392 1.1 ± 0.10.4 ± 0.0336
SID-17413034 1.6 ± 0.61.7 ± 0.6106
SID-4242079 9.0 ± 0.74.5 ± 0.350
SID-22413249 1.3 ± 0.31.5 ± 0.2115
BIIE-0246 12 ± 20.2 ± 0.12

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