CCT251236

Protein target name:

PIR

Mechanism of action:

Unknown

Recommended Concentration for use in cells:

10-100 nM

Probe Availability

MedChemExpress

Probe Information

Target and Probe Information

Target Details

Target class:

Other

Define other target class:

Transcription factor regulator

Subclass:

Cupin

HUGO ID:

HGNC:30048

Entrez gene ID:

8544

UNIPROT ID:

O00625

Target alias:

Pirin, iron-binding nuclear protein,

Probe Details

Chemical Probes Portal ID:

CPP955

PubChem CID:

117996749

SMILES string:

CC1=CC=C(NC(C2=CC(OCCO3)=C3C=C2)=O)C=C1NC(C4=CC(C=CC(OCCN5CCCC5)=N6)=C6C=C4)=O

InChi Key:

KLHOCHQJHXNKAS-UHFFFAOYSA-N

Control compound:

N-(5-(2,3-dihydrobenzo[b][1,4]dioxine-5-carboxamido)-2-methylphenyl)-2-(2-(pyrrolidin-1-yl)ethoxy)quinoline-6-carboxamide

Physico-chemical properties

Solubility - preferred solvent:

DMSO

Solubility - concentration:

10 mM

Potential reactivities:

Oxidation of the solubilizing group

NMR:

H NMR (500 MHz, DMSO-d6) δ 10.07 (s, 2H), 8.57 (d, J = 2.0 Hz, 1H), 8.38 (d, J = 8.8 Hz, 1H), 8.22 (dd, J = 8.7, 2.0 Hz, 1H), 7.88− 7.84 (m, 2H), 7.58 (dd, J = 8.3, 2.2 Hz, 1H), 7.54 (d, J = 2.1 Hz, 1H), 7.51 (dd, J = 8.5, 2.2 Hz, 1H), 7.24 (d, J = 8.5 Hz, 1H), 7.12 (d, J = 8.8 Hz, 1H), 6.98 (d, J = 8.4 Hz, 1H), 4.57 (t, J = 5.8 Hz, 2H), 4.34−4.27 (m, 4H), 2.89 (br s, 2H), 2.58 (br s, 4H), 2.23 (s, 3H), 1.70 (br s, 4H). 13C NMR (126 MHz, DMSO-d6) δ 165.39, 164.81, 163.15, 148.00, 146.80, 143.39, 140.72, 137.76, 136.79, 130.57, 130.52, 129.23, 128.88, 128.58, 128.15, 127.21, 124.49, 121.65, 119.08, 118.58, 117.30, 117.12, 114.48, 65.03, 64.86, 64.48, 54.58, 54.42, 40.32, 40.15, 23.61, 17.95

Storage Recommendations

Storage (dry powder):

4o C under an inert atmosphere for >6 months. Stable on the bench <6 months.

Storage (solution):

Stable in DMSO for <3 months

Validation in Cells and Model Organisms

In vitro validation

On target activity

Potency:

44 nM

Potency assay:

Surface Plasmon Resonance

PDB ID for probe-target interaction (3D structure):

5JCT

Structure-activity relationship:

SAR for 8 analogues are available as affinities measured by SPR. The SAR is consistent with the binding mode described in by the crystal structure.

Off target activity

Potency assay (off target):

There are no known proteins that are family members with pirin. Pirin is a member of the cupin superfamily of proteins which all possess a similar structural feature, the core beta-barrel but retain no sequence or binding site homology and no common endogenous ligand.

Probe Selectivity in Vitro:

Kinomescan assay of 442 kinases for the chemical probes parent compound yielded 4 kinases that were inhibited >90% at 1 uM (KIT, PDGFRA, PFGFRB, BRAF). IC50s for KIT, PDGFRA and PDGFRB were >10,000 nM. IC50 for BRAF=420 nM; however, assessment of chemically unrelated BRAF inhibitors yielded no activity in the phenotypic screen, indicating that CCT251236 activity in the screen was not attributable to BRAF.
Cerep Diversity Screen of 98 molecular targets gave 4 hits which displayed >80% inhibition at 10 uM, adenosine A2A and A3 receptors, histamine H2 and H3 receptors, muscarinic receptor and acetylcholine esterase.

In cell validation

On target activity in cells

Potency in cells:

IC50

19 nM

Potency assay (cells):

In SK-OV-3 cells, CCT251236 inhibited 17-AAG-induced HSP72 expression (assayed by ELISA). This assay was designed as an HSF1 phenotypic screen.

Target engagement assay (cells):

Using a compound adapted for SILAC mass-spec chemical proteomics, we identified pirin (Ki=28 nM) as the only high affinity protein target from SK-OV-3 cell lysate.

Off target activity in cells

Potency assay, off target (cells):

GSK3beta and PDE6D were also identified in the proteomic assays but ruled out as targets in follow-up experiments.

Probe Selectivity in Cell:

Not available

Toxicity

Cytoxicity assay:

Yes

Notes on cytotoxicity:

The parent compound of the chemical probe demonstrates potent (<100 nM in 435 cell lines) in 435 and wide-ranging antiproliferative activity in a panel of 635 genetically diverse cancer cells (Genomics of Drug Sensitivity in Cancer).

In vivo validation

Organism:

NCr-Fox1nu

Dose:

20 mg/kg

Route of delivery:

Oral

Plasma half life:

5.4 h

Ratio of probe in organ to plasma (O:P):

Not available

Fraction of probe bound to plasma protein:

97.5

Cmax:

480 nM

Tmax:

1 hour

Organ of Interest

Organ (O):

SK-OV-3 xenograft

Target engagement assay:

Not available

Primary Reference

Reference (doi):

10.1021/acs.jmedchem.6b01055

Reference (PMID):

28004573

Endorsed probe

The Chemical Probes Portal endorses this compound as chemical probe for use in cells and in model organisms.

SAB Rating

Rating for use in Cells

3 review(s)

Rating for use in Model Organisms

3 review(s)

SAB Members

SAB Comments

This is a useful probe for Pirin - useful to study Pirin function both in cells and in vivo. There is a correlation of molecule binding to Pirin and cellular activity. Active and inactive versions of the molecule have been reported, and the inactive version should be used as a control.

Mar 27 2017 - 3:21pm

CCT251236 has been thoroughly investigated and characterized as a cellular probe for Pirin (target) and HSF (pathway) and is supported by a robust and transparent set of data. My only concern is for possible in vivo use where there is an apparent non-linear pharcacokinetic response in mouse (20 mg/kg, oral dose gave a 3-fold lower AUC than a 5 mg/kg dose, albeit in a different mouse strain). I suggest users run a pilot PK study in their strain of interest to ensure the required exposures can be achieved with their dose.

Jul 24 2017 - 11:44am

While CCT251236 clearly shows potent binding (SPR) to human pirin, displays potent inhibition of Hsp72 induction in a cell-based assay (ELISA), and shows activity in an SK-OV-3 mouse xenograft study, this chemical probe may well be active against other targets, since the anti-proliferative activity against a panel of cancer cell lines is often more potent than the potency measured by the assays outlined above. The enduser should thus use caution when interpreting cell-based or in vivo results obtained using CCT251236.

Mar 12 2021 - 11:15am

CCT251236

Probe Availability

Probe Information

Target Details

Pirin, iron-binding nuclear protein,

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

Toxicity

In vivo validation

Primary Reference

Supporting Organizations

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CCT251236

Probe Availability

Probe Information

Target Details

Pirin, iron-binding nuclear protein,

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

Toxicity

In vivo validation

Primary Reference

Supporting Organizations

Interested in supporting the portal?