NI-57

Protein target name:

BRPF1, BRD1, BRPF3

Mechanism of action:

Antagonist

Recommended Concentration for use in cells:

Up to 500 nM

Probe Information

Target and Probe Information

Target Details

Target class:

Epigenetics

Subclass:

Bromodomain

HUGO ID:

HGNC:14255 HGNC:1102 HGNC:14256

Entrez gene ID:

7862 23774 27154

UNIPROT ID:

P55201 O95696 Q9ULD4

Target alias:

BR140, IDDDFP, BRL, BRPF1, BRPF2, BRD1, BRPF3

Probe Details

Chemical Probes Portal ID:

CPP68

Probe alias:

GTPL8573

PubChem CID:

91827373

ChEMBL ID:

CHEMBL3752151

SMILES string:

CC1=CC2=C(C=CC(=C2)NS(=O)(=O)C3=C(C=C(C=C3)C#N)OC)N(C1=O)C

InChi Key:

UEMQPCYDWCSVCU-UHFFFAOYSA-N

Control compound:

NI-198

Orthogonal probe:

OF-1GSK6853

Validation in Cells and Model Organisms

In vitro validation

On target activity

Potency:

BRPF1: 31 nM, BRD1: 110 nM, BRPF3: 410 nM

Potency assay:

ITC

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

5MYG

Structure-activity relationship:

Yes, see J. Med Chem paper

Off target activity

Potency (off target):

Off target protein and potency:

BRD9: 1,000 nM

Potency assay (off target):

NI-57 was screened for binding by DSF against a panel of 47 Bromodomains, and it showed excellent selectivity. All activity was confined to the class IV family of BRDs with ∆Tm < 1 oC stabilization for all non-class IV BRDs except TRIM24 (2.0), CREBBP (1.1) and EP300 (1.1). NI-57 was also tested for Bromodomain selectivity in the BROMOscan panel of 46 binding assays, which showed all potent activity was confined to the class IV family. Once again, TRIM24 was identified as the non-class IV BRD with most affinity (IC50=1,600 nM), but with a 500-fold window of selectivity.

Probe Selectivity in Vitro:

NI-57 was screened for off-target pharmacology against a panel of 55 representative GPCRs, ion channels and enzymes (Eurofins, ExpressProfile) and was found to have minimal inhibition (<40 % at 10 uM).

In cell validation

On target activity in cells

Potency assay (cells):

Cellular ‘on-target’ activity for NI-57 was assessed in a BRPF1B-Histone3.3 nanoBRET assay which showed dose-dependent displacement of BRPF1B (but not BRPF1A) from Histone H3.3 with an estimated IC50 of 0.07 ± 0.0034 uM. These results were verified in a FRAP assay where NI-57 displaced a triple BRD-BRPF1B-GFP fusion construct, as well as full length BRD1-GFP, which was demonstrated by significant reduction in recovery times in the presence of the inhibitor. Hence, the nanoBRET and FRAP experiments confirm NI-57 inhibit BRPF BRDs in the nucleus.

Target engagement assay (cells):

Yes, nanoBRET and FRAP assays

Toxicity

Cytoxicity assay:

Yes

Notes on cytotoxicity:

No general cyctoxicity was observed in cellular models of cancer (up to 30 uM as top dose) and inflammation (up to 10 uM as top dose).

In vivo validation

Organism:

Female CD-1 mouse

Dose:

3 mg/kg (oral), 1 mg/kg (IV)

Route of delivery:

OralIntravenous

Plasma half life:

1.6 h

Systemic clearance:

21 mL/min/kg (IV)

Cmax:

0.63 uM (oral)2.3 uM (IV)

Tmax:

2.0 h (oral)

Organ of Interest

Organ (O):

Blood

Primary Reference

Reference (doi):

10.1021/acs.jmedchem.7b00611 10.1021/acschembio.7b00481

Reference (PMID):

28714688 28849908

Endorsed probe

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

SAB Rating

Rating for use in Cells

3 review(s)

Rating for use in Model Organisms

3 review(s)

SAB Members

SAB Comments

NI-57 is a selective agent to probe BRPF1, BRD1 and BRPF3. The recommended concentration of 500 nM in cells appears appropriate, but the caveat is that the bromodomain selectivity in-cell through target engagement studies such as those used for BRPF1B is unknown. The probe is suitable for oral in vivo use at the dose stated, but investigators should appreciate that the free drug levels may be significantly lower than those required (depending on the specific question being asked). Although aqueous solubility is quoted as being reasonable, the investigator should appreciate that thermodynamic solubility may differ considerably.

May 16 2020 - 8:26am

NI-57 is reported as a pan BRFP inhibitor.

At the recommended cellular compound concentration (up to 500nM), the cellular effect observed will be mainly due to BRPF1 inhibition and maybe to some weaker extent to BRD1 (BRPF2) inhibition.
Of note the compound shows similar activity on BRPF3 and BRD7 (IC50=140nM, DiscoveRx bromoScan panel).
In vivo, NI-57 is a low clearance compound with a moderate volume of distribution resulting in a moderate half-life of 1.2 hour in the female mouse following a 1 mg/kg solution IV dose. Oral bioavailability following a 3 mg/kg oral dose of NI-57 was 29% with a Cmax of 0.63 µM and an AUC0-t of 651 ng.h/mL. The oral half-life was 1.6 h. No in vivo tolerability data is available.

May 16 2020 - 8:27am

Context and in vitro properties:
The chemical probe NI-57 is a pan-BRPF chemical probe of the bromodomain of BRPF1, BRPF2 (BRD1), and BRPF3 (Igoe et al., J. Med Chem, 2017). It is closely related to a previously reported NI-42, but should be considered an optimized and preferred compound due ot slightly increased potency and cellular activity. NI-57 binding affinity (Kd) measured by ITC for BRPF bromodomains is 30-400 nM. Its most potent off-targets determined in the Igoe et al. study are BRD9 and TRIM24, which are inhibited w/ ~100-500-fold less potency depending on BRPF member, although this exact selectivity appears to be assay format dependent.

Off target profiling:
The authors perform additional pharmacological profiling to establish the broader selectivity and biopharmaceutical properties, assaying for off-target effects against a panel of 55 representative GPCRs, ion channels, and enzymes (Eurofins, ExpresSProfile) as well as 8 cardiac ion channels. In these assays, NI-57 displays <40% inhibition against of any of these members at 10 and 40 uM, respectively.

Cellular activity and evidence of target engagement:
Cellular target engagement was verified using a BRPF1B-Histone3.3 nanoBRET assay, where NI-57 demonstrated an IC50 of ~70 nM. The authors indicate this was verified in FRAP studies using a triple BRD-BRPF1B-GFP fusion construct, although this finding cites unpublished results. NI-57 affects cytokine production in alveolar macrophages. Of note, this activity is observed at concentrations considerably higher than required for pan-BRPF target engagement via nano-BRET, and the authors are careful to cite this as evidence of activity, not selectivity.

Pharmacokinetics:
Clearlance half-life injection following a single IP injection was ~1.2 hours. Mouse pharmacokinetic data indicates favorable availability upon oral bioadministration (oral bioavailability 29%). Fraction absorbed was calculated to be 35%.

Overall, this appears to be a useful pan-BRPF inhibitor for in vitro and in vivo studies. Further studies of target engagement will be required to understand whether the phenotypic effects of the compound (i.e. effects on macrophages, unpublished results showing effects on osteoclast differentiation) are due to inhibition of BRPF or (potentially) partial engagement of off-targets. According to authors additional characterization data is forthcoming, and should be added to this record (8/25/17).

Jun 2 2020 - 1:52pm

NI-57

Probe Information

Target Details

BR140, IDDDFP, BRL, BRPF1, BRPF2, BRD1, BRPF3

Probe Details

In vitro validation

In cell validation

Toxicity

In vivo validation

Primary Reference

Supporting Organizations

Interested in supporting the portal?

Search form

NI-57

Probe Information

Target Details

BR140, IDDDFP, BRL, BRPF1, BRPF2, BRD1, BRPF3

Probe Details

In vitro validation

In cell validation

Toxicity

In vivo validation

Primary Reference

Supporting Organizations

Interested in supporting the portal?