NI-57

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.

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.

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).