MBM-55

Protein target name:

NEK2

Mechanism of action:

Inhibitor

Recommended Concentration for use in cells:

~~up to 1 uM~~

Reviewer suggested Concentration for use in cells:

up to 200 nM

Probe Availability

MedKoo Bioscienes, Inc

Probe Information

Target and Probe Information

Target Details

Target class:

Protein kinase

Subclass:

Ser/Thr Protein Kinase

HUGO ID:

HGNC:7745

Entrez gene ID:

4751

UNIPROT ID:

P51955

Target alias:

HsPK21, NEK2A, NLK1, PPP1R111, RP67

Probe Details

Chemical Probes Portal ID:

CPP2057

Probe alias:

cpd 42g

PubChem CID:

137636872

ChEMBL ID:

CHEMBL4062453

SMILES string:

NC(C(C=C1)=C(OCC2=CC=CC(F)=C2)C=C1C3=CN=C4N3C=CC(C5=CN(CCN(C)C)N=C5)=C4)=O

InChi Key:

CGECJCJUHCZZGO-UHFFFAOYSA-N

Control compound:

cpd 28a

PAINs evaluation:

PAINs Free

Physico-chemical properties

NMR:

1H NMR (400 MHz, CDCl3 ) δ 8.39 (d, J = 8.1 Hz, 1H), 8.16 (d, J = 7.1 Hz, 1H), 7.86 (s, 2H), 7.76 – 7.69 (m, 2H), 7.66 (s, 1H), 7.48 – 7.39 (m, 1H), 7.34 (dd, J = 8.1, 1.3 Hz, 1H), 7.25 (s, 1H),7.21 – 7.08 (m, 3H), 6.96 (dd, J = 7.2, 1.6 Hz, 1H), 5.79 (s, 1H), 5.29 (s, 2H), 4.33 (t, J = 6.4 Hz, 2H), 2.88 (t, J = 6.4 Hz, 2H), 2.34 (s, 6H).

13C NMR (100 MHz, DMSO-d6 ) δ 166.07, 162.24 (d, JC-F = 243.9 Hz), 156.18, 146.41, 139.52 (d, JC-F = 7.6 Hz), 137.66, 133.57, 132.30, 131.58, 130.70 (d, JC-F = 8.2 Hz), 129.41, 128.98, 124.67, 124.23, 123.59 (d, JC-F = 2.4 Hz), 122.68, 120.33, 119.27, 114.89 (d, JC-F = 20.9 Hz), 114.33 (d, JC-F = 22.0 Hz), 111.74, 111.66, 110.86, 69.13, 55.77, 46.35, 42.87.

Validation in Cells and Model Organisms

In vitro validation

On target activity

Potency:

IC50

1 nM

Potency assay:

HTRF® KinEASE™-STK (Cisbio Assays)

Structure-activity relationship:

yes (modelling)

Off target activity

Potency (off target):

IC50

Off target protein and potency:

RSK1 5.4 nM, DYRK1 6.5 nM

Potency assay (off target):

HTRF® KinEASE™-STK (Cisbio Assays)

Probe Selectivity in Vitro:

Selectivity against a panel of 11 kinases (IC50 nM): AurA 5300, AKT1 608, ABL 20, RSK1 5.4, DYRK1a 6.5, CHK1 57 MAPKAPK2 >10000, GSK-3β 91, PI3Ka 6226, P38a >10000, CDK2 370, CDK4 441

In cell validation

On target activity in cells

Potency in cells:

IC50

530 nM (MGC-803)840 nM (HCT-116)7130 nM (Bel-7402)

Potency assay (cells):

cell cycle analysis by flow cytometry

In vivo validation

Organism:

Male Sprague Dawley (SD) rats

Dose:

1 mg/Kg

Route of delivery:

Intravenous

Plasma half life:

1.72 h

Systemic clearance:

33.3 mL/min/Kg

Primary Reference

Reference (doi):

10.1016/j.ejmech.2016.12.026

Reference (PMID):

28039836

Under Review

This probe is in the process of SAB review. Please continue to check back for new reviews and commentary.

SAB Rating

Rating for use in Cells

2 review(s)

Rating for use in Model Organisms

2 review(s)

SAB Members

SAB Comments

Additional validation is required to determine whether MBM-55 engages NEK2 in cells. The cell-based data is simply antiproliferative data and the compound does not elicit significant potency in these assays. As for animal-based studies, only limited pharmacokinetic profiling was done in rats. MBM-55 demonstrated a short half-life and was efficiently cleared. Additional optimization is required to furnish a NEK2 inhibitor with more optimal metabolic stability.

Dec 8 2020 - 11:35am

Missing connecting PK data weakens the strength of recommendation. The PK was done i.v in rat at 1mg/kg, but the model in mouse i.p. at 20mg/kg so mouse exposure is not clear. However, the biggest issue is that the in vitro Nek2 IC50 does not seem to correlate with cell IC50. For example, the Nek2 IC50 of 42g is 1nM and of 42e is 386 nM (so 386-fold difference), yet cell-based MGC-803 IC50 values are 0.53uM and 1.46 uM respectively (3-fold difference), but both are physicochemically very similar. So 42e is a much better negative control than 28a and suggests cell-based activity might be driven by something else. Noting that 42g, against an 11-kinase panel, was active at under 10 nM in more than 20% (RSK1 IC50= 5.4nM and DRK1a IC50= 6.5 nM, with IC50 values of 20nM, 57nM, 91nM against ABL, CHK1, GSK3beta, evidence is insufficient that observed cell-based activity of 42g is driven by Nek2 inhibition. This does not mean that 42g (=MBM-55) is unsuitable for use as a cell-based probe, but that perhaps the most appropriate cell-based readout has yet to be discovered. One might hope for a Nek2-based cell-based IC50 value to be closer to the region of 100nM, and where 42e should be >100x less active. In any such assay, RSK1 and DRK1a-driven activity will need to be ruled out. Furthermore, given these data and the fact that 42g is a kinase inhibitor, a full kinase panel should be undertaken to find other potent off-targets that could drive cell-based activity.

Apr 10 2021 - 1:19pm

MBM-55

Probe Availability

Probe Information

Target Details

HsPK21, NEK2A, NLK1, PPP1R111, RP67

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

In vivo validation

Primary Reference

Supporting Organizations

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MBM-55

Probe Availability

Probe Information

Target Details

HsPK21, NEK2A, NLK1, PPP1R111, RP67

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

In vivo validation

Primary Reference

Supporting Organizations

Interested in supporting the portal?