GSK-3 inhibitor

Target protein: 
GSK-3
Probe Name: 
BI-5521
MOLECULAR WEIGHT [DA]: 
419.5
In stock: 
351

Chemical structure

Figure 1: 2-D structure of BI-5521, a potent and selective GSK-3 inhibitor

Highlights

Our non-covalent ATP-competitive inhibitor of glycogen synthase kinase (GSK-3) BI-5521(1) is active on both isoforms GSK-3α and GSK-3β with single digit nM potency. We could demonstrate glucose lowering efficacy of orally administered BI-5521 in both acute and subchronic settings in rodents. High selectivity and decent PK properties make BI-5521 a suitable tool for in vivo validation of GSK-3 as a therapeutic target. We also offer the N-alkylated analog BI-4481 as negative control.

Figure 2: BI-5521, 3D conformation

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Target information

Glycogen synthase kinase (GSK-3) is a constitutively active serine/threonine kinase that phosphorylates a large number of proteins in a variety of different pathways. In mammalian tissues, GSK-3 exists as two isoforms (GSK-3α and GSK-3β) that share 98% homology of their kinase domains. GSK-3β has been implicated in various diseases such as diabetes, inflammation, cancer, amyotrophic lateral sclerosis (ALS), Alzheimer’s and Parkinson’s diseases, and bipolar disorders.(2-5) Moreover, GSK-3 inhibitors serve as tools for regenerative medicine through increasing self-renewal and/or differentiation of stem cells.(5)

Figure 3: Crystal structure of GSK-3 complexed with BI-91BS, a close analog of BI-5521 (PDB code 6GJO).

Figure 3: Crystal structure of GSK-3 complexed with BI-91BS, a close analog of BI-5521 (PDB code 6GJO).

In vitro Activity

BI-5521 inhibits GSK-3βa with an IC50 of 1.1 nM. The N-ethylated analog BI-4481, which can be used as a negative control, shows an activity of >10,000 nM in this assay.

PROBE NAME / NEGATIVE CONTROL

BI-5521

BI-4481

MW [Da]

419

448

Inhibiton of GSK-3β (IC50) [nM]a

1.1

>10,000

Inhibiton of GSK-3α (IC50) [nM]b

2

n.d.

Inhibiton of GSK-3β (DC50) [nM]b

5

n.d.

Stimulation of Glocogen Synthesis rate in C3A cells (EC50) [nM]

3

n.d.

Cytotoxicity (IC50) [nM]c

390

>50,000

a Invitrogen data, Invitrogen panel 2017

b Inhouse assay: recombinant human GSK-3 was incubated with 33P-ATP and a 21 AA-peptide from Glycogen Synthase bearing the phosphorylation sites for GSK-3. The radiolabelled P-GS peptide was absorbed using a filter and was quantified in a counter

cIn vitro cytotoxicity assay using the U937 cell line and the colorimetric EZ4U assay

In vitro DMPK and CMC parameters

PROBE NAME / NEGATIVE CONTROL

BI-5521

BI-4481

Solubility @ pH 6.8 [µg/ml]

76

90

CACO permeability @pH7.4 [*10-6 cm/s]

10

30

CACO efflux ratio

4.6

0.7

Microsomal stability (human/mouse/rat) [% QH]

26

48

34

52

>88

83

CYP 3A4 (IC50) [µM]

>50

n.a.

CYP 1A2 (IC50) [µM]

>50

n.a.

CYP 2C9 (IC50) [µM]

>50

n.a.

CYP 2C19 (IC50) [µM]

12.9

n.a.

CYP 2D6 (IC50) [µM]

>50

n.a.

In vivo DMPK parameters

Pharmacokinetic parameters of BI-5521 in rats

BI-5521

Rat

Clearance [mL/min/kg]a

32

Mean residence time after iv dose [h]a

2.90

tmax [h]b

4

Cmax [nM]b

15.5

Vss [l/kg]a

5.6

AUC0-inf [nMh]a

519

a IV application of 4.20 mg/kg

b Oral application of 0.42 mg/kg

In vivo pharmacology

BI-5521 showed acute efficacy in ZDF rats

Fig. 4: Decreased plasma glucose levels during an oral glucose tolerance test in ZDF rats.

Fig. 4: Decreased plasma glucose levels during an oral glucose tolerance test in ZDF rats.

BI-5521 showed subchronic efficacy in db/db mice

Fig. 5: Decreased plasma glucose levels over 4 weeks in db/db mice.

Fig. 5: Decreased plasma glucose levels over 4 weeks in db/db mice.

Negative control

MOLECULAR WEIGHT OF NEGATIVE CONTROL [DA]: 
447.5

The N-ethylated analog BI-4481 can be used as negative control (IC50 GSK-3β >10,000 nM).

Figure 4: BI-4481 which serves as a negative control.

Figure 4: BI-4481 which serves as a negative control.

Selectivity

BI-5521 is similarly active on both isoforms GSK-3α and GSK-3β.

 

Selectivity against other protein kinases:

- No selectivity against DYRK1A (99% inhibition @500 nM)

- ≥100fold selectivity against CDK2/CyclinA, MAP3K7_K7IP1, MAPKAPK1A, ROCK1

- >1000fold selectivity against all other targets tested

 

Selectivity against non-kinase targets:

- >500 fold selectivity against all targets of a 62 target panel (please see Suppplementary Data for detailed information)

BI-5521

SELECTIVITY DATA AVAILABLE

Cerep®

no

Panlabs®

yes

Invitrogen®

yes

DiscoverX®

no

Dundee

yes

Download selectivity data: 

Co-crystal structure of the BI probe compound and the target protein

A crystal structure of GSK-3 complexed with BI-91BS a close analog of BI-5521 is available (PDB code 6GJO).

reference molecules

Pharmacological inhibitors of GSK-3 described in the literature can be classified as either ATP competitive, non-ATP-competitive (allosteric) or substrate competitive inhibitors. Among these, three molecules so far reached clinical trials: the ATP competitive inhibitors AZD1080 and LY2090314 and Tideglusib with a non-ATP-competitive binding mode.(2,4)

Summary

Our GSK-3 inhibitor BI-5521 with shown in vivo efficacy is characterized by high potency and good selecticvity.

Supplementary data

References

  1. US Patent 2005/0203104

    1. US Patent 2005/0203104, A. Heckel, G. J. Roth, J. Kley, S. Hoerer, I. Uphues. (BI-5521 corresponds to example 1)

  2. Glycogen synthase kinase 3 (GSK-3) inhibitors: a patent update (2014-2015)

    2. V. Palomo, A. Martinez, Glycogen synthase kinase 3 (GSK-3) inhibitors: a patent update (2014-2015), Expert Opinion on Therapeutic Patents, 2017, 27(6), 657-666.

  3. Glycogen Synthase Kinase 3: A Kinase for All Pathways?

    3. P. Patel, J. R. Woodgett, Glycogen Synthase Kinase 3: A Kinase for All Pathways?, Current Topics in Developmental Biology 2017, 123, 277-302.

  4. Natural and synthetic bioactive inhibitors of glycogen synthase kinase

    4. I. Khan, M. A. Tantray, M. S. Alam, H. Hamid, Natural and synthetic bioactive inhibitors of glycogen synthase kinase, European Journal of Medicinal Chemistry 2017, 125, 464-477.

  5. Subtly Modulating Glycogen Synthase Kinase 3 β: Allosteric Inhibitor Development and Their Potential for the Treatment of Chronic Diseases

    5. V. Palomo, D. I. Perez, C. Roca, C. Anderson, N. Rodríguez-Muela, C. Perez, J. A. Morales-Garcia, J. A. Reyes, N. E. Campillo, A. M. Perez-Castillo, L. L. Rubin, L. Timchenko, C. Gil, A. Martinez, Subtly Modulating Glycogen Synthase Kinase 3 β: Allosteric Inhibitor Development and Their Potential for the Treatment of Chronic Diseases, J. Med. Chem. 2017, 60, 4983−5001.