PTK2 PROTAC | BI-3663

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Target protein: 
PTK2
Probe Name: 
BI-3663
MOLECULAR WEIGHT [DA]: 
917.8
In stock: 
100

Chemical structure

Highlights

Inducing macromolecular interactions with small molecules to activate cellular signalling is a challenging goal. PROTACs (proteolysis-targeting chimeras) are a new exciting therapeutic modality that allow probing the scaffold function of a given protein by degrading it beyond classical functional interference by recruiting the target protein in proximity to an E3 ubiquitin ligase to trigger protein degradation. BI-36631 is a first in class low molecular weight degrader that tethers a highly selective PTK2 Kinase inhibitor to a CRL4CRBN E3 ubiquitin ligase ligand, aimed at triggering the intracellular destruction of the PTK2 protein. BI-3663 potently and rapidly induces reversible, long-lasting, and preferential removal of PTK2 in cells.

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Target information

Focal adhesion tyrosine kinase (PTK2) is a cytoplasmic protein tyrosine kinase that is overexpressed and activated in many types of advanced-stage solid cancers. PTK2 has been shown to play an important role in adhesion, spreading, motility, invasion, metastasis, survival, angiogenesis, epithelial to mesenchymal transition (EMT), cancer stem cells and the tumor microenvironment2, 3. Overexpression and activation of PTK2 is associated with several human malignant diseases, and is correlated with poor overall patient survival4-6. The focal adhesion tyrosine kinase (PTK2) is often over-expressed in human hepatocellular carcinoma (HCC) and several reports have linked PTK2 depletion and/or pharmacological inhibition to reduced tumorigenicity7, 8. However, the clinical relevance of targeting PTK2 remains to be proven. Traditionally small molecules have been used to inhibit the action of a target protein by occupying and blocking a functional region of the protein. However, the disconnect between modulation of intracellular PTK2 autophosphorylation and growth inhibition as well as the often suboptimal selectivity profile of the inhibitors used makes it difficult to link the reported blockade of HCC tumour initiation and maintenance to PTK2 inhibition.
An alternative innovative approach is the development of proteolysis targeting chimeras (PROTACs), i.e. hetero bifunctional compounds consisting of one moiety that binds a Cullin RING E3 ubiquitin ligase linked to another that binds a desired protein of interest (POI), bringing the ligase and the POI into close spatial proximity This hijacks the intrinsic catalytic activity of the E3 ligase and directs it toward the POI as a neo-substrate, triggering its poly-ubiquitination and subsequent proteasome-dependent degradation. As a result, a PROTAC acts as a degrader of the target as opposed to just an inhibitor, enabling the effective post-translational elimination of a target gene product in living organisms9. This approach presents many advantages compared to conventional target inhibition. One of the most attractive features of the approach is that a PROTAC molecule acts sub-stoichiometrically, i.e. it only needs to bind a molecule of target once to induce its degradation, and then is released and set free to bind another molecule of target and carry on, as in a catalytic cycle. For this reason, the concentrations required for PROTACs to be active in cells tend to be much lower compared to those needed to be reached and maintained with inhibitors, which can lead to fewer off-target effects and a more selective chemical intervention on the desired target.

Structure of BI-4464 (ligand in BI-3663) bound to PTK2 (PDB ID 6I8Z)

Structure of BI-4464 (ligand in BI-3663) bound to PTK2 (PDB ID 6I8Z). Hydrogen bonds to cysteine 502 and aspartic acid 564 are depicted in blue.

In vitro Activity

The binary affinities for BI-3663 to PTK2 and to CRBN (cereblon (CRBN) complex CRL4CRBN) are 18 and 877 nM respectively. BI-3663 degrades the PTK2 protein in A549 cells with a DC50 of 25 nM (Table 1). BI-3663 (cereblon-based) degrades PTK2 with a median DC50 of 30 nM to > 80 % across a panel of eleven HCC cell lines (Table 2).

Table 1. Binary affinities of BI-4464 (PTK2 inhibitor) and BI-3663 for PTK2 and the respective PTK2 degradation data in A549 cells.

 

BI-4463 (PKT2 inhibitor)

BI-3663 (PTK2 degrader)

E3 ligase ligand

-

POMA

PTK2 pIC50*

7.8 ± 0.1

7.7 ± 0.1

CRBN-DDB1 TR-FRET pIC50

<4

6.1

A549 cells, 18h, pDC50**

<4

7.6 ± 0.1

A549 cells, 18h, Dmax[%]**

-

95 ± 4

* Thermo Fisher selectScreen Kinase Profiling Services, Z´-Light, ATP@Km, pIC50 ± STDEV

** Degradation activity is reported as concentration needed to achieve 50 % PTK2 protein degradation (pDC50 ± STDEV) and maximal achievable protein degradation (Dmax) relative to DMSO. PTK2 levels were determined by protein capillary electrophoresis and normalized to GAPDH. (N = 3)

 

Table 2. Degradation characteristics and effect on proliferation of BI-3663 and BI-4464 in HCC lines.

Cell Line

BI-4463 (PKT2 inhibitor)

BI-3663 (PTK2 degrader)

pDC50

Dmax [%]

pIC50 (proliferation)

pIC50 (proliferation)

SNU-387

7.6

90.0

<4.6

5.2

HUH-1

6.6

50.0

4.6

5.4

Hep3B2.1-7

7.9

96.0

4.6

5.3

HepG2

7.5

89.0

<4.6

5.5

SK-Hep-1

7.5

89.0

5.8

5.2

HLF

6.4

30.0

<4.6

5.4

SNU-398

8.5

95.0

<4.6

5.2

HUCCT1

7.9

90.0

<4.6

5.2

HLE

6.8

79.0

<4.6

5.1

HuH-7

7.3

93.0

<4.6

5.4

SNU-423

7.9

93.0

4.7

5.4

 

In vitro DMPK and CMC parameters

BI-3663 is poorly soluble at physiological pH, and is a PGP substrate (high Caco2 efflux ratio). Of note, the CRBN-based PROTAC BI-3663 was considerably less stable in cell assay buffer containing 10% FCS (M+18 and +32 observed) than the VHL-based PROTAC BI-0319. BI-3663 was found to be stable as a solid and in DMSO stock solution (>3 month, data not shown). Despite this previously reported instability of CRBN based PROTACs BI-3663 showed comparable maximal degradation of PTK2 after 18 h and 72h days incubation.

Probe name / negative Control

BI-3663

BI-4206

MW [Da]

918

1061

Solubility @ pH 6.8 [µg/ml]

<1

<1

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

17

0.4

CACO efflux ratio

23

61

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

>95/>95/>95

>95/90/54

Half-life (EMEM, 10 % FCS) [h]

13

-

Plasma protein binding (human/mouse/rat/10%FCS) [%fu]

99.4/>99.5/>99.7/88.7

>99.8/>99.9/>99.8/-

 

Negative control

MOLECULAR WEIGHT OF NEGATIVE CONTROL [DA]: 
1061

cisVHL, BI-4206 is the (S) hydroxy diastereoisomer of BI-0319 and can also serve as a negative control for BI-3663. While exhibiting BI-3663 and BI-0319 comparable PTK2 binding affinity it no longer is able to bind and recruit VCB and therefore is not degrading PTK2 proteins in cells.

BI-4206 which serves as a negative control

cisVHL, BI-4206 which serves as a negative control

Selectivity

In a 397 kinase kinase panel BI-0319 beside PTK2 only inhibited LRRK2 and FES by more than 50 % at 1 µM. Interestingly, BI-0319 is more selective than the already highly selective PTK2 TKI BI-4464. We assume that the kinase selectivity panel for BI-3663 is comparable to BI-0319 since both exit vector and linker are identical for a distance of nine atoms from the piperidine moiety of the PTK2 ligand.

Multiplexed isobaric tagging mass spectrometry was employed to assess the cellular selectivity of BI-3663 for PTK2 degradation and identify potential degradation off-targets in a quantitative and unbiased manner. Amongst the 6,008 proteins quantified in this analysis in A549 cells, PTK2 showed a distinct and significant change in abundance upon treatment with BI-3663. BI-3663 did not induce any significant changes in abundance of other detectable kinases, thus confirming the high selectivity of both degraders within the kinase family. Of note, the two most prominent kinase off-targets of the inhibitor (LRRK2 and FES) were not detected in this dataset.

Total proteome analysis of A549 cells treated with BI-3663 for 18 h and compared to DMSO controls

Total proteome analysis of A549 cells treated with BI-3663 for 18 h and compared to DMSO controls. Samples were run in biological triplicates and analyzed by mass spectrometry. Volcano plot displays log2 of fold-change in abundance versus –log10 of adjusted p value (N = 3).

BI-3663

Selectivity data available

Cerep®

No

Panlabs®

No

Invitrogen®

Yes

DiscoverX®

No

Dundee

No

reference molecules

There are currently no PTK2 PROTACs with the benchmarked selectivity

Summary

BI-3663 is a first in class low molecule weight degrader that tethers a highly selective PTK2 Kinase inhibitor BI-4464 to a CRL4CRBN E3 ubiquitin ligase ligand, aimed at triggering the intracellular destruction of the PTK2 protein. BI-3663 potently and rapidly induces reversible, long-lasting, and preferential removal of PTK2 in cells. We believe that BI-3663 is a valuable tool to effectively reduce PTK2 protein levels in cell lines to reveal and differentiate between kinase- dependent and -independent functions of PTK2.

Supplementary data

References

  1. Highly Selective PTK2 Proteolysis Targeting Chimeras to Probe Focal Adhesion Kinase Scaffolding Functions

    Popow J., Arnhof H., Bader G., Berger H., Ciulli A., Covini D., Dank C., Gmaschitz T., Greb P., Karolyi-Ozguer J., Koegl M., McConnell D. B., Pearson M., Rieger M., Rinnenthal J., Roessler V., Schrenk A., Spina M., Steurer S., Trainor N., Traxler E., Wieshofer, C. Zoephel A., Ettmayer P.

    J. Med. Chem. 2019, 62, 2508-2520.

  2. Targeting FAK in human cancer: from finding to first clinical trials

    Golubovskaya V. M.

    Front Biosci (Landmark Ed) 2014, 19, 687-706.

  3. FAK in cancer: mechanistic findings and clinical applications

    Sulzmaier F. J., Jean C., Schlaepfer D. D.

    Nat Rev Cancer 2014, 14, 598-610.

  4. Overexpression of focal adhesion kinase, a protein tyrosine kinase, in ovarian carcinoma

    Judson P. L., He X., Cance W. G., Van Le L.

    Cancer 1999, 86, 1551-1556.

  5. Overexpression of focal adhesion kinase in primary colorectal carcinomas and colorectal liver metastases: immunohistochemistry and real-time PCR analyses

    Lark A. L., Livasy C. A., Calvo B., Caskey L., Moore D. T., Yang X., Cance W. G.

    Clin Cancer Res 2003, 9, 215-222.

  6. FAK overexpression is correlated with tumour invasiveness and lymph node metastasis in oesophageal squamous cell carcinoma

    Miyazaki T., Kato H., Nakajima M., Sohda M., Fukai Y., Masuda N., Manda R., Fukuchi M., Tsukada K., Kuwano H.

    Br J Cancer 2003, 89, 140-145.

  7. 3'3-Diindolylmethane inhibits migration, invasion and metastasis of hepatocellular carcinoma by suppressing FAK signaling

    Li W. X., Chen L. P., Sun M. Y., Li J. T., Liu H. Z., Zhu W.

    Oncotarget 2015, 6, 23776-23792.

  8. Focal adhesion kinase depletion reduces human hepatocellular carcinoma growth by repressing enhancer of zeste homolog 2

    Gnani D., Romito I., Artuso S., Chierici M., De Stefanis C., Panera N., Crudele A., Ceccarelli S., Carcarino E., D'Oria V., Porru M., Giorda E., Ferrari K., Miele L., Villa E., Balsano C., Pasini D., Furlanello C., Locatelli F., Nobili V., Rota R., Leonetti C., Alisi A.

    Cell Death Differ 2017, 24, 889-902.

  9. Catalytic in vivo protein knockdown by small-molecule PROTACs

    Bondeson D. P., Mares A., Smith I. E., Ko E., Campos S., Miah A. H., Mulholland K. E., Routly N., Buckley D. L., Gustafson J. L., Zinn N., Grandi P., Shimamura S., Bergamini G., Faelth-Savitski M., Bantscheff M., Cox C., Gordon D. A., Willard R. R., Flanagan J. J., Casillas L. N., Votta B. J., den Besten W., Famm K., Kruidenier L., Carter P. S., Harling J. D., Churcher I., Crews C. M.

    Nat Chem Biol 2015, 11, 611-617.

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