STEP activator | BI-0314

Target protein: 
STEP
Probe Name: 
BI-0314
MOLECULAR WEIGHT [DA]: 
286.3
In stock: 
15

Chemical structure

Figure 1: 2-D structure of BI-0314, a STEP activator

Highlights

With BI-0314 we report the discovery of the first small molecule allosteric modulator for STEP (STriatal-Enriched protein tyrosine Phosphatase), that binds to the phosphatase domain and upregulates the catalytic activity of STEP. Its activity has been demonstrated in enzymatic assays showing an activation by ~30% at 100µM and ~60% at 500µM in complementary readouts and on different STEP constructs. The selectivity against PTP1B and TCPTP has been tested and no activation at 500µM could be seen. To elucidate the mode of action, an X-ray structure with BI-0314 bound STEP has been solved demonstrating remote site binding ~20 Å away from the active phosphatase site. The allosteric binding site could be confirmed also in solution by 15N TROSY NMR. Long range allosteric mechanisms have been confirmed by extensive molecular dynamics simulations. The identification of a druggable allosteric pocket provides new opportunities for the discovery of selective STEP modulators as treatment options for CNS disorders.

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Target information

STEP is a multi-domain tryrosine phosphatase which exists as two splice variants, the membrane anchored longer isoform STEP61 and the cytosolic STEP46. Both isoforms share the identical kinase interaction motif (KIM) and the protein tyrosine phosphatase (PTP) domain with the phosphatase consensus motif C(X)5R. The KIM domain mediates binding to various target kinases with high affinity, while the PTP domain catalyzes their subsequent dephosphorylation. To avoid developing ligands which potentially suffer from substrate specificity, we preferred targeting the PTP domain over the KIM domain. The PTP domain bears various conserved structural motifs, such as the WPD loop, which is crucial for the catalytic step, as its aspartate (D461) mediates proton transfer to the phosphate leaving group.

Figure 3: STEP structure and allosteric binding site with bound ligand (orange sticks). PDB code: 6H8S.

Figure 3: STEP structure and allosteric binding site with bound ligand (orange sticks). PDB code: 6H8S.

In vitro Activity

BI-0314 displays an activation of STEP of ~60% at 500µM on the dephosphorylation of a pFYN derived peptide.

Probe name / Negative control BI-0314
MW [Da] (Free base) b 286.3
Enzyme Substrate Assay technology Effect of BI-0314 in assay
hSTEP46a pFYN-peptide AlphaLISA Activating
56% ± 5 % at 500 µM, (n=8)
33% ± 12 % at 100 µM, (n=12)
hSTEP46a pFYN-peptide RapidFire (MS) Activating
28% ± 5 % at 100 µM, (n=4)
PTP domain of
hSTEPa
pFYN-peptide AlphaLISA Activating
61% ± 6 % at 500 µM, (n=10)
hSTEP46a DiFMUP Fluorescence Activating
48% ± 8 % at 1000 µM, (n=3)
27% ± 5 % at 300 µM, (n=3)

a for detailed assay conditions see Ref. 3

b will be shipped as salt (for MW of the salt and salt form please refer to vail-label).

In vitro DMPK and CMC parameters

Not determined.

In vivo DMPK parameters

Not determined.

In vivo pharmacology

Not determined.

Negative control

Not available.

Selectivity

The phylogenetically closest enzyme (TCPTP) and the “generic” tyrosine phosphatase PTP1B have been investigated and at concentrations up to 500 µM of BI-0314 no signs of activation could be observed. No other panels have been tested.

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

The Xray crystal structure of target in complex with BI-0314 is available (PDB code: 6H8S)

reference molecules

No other STEP activators are described so far. However, there are quite potent orthosteric inhibitors described, which only show moderate selectivity over other phosphatases. (Xu 20141, Witten 20172)

Summary

With BI-0314 the first allosteric activator of tyrosine phosphatases is described. We hope to spark the design of selective allosteric ligands of phosphatases with the disclosure of the X-ray structure, and the elucidation of the mode of action of BI-0314 on STEP.

Supplementary data

References

  1. Inhibitor of the Tyrosine Phosphatase STEP Reverses Cognitive Deficits in a Mouse Model of Alzheimer's Disease

    Jian Xu, Manavi Chatterjee, Tyler D. Baguley, Jonathan Brouillette, Pradeep Kurup, Debolina Ghosh, Jean Kanyo, Yang Zhang, Kathleen Seyb, Chimezie Ononenyi, Ethan Foscue, George M. Anderson, Jodi Gresack, Gregory D. Cuny,
    Marcie A. Glicksman, Paul Greengard, TuKiet T. Lam, Lutz Tautz, Angus C. Nairn, Jonathan A. Ellman, Paul J. Lombroso

    PLOS Biology, 2014, 12 (8), e1001923.

  2. X-ray Characterization and Structure-Based Optimization of Striatal-Enriched Protein Tyrosine Phosphatase Inhibitors

    Michael R. Witten, Lisa Wissler, Melanie Snow, Stefan Geschwindner, Jon A. Read∥, Nicholas J. Brandon, Angus C. Nairn, Paul J. Lombroso, Helena Käck , and Jonathan A. Ellman

    J. Med. Chem., 2017, 60 (22), 9299–9319.

  3. Allosteric activation of the protein tyrosine phosphatase STEP by a fragment-like molecule

    C.S. Tautermann et al.

    J. Med. Chem., ASAP, 2018.

  4. Therapeutic Implications for Striatal-Enriched Protein Tyrosine Phosphatase (STEP) in Neuropsychiatric Disorders

    Susan M. Goebel-Goody, Matthew Baum, Constantinos D. Paspalas, Stephanie M. Fernandez, Niki C. Carty, Pradeep Kurup, and Paul J. Lombroso

    Pharmacological Reviews 2012, 64, 65-87.

  5. Genetic reduction of striatal-enriched tyrosine phosphatase (STEP) reverses cognitive and cellular deficits in an Alzheimer’s disease mouse model

    Yongfang Zhang, Pradeep Kurup, Jian Xu, Nikisha Carty, Stephanie M. Fernandez, Haakon B. Nygaard, Christopher Pittenger, Paul Greengard, Stephen M. Strittmatter, Angus C. Nairn, and Paul J. Lombroso

    Proceedings of the National Academy of Sciences 2010, 107, 19014-19019.

  6. STEP61 is a substrate of the E3 ligase parkin and is upregulated in Parkinson’s disease

    Pradeep K. Kurup, Jian Xu, Rita Alexandra Videira, Chimezie Ononenyi, Graça Baltazar, Paul J. Lombroso, and Angus C. Nairn

    Proceedings of the National Academy of Sciences 2015, 112, 1202-1207.

  7. The tyrosine phosphatase STEP: implications in schizophrenia and the molecular mechanism underlying antipsychotic medications

    NC Carty, J Xu, P Kurup, J Brouillette, SM Goebel-Goody, DR Austin, P Yuan, G Chen, PR Correa, V Haroutunian, C Pittenger and PJ Lombroso

    Transl Psychiatry 2012, 2, e137.