Structural analysis of PI3-kinase isoforms: Identification of residues enabling selective inhibition by small molecule ATP-competitive inhibitors

4-Nitrophenyl phosphate (p-nitrophenyl phosphate, pNPP) is widely used as a small molecule phosphotyrosine-like substrate in activity assays for protein tyrosine phosphatases. It is a colorless substrate that upon hydrolysis is converted to a yellow 4-nitrophenolate ion that can be monitored by absorbance at 405 nm. Therefore, the pNPP assay has been widely adopted as a quick and simple method to assess phosphatase activity and is also commonly used in assays to screen for inhibitors. Here, the first crystal structure is presented of a dual-specificity phosphatase, human dual-specificity phosphatase 22 (DUSP22), in complex with pNPP. The structure illuminates the molecular basis for substrate binding and may also facilitate the structure-assisted development of DUSP22 inhibitors.

Download Full-text

Structural Analysis of Small-Molecule Binding to the BAZ2A and BAZ2B Bromodomains

ChemMedChem ◽

10.1002/cmdc.201800234 ◽

2018 ◽

Vol 13 (14) ◽

pp. 1479-1487 ◽

Cited By ~ 2

Author(s):

Andrea Dalle Vedove ◽

Dimitrios Spiliotopoulos ◽

Vito G. D'Agostino ◽

Jean-Rémy Marchand ◽

Andrea Unzue ◽

...

Keyword(s):

Structural Analysis ◽

Small Molecule

Download Full-text

Discovery and Design of Novel Small Molecule GSK-3 Inhibitors Targeting the Substrate Binding Site

International Journal of Molecular Sciences ◽

10.3390/ijms21228709 ◽

2020 ◽

Vol 21 (22) ◽

pp. 8709

Author(s):

Ido Rippin ◽

Netaly Khazanov ◽

Shirley Ben Joseph ◽

Tania Kudinov ◽

Eva Berent ◽

...

Keyword(s):

Binding Site ◽

Small Molecule ◽

Substrate Binding ◽

Pharmacophore Model ◽

Drug Induced ◽

Substrate Binding Site ◽

Zinc Database ◽

Competitive Inhibitors ◽

Ic50 Values ◽

Critical Binding

The serine/threonine kinase, GSK-3, is a promising drug discovery target for treating multiple pathological disorders. Most GSK-3 inhibitors that were developed function as ATP competitive inhibitors, with typical limitations in specificity, safety and drug-induced resistance. In contrast, substrate competitive inhibitors (SCIs), are considered highly selective, and more suitable for clinical practice. The development of SCIs has been largely neglected in the past because the ambiguous, undefined nature of the substrate-binding site makes them difficult to design. In this study, we used our previously described structural models of GSK-3 bound to SCI peptides, to design a pharmacophore model and to virtually screen the “drug-like” Zinc database (~6.3 million compounds). We identified leading hits that interact with critical binding elements in the GSK-3 substrate binding site and are chemically distinct from known GSK-3 inhibitors. Accordingly, novel GSK-3 SCI compounds were designed and synthesized with IC50 values of~1–4 μM. Biological activity of the SCI compound was confirmed in cells and in primary neurons that showed increased β-catenin levels and reduced tau phosphorylation in response to compound treatment. We have generated a new type of small molecule GSK-3 inhibitors and propose to use this strategy to further develop SCIs for other protein kinases.

Download Full-text