TY - JOUR
T1 - Probing an Allosteric Pocket of CDK2 with Small Molecules
AU - Christodoulou, Michael S.
AU - Caporuscio, Fabiana
AU - Restelli, Valentina
AU - Carlino, Luca
AU - Cannazza, Giuseppe
AU - Costanzi, Elisa
AU - Citti, Cinzia
AU - Lo Presti, Leonardo
AU - Pisani, Pasquale
AU - Battistutta, Roberto
AU - Broggini, Massimo
AU - Passarella, Daniele
AU - Rastelli, Giulio
PY - 2017/1/5
Y1 - 2017/1/5
N2 - The availability of well-characterized allosteric modulators is crucial for investigating the allosteric regulation of protein function. In a recently identified inactive conformation of cyclin-dependent kinase 2 (CDK2), an open allosteric pocket was detected and proposed as a site to accommodate allosteric inhibitors. Previous structure-based approaches allowed the identification of a hit compound expected to bind to this pocket. Herein we report the characterization of this compound by X-ray crystallography, which surprisingly provided a chemical structure different from that previously reported. Therefore, the compound was synthesized and completely characterized. X-ray structures of the synthesized and purchased compounds were found to be superimposable. A reaction mechanism was proposed to explain the formation of the structure indicated by crystallography. Moreover, a stereoselective synthesis was developed to evaluate the biological activity of the pure stereoisomers. Modeling studies were performed to unveil the details of the interaction with CDK2. The activity of the obtained compounds was evaluated with various biological assays. Mutagenesis experiments confirmed binding to the allosteric pocket. Finally, the allosteric ligands were shown to inhibit the growth of lung (A549) and ovarian (SKOV3) cancer cell lines. Therefore, this report presents a thorough chemical and biological characterization of the first small-molecule ligands to be used as probes to study the allosteric modulation of CDK2 activity.
AB - The availability of well-characterized allosteric modulators is crucial for investigating the allosteric regulation of protein function. In a recently identified inactive conformation of cyclin-dependent kinase 2 (CDK2), an open allosteric pocket was detected and proposed as a site to accommodate allosteric inhibitors. Previous structure-based approaches allowed the identification of a hit compound expected to bind to this pocket. Herein we report the characterization of this compound by X-ray crystallography, which surprisingly provided a chemical structure different from that previously reported. Therefore, the compound was synthesized and completely characterized. X-ray structures of the synthesized and purchased compounds were found to be superimposable. A reaction mechanism was proposed to explain the formation of the structure indicated by crystallography. Moreover, a stereoselective synthesis was developed to evaluate the biological activity of the pure stereoisomers. Modeling studies were performed to unveil the details of the interaction with CDK2. The activity of the obtained compounds was evaluated with various biological assays. Mutagenesis experiments confirmed binding to the allosteric pocket. Finally, the allosteric ligands were shown to inhibit the growth of lung (A549) and ovarian (SKOV3) cancer cell lines. Therefore, this report presents a thorough chemical and biological characterization of the first small-molecule ligands to be used as probes to study the allosteric modulation of CDK2 activity.
KW - allosteric modulators
KW - computational chemistry
KW - cyclin-dependent kinase 2
KW - protein kinases
KW - structure elucidation
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U2 - 10.1002/cmdc.201600474
DO - 10.1002/cmdc.201600474
M3 - Article
AN - SCOPUS:85007009595
VL - 12
SP - 33
EP - 41
JO - ChemMedChem
JF - ChemMedChem
SN - 1860-7179
IS - 1
ER -