title: Probing an Allosteric Pocket of CDK2 with Small Molecules.
abstract: 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.
authors.affiliation: Dipartimento di Scienze della Vita, Università degli Studi di Modena e Reggio Emilia, Via Campi 103, 41125, Modena, Italy.|Dipartimento di Chimica, Università degli Studi di Milano, Via Golgi 19, 20133 Milano, Italy.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Christodoulou
authors.affiliation: Dipartimento di Scienze della Vita, Università degli Studi di Modena e Reggio Emilia, Via Campi 103, 41125, Modena, Italy.
authors.forename: Fabiana
authors.initials: F
authors.lastname: Caporuscio
authors.affiliation: Istituto di Ricerche Farmacologiche Mario Negri, Via La Masa 19, 20156, Milano, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Restelli
authors.affiliation: Dipartimento di Scienze della Vita, Università degli Studi di Modena e Reggio Emilia, Via Campi 103, 41125, Modena, Italy.
authors.forename: Luca
authors.initials: L
authors.lastname: Carlino
authors.affiliation: Dipartimento di Scienze della Vita, Università degli Studi di Modena e Reggio Emilia, Via Campi 103, 41125, Modena, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Cannazza
authors.affiliation: Dipartimento di Scienze Chimiche, Università degli Studi di Padova, Via Marzolo 1, 35131, Padova, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Costanzi
authors.affiliation: Dipartimento di Scienze e Tecnologie Biologiche e Ambientali, Università del Salento, Via per Monteroni, 73100, Lecce, Italy.
authors.forename: Cinzia
authors.initials: C
authors.lastname: Citti
authors.affiliation: Dipartimento di Chimica, Università degli Studi di Milano, Via Golgi 19, 20133 Milano, Italy.
authors.forename: Leonardo
authors.initials: L
authors.lastname: Lo Presti
authors.affiliation: Dipartimento di Scienze della Vita, Università degli Studi di Modena e Reggio Emilia, Via Campi 103, 41125, Modena, Italy.
authors.forename: Pasquale
authors.initials: P
authors.lastname: Pisani
authors.affiliation: Dipartimento di Scienze Chimiche, Università degli Studi di Padova, Via Marzolo 1, 35131, Padova, Italy.
authors.forename: Roberto
authors.initials: R
authors.lastname: Battistutta
authors.affiliation: Istituto di Ricerche Farmacologiche Mario Negri, Via La Masa 19, 20156, Milano, Italy.
authors.forename: Massimo
authors.initials: M
authors.lastname: Broggini
authors.affiliation: Dipartimento di Chimica, Università degli Studi di Milano, Via Golgi 19, 20133 Milano, Italy.
authors.forename: Daniele
authors.initials: D
authors.lastname: Passarella
authors.affiliation: Dipartimento di Scienze della Vita, Università degli Studi di Modena e Reggio Emilia, Via Campi 103, 41125, Modena, Italy.
authors.forename: Giulio
authors.identifier: 0000-0002-2474-0607
authors.initials: G
authors.lastname: Rastelli
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects; D000495:Allosteric Site / Q000187:drug effects*; D000970:Antineoplastic Agents / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D018360:Crystallography, X-Ray; D051357:Cyclin-Dependent Kinase 2 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004354:Drug Screening Assays, Antitumor; D006801:Humans; D008958:Models, Molecular; D015394:Molecular Structure; D011804:Quinolines / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D054852:Small Molecule Libraries / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D000970:Antineoplastic Agents; D011804:Quinolines; D054852:Small Molecule Libraries; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2
keywords: allosteric modulators; computational chemistry; cyclin-dependent kinase 2; protein kinases; structure elucidation