title: An antibody-free strategy for screening putative HDM2 inhibitors using crude bacterial lysates expressing GST-HDM2 recombinant protein.
abstract: Targeting the interaction of p53 with its natural inhibitor MDM2 by the use of small synthetic molecules has emerged as a promising pharmacological approach to restore p53 oncosuppressor function in cancers retaining wild-type p53. The first critical step in the experimental validation of newly synthesized small molecules developed to inhibit MDM2-p53 interaction is represented by the evaluation of their efficacy in preventing the formation of the MDM2-p53 complex. This can be achieved using the in vitro reconstructed recombinant MDM2-p53 complex in cell-free assays. A number of possible approaches have been proposed, which are however not suitable for screening large chemical libraries, due to the high costs of reagents and instrumentations, or the need of large amounts of highly pure recombinant proteins. Here we describe a rapid and cheap method for high-throughput screening of putative inhibitors of MDM2-p53 complex formation--based on the use of GST-recombinant proteins--that does not require antibodies and recombinant protein purification steps from bacterial cell lysates.
authors.affiliation: Department of Pharmacy, University of Pisa, Italy.
authors.forename: Barbara
authors.initials: B
authors.lastname: Costa
authors.forename: Agostina Francesca
authors.initials: AF
authors.lastname: Grillone
authors.forename: Alessandra
authors.initials: A
authors.lastname: Salvetti
authors.forename: Silvia
authors.initials: S
authors.lastname: Rocchiccioli
authors.forename: Paola
authors.initials: P
authors.lastname: Iacopetti
authors.forename: Simona
authors.initials: S
authors.lastname: Daniele
authors.forename: Eleonora
authors.initials: E
authors.lastname: Da Pozzo
authors.forename: Pietro
authors.initials: P
authors.lastname: Campiglia
authors.forename: Ettore
authors.initials: E
authors.lastname: Novellino
authors.forename: Claudia
authors.initials: C
authors.lastname: Martini
authors.forename: Leonardo
authors.initials: L
authors.lastname: Rossi
mesh_terms: D004353:Drug Evaluation, Preclinical / Q000379:methods; D015964:Gene Expression Regulation, Bacterial; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D011485:Protein Binding / Q000502:physiology; D051736:Proto-Oncogene Proteins c-mdm2 / Q000037:antagonists & inhibitors* / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis
chemical_list: D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2
keywords: MDM2; MDM2 inhibitors; p53; recombinant proteins