RESUMO
Identifying ligand binding sites on proteins is a critical step in target-based drug discovery. Current approaches to this require resource-intensive screening of large libraries of lead-like or fragment molecules. Here, we describe an efficient and effective experimental approach to mapping interaction sites using a set of halogenated compounds expressing paired hydrogen-bonding motifs, termed FragLites. The FragLites identify productive drug-like interactions, which are identified sensitively and unambiguously by X-ray crystallography, exploiting the anomalous scattering of the halogen substituent. This mapping of protein interaction surfaces provides an assessment of druggability and can identify efficient start points for the de novo design of hit molecules incorporating the interacting motifs. The approach is illustrated by mapping cyclin-dependent kinase 2, which successfully identifies orthosteric and allosteric sites. The hits were rapidly elaborated to develop efficient lead-like molecules. Hence, the approach provides a new method of identifying ligand sites, assessing tractability and discovering new leads.
Assuntos
Halogenação , Sítios de Ligação , Cristalografia por Raios X , Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos , Ligantes , Bibliotecas de Moléculas Pequenas/químicaRESUMO
Non-genotoxic reactivation of the p53 pathway by MDM2-p53 binding antagonists is an attractive treatment strategy for wild-type TP53 cancers. To determine how resistance to MDM2/p53 binding antagonists might develop, SJSA-1 and NGP cells were exposed to growth inhibitory concentrations of chemically distinct MDM2 inhibitors, Nutlin-3 and MI-63, and clonal resistant cell lines generated. The p53 mediated responses of parental and resistant cell lines were compared. In contrast to the parental cell lines, p53 activation by Nutlin-3, MI-63 or ionizing radiation was not observed in either the SJSA-1 or the NGP derived cell lines. An identical TP53 mutation was subsequently identified in both of the SJSA-1 resistant lines, whilst one out of three identified mutations was common to both NGP derived lines. Mutation specific PCR revealed these mutations were present in parental SJSA-1 and NGP cell populations at a low frequency. Despite cross-resistance to a broad panel of MDM2/p53 binding antagonists, these MDM2-amplified and TP53 mutant cell lines remained sensitive to ionizing radiation (IR). These results indicate that MDM2/p53 binding antagonists will select for p53 mutations present in tumours at a low frequency at diagnosis, leading to resistance, but such tumours may nevertheless remain responsive to alternative therapies, including IR.
Assuntos
Resistencia a Medicamentos Antineoplásicos/fisiologia , Resistencia a Medicamentos Antineoplásicos/efeitos da radiação , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteína Supressora de Tumor p53/genética , Linhagem Celular Tumoral , Humanos , Mutação , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteína Supressora de Tumor p53/antagonistas & inibidoresRESUMO
Two libraries of substituted benzimidazoles were designed using a 'scaffold-hopping' approach based on reported MDM2-p53 inhibitors. Substituents were chosen following library enumeration and docking into an MDM2 X-ray structure. Benzimidazole libraries were prepared using an efficient solution-phase approach and screened for inhibition of the MDM2-p53 and MDMX-p53 protein-protein interactions. Key examples showed inhibitory activity against both targets.