nutlin-3a and nutlin-1

Topics: Animals; Anticarcinogenic Agents; Antineoplastic Agents; Carrier Proteins; Humans; Imidazoles; Models, Molecular; Neoplasms; Piperazines; Protein Binding; Tumor Suppressor Protein p53

Analyzing the effect of ligands on protein-protein interactions is important to better understand the cellular processes. In vitro characterization of these modulations remains challenging because of the drawbacks associated with the analysis of noncovalent interactions. To facilitate the analysis, stabilization of the protein complex by chemical cross-linking followed by High-Mass MALDI mass spectrometry is a recently developed method offering several advantages: No need for immobilization or special tags, the analysis is possible directly on wild-type protein complexes, no need for buffer exchange, large applicability range for any type of protein complex from 0 to 1,500 kDa. Using this method, we analyzed the effect of the inhibitors Nutlin-3a and Nutlin-3b on the protein complex MDM2-p53. Using this fast and sensitive method, the IC(50) values of these inhibitors have been determined.

Topics: Cross-Linking Reagents; Imidazoles; Inhibitory Concentration 50; Ligands; Molecular Weight; Piperazines; Protein Binding; Protein Interaction Maps; Proto-Oncogene Proteins c-mdm2; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Statistics as Topic; Tumor Suppressor Protein p53

We have developed a heterobifunctional all-small molecule PROTAC (PROteolysis TArgeting Chimera) capable of inducing proteasomal degradation of the androgen receptor. This cell permeable PROTAC consists of a non-steroidal androgen receptor ligand (SARM) and the MDM2 ligand known as nutlin, connected by a PEG-based linker. The SARM-nutlin PROTAC recruits the androgen receptor to MDM2, which functions as an E3 ubiquitin ligase. This leads to the ubiquitination of the androgen receptor, and its subsequent degradation by the proteasome. Upon treatment of HeLa cells with 10microM PROTAC for 7h, we were able to observe a decrease in androgen receptor levels. This degradation is proteasome dependent, as it is mitigated in cells pre-treated with 10microM epoxomicin, a specific proteasome inhibitor. These results have implications for the potential study and treatment of various cancers with increased androgen receptor levels.

Topics: Blotting, Western; Cell Line, Tumor; Cell Membrane Permeability; Cell Proliferation; Cell Survival; Humans; Imidazoles; Indicators and Reagents; Piperazines; Proteins; Proteomics; Receptors, Androgen; Ubiquitin; Ubiquitin-Protein Ligases

Recent studies have shown that activation of cell cycle checkpoints can protect normal proliferating cells from mitotic inhibitors by preventing their entry into mitosis. These studies have used genotoxic agents that act, at least in part, by activation of the p53 pathway. However, genotoxic drugs are known also to have p53-independent activities and could affect the sensitivity of tumor cells to antimitotic agents. Recently, we have developed the first potent and selective small-molecule inhibitors of the p53-MDM2 interaction, the nutlins, which activate the p53 pathway only in cells with wild-type but not mutant p53. Using these compounds, we show that p53 activation leads to G1 and G2 phase arrest and can protect cells from mitotic block and apoptosis caused by paclitaxel. Pretreatment of HCT116 and RKO colon cancer cells (wild-type p53) or primary human fibroblasts (1043SK) with nutlins for 24 hours followed by incubation with paclitaxel for additional 48 hours did not increase significantly their mitotic index and protected the cells from the cytotoxicity of paclitaxel. Cancer cells with mutant p53 (MDA-MB-435) responded to the same treatment with mitotic arrest and massive apoptosis. These results have two major implications for cancer therapy. First, p53-activating therapies may have antagonistic effect when combined with mitotic poisons. Second, pretreatment with MDM2 antagonists before chemotherapy of tumors with mutant p53 may offer a partial protection to proliferating normal tissues.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Breast Neoplasms; Cell Proliferation; Colonic Neoplasms; Female; G1 Phase; G2 Phase; Humans; Imidazoles; Mitosis; Mutation; Nuclear Proteins; Paclitaxel; Piperazines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Tumor Cells, Cultured; Tumor Suppressor Protein p53

MDM2 binds the p53 tumor suppressor protein with high affinity and negatively modulates its transcriptional activity and stability. Overexpression of MDM2, found in many human tumors, effectively impairs p53 function. Inhibition of MDM2-p53 interaction can stabilize p53 and may offer a novel strategy for cancer therapy. Here, we identify potent and selective small-molecule antagonists of MDM2 and confirm their mode of action through the crystal structures of complexes. These compounds bind MDM2 in the p53-binding pocket and activate the p53 pathway in cancer cells, leading to cell cycle arrest, apoptosis, and growth inhibition of human tumor xenografts in nude mice.

Topics: Animals; Apoptosis; Binding Sites; Cell Cycle; Cell Division; Cell Line; Cell Line, Tumor; Cell Survival; Crystallization; Crystallography, X-Ray; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Dose-Response Relationship, Drug; Gene Expression; Genes, p53; Humans; Hydrophobic and Hydrophilic Interactions; Imidazoles; Mice; Mice, Nude; Models, Molecular; Molecular Weight; Neoplasm Transplantation; Neoplasms, Experimental; NIH 3T3 Cells; Nuclear Proteins; Phosphorylation; Piperazines; Protein Conformation; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Stereoisomerism; Transplantation, Heterologous; Tumor Suppressor Protein p53