ABSTRACT
Here we report corin, a synthetic hybrid agent derived from the class I HDAC inhibitor (entinostat) and an LSD1 inhibitor (tranylcypromine analog). Enzymologic analysis reveals that corin potently targets the CoREST complex and shows more sustained inhibition of CoREST complex HDAC activity compared with entinostat. Cell-based experiments demonstrate that corin exhibits a superior anti-proliferative profile against several melanoma lines and cutaneous squamous cell carcinoma lines compared to its parent monofunctional inhibitors but is less toxic to melanocytes and keratinocytes. CoREST knockdown, gene expression, and ChIP studies suggest that corin's favorable pharmacologic effects may rely on an intact CoREST complex. Corin was also effective in slowing tumor growth in a melanoma mouse xenograft model. These studies highlight the promise of a new class of two-pronged hybrid agents that may show preferential targeting of particular epigenetic regulatory complexes and offer unique therapeutic opportunities.
Subject(s)
Benzamides/pharmacology , Co-Repressor Proteins/antagonists & inhibitors , Histone Deacetylase Inhibitors/pharmacology , Melanoma/drug therapy , Nerve Tissue Proteins/antagonists & inhibitors , Pyridines/pharmacology , Tranylcypromine/pharmacology , Aged , Animals , Antineoplastic Agents , Cell Line, Tumor , Cell Proliferation , Co-Repressor Proteins/metabolism , Drug Design , Drug Screening Assays, Antitumor , Female , Histone Deacetylases/chemistry , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Middle Aged , Nerve Tissue Proteins/metabolism , Oligonucleotide Array Sequence Analysis , Repressor Proteins/antagonists & inhibitors , Repressor Proteins/metabolism , Skin Neoplasms/drug therapy , Xenograft Model Antitumor AssaysABSTRACT
Recent proteomic studies have identified a novel histone deacetylase complex that is upregulated during mitosis and is associated with cyclin A. This complex is conserved from nematodes to man and contains histone deacetylases 1 and 2, the MIDEAS corepressor protein and a protein called DNTTIP1 whose function was hitherto poorly understood. Here, we report the structures of two domains from DNTTIP1. The amino-terminal region forms a tight dimerization domain with a novel structural fold that interacts with and mediates assembly of the HDAC1:MIDEAS complex. The carboxy-terminal domain of DNTTIP1 has a structure related to the SKI/SNO/DAC domain, despite lacking obvious sequence homology. We show that this domain in DNTTIP1 mediates interaction with both DNA and nucleosomes. Thus, DNTTIP1 acts as a dimeric chromatin binding module in the HDAC1:MIDEAS corepressor complex.
Subject(s)
Carrier Proteins/chemistry , Carrier Proteins/metabolism , Histone Deacetylase 1/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Nucleosomes/metabolism , Cell Cycle , Co-Repressor Proteins/metabolism , DNA/metabolism , DNA-Binding Proteins , HEK293 Cells , Histone Deacetylase 2/metabolism , Humans , Models, Molecular , Protein Binding , Protein Interaction Domains and Motifs , Protein Multimerization , Protein Structure, Tertiary , Transcription FactorsABSTRACT
The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293 F cells.
