CtBPs (CtBP1 and CtBP2) act in the nucleus as transcriptional corepressors and in the cytoplasm as regulators of Golgi apparatus fission. Studies in which the expression or function of CtBPs has been inhibited have independently identified roles for CtBPs in both suppressing apoptosis and promoting cell cycle progression. Here, we have analyzed the consequences of ablating CtBP expression in breast cancer-derived cell lines. We found that loss of CtBP expression suppresses cell proliferation through a combination of apoptosis, reduction in cell cycle progression, and aberrations in transit through mitosis. The third phenotype includes errors in mitotic chromosome segregation that are associated with decreased association of the chromosome passenger protein aurora B with mitotic chromatin and that are likely to be a primary cause of the proapoptotic and antiproliferative effects of CtBP loss. We also show that loss of CtBP expression results in the activation of the transcription factor p53 and that loss of p53 function renders cells more susceptible to CtBP small interfering RNA-induced apoptosis.

Identification of direct modulators of transcription factor protein–protein interactions is a key challenge for ligand discovery that promises to significantly advance current approaches to cancer therapy. Here, we report an inhibitor of NADH-dependent dimerization of the C-terminal binding protein (CtBP) transcriptional repressor, identified by screening genetically encoded cyclic peptide libraries of up to 64 million members. CtBP dimers form the core of transcription complexes associated with epigenetic regulation of multiple genes that control many characteristics of cancer cells, including proliferation, survival and migration. CtBP monomers also have distinct and critical cellular function, thus current experimental tools that deplete all forms of a targeted protein (e.g. siRNA) do not allow the cellular consequences of this metabolically regulated transcription factor to be deciphered. The most potent inhibitor from our screen (cyclo-SGWTVVRMY) is demonstrated to disrupt CtBP dimerization in vitro and in cells. This compound is used as a chemical tool to establish that the NADH-dependent dimerization of CtBPs regulates the maintenance of mitotic fidelity in cancer cells. Treatment of highly glycolytic breast cancer cell lines with the identified inhibitor significantly reduced their mitotic fidelity, proliferation and colony forming potential, whereas the compound does not affect mitotic fidelity of cells with lower glycolytic flux. This work not only links the altered metabolic state of transformed cells to a key determinant of the tumor cell phenotype, but the uncovered compound also serves as the starting point for the development of potential therapeutic agents that target tumors by disrupting the CtBP chromatin-modifying complex.

Aminoimidazole carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase (ATIC) is a bifunctional homodimeric enzyme that catalyses the last two steps of de novo purine biosynthesis. Homodimerization of ATIC, a protein-protein interaction with an interface of over 5000 Å2, is required for its aminoimidazole carboxamide ribonucleotide (AICAR) transformylase activity, with the active sites forming at the interface of the interacting proteins. Here, we report the development of a small-molecule inhibitor of AICAR transformylase that functions by preventing the homodimerization of ATIC. The compound is derived from a previously reported cyclic hexa-peptide inhibitor of AICAR transformylase (with a Ki of 17 μM), identified by high-throughput screening. The active motif of the cyclic peptide is identified as an arginine-tyrosine dipeptide, a capped analogue of which inhibits AICAR transformylase with a Ki of 84 μM. A library of non-natural analogues of this dipeptide was designed, synthesized, and assayed. The most potent compound inhibits AICAR transformylase with a Ki of 685 nM, a 25-fold improvement in activity from the parent cyclic peptide. The potential for this AICAR transformylase inhibitor in cancer therapy is assessed by studying its effect on the proliferation of a model breast cancer cell line. Using a non-radioactive proliferation assay and live cell imaging, a dose-dependent reduction in cell numbers and cell division rates was observed in cells treated with our ATIC dimerization inhibitor.

Click DNA ligation promises an alternative to the current enzymatic approaches for DNA assembly, with the ultimate goal of using efficient chemical reactions for the total chemical synthesis and assembly of genes and genomes. Such an approach would enable the incorporation of various chemically modified bases throughout long stretches of DNA, a feat not possible with current polymerase-based methods. An unequivocal requirement for this approach is the biocompatibility of the resulting triazole-linked DNA. The correct function of this unnatural DNA linker in human cells is demonstrated here by using a click-linked gene encoding the fluorescent protein mCherry. Reverse transcription of mRNA isolated from these cells and subsequent sequencing of the mCherry cDNA shows error-free transcription. Nucleotide excision repair (NER) is shown to not play a role in the observed biocompatibility by using a NER-deficient human cell line. This is the first example of a non-natural DNA linker being functional in a eukaryotic cell.