Abstract
BCL6 is a transcriptional repressor that is over-expressed due to chromosomal translocations, or other abnormalities, in ∼40% of diffuse large B-cell lymphoma. BCL6 interacts with co-repressor, SMRT, and this is essential for its role in lymphomas. Peptide or small molecule inhibitors, which prevent the association of SMRT with BCL6, inhibit transcriptional repression and cause apoptosis of lymphoma cells in vitro and in vivo. In order to discover compounds, which have the potential to be developed into BCL6 inhibitors, we screened a natural product library. The ansamycin antibiotic, rifamycin SV, inhibited BCL6 transcriptional repression and NMR spectroscopy confirmed a direct interaction between rifamycin SV and BCL6. To further determine the characteristics of compounds binding to BCL6-POZ we analyzed four other members of this family and showed that rifabutin, bound most strongly. An X-ray crystal structure of the rifabutin-BCL6 complex revealed that rifabutin occupies a partly non-polar pocket making interactions with tyrosine58, asparagine21 and arginine24 of the BCL6-POZ domain. Importantly these residues are also important for the interaction of BLC6 with SMRT. This work demonstrates a unique approach to developing a structure activity relationship for a compound that will form the basis of a therapeutically useful BCL6 inhibitor.
Highlights
BCL6 is a transcriptional repressor [1] that accomplishes its effects by binding to DNA through carboxy-terminal zinc fingers and recruitment of co-repressors to its mid-portion and aminoterminus (Figure 1A)
We screened a commercial natural product library consisting of 480 compounds for ability to prevent BCL6 induced transcriptional repression in the Burkitt’s lymphoma cell line DG75 (Figure 1B)
Luciferase expression was repressed by BCL6 and this was relieved by the addition of rifamycin SV (Figure 1D), which was detected in the initial library screen (Figure 1C), but not by the other eight compounds
Summary
BCL6 is a transcriptional repressor [1] that accomplishes its effects by binding to DNA through carboxy-terminal zinc fingers and recruitment of co-repressors to its mid-portion and aminoterminus (Figure 1A). Co-repressors NCoR (NCOR1), BCoR (BCOR) and SMRT (NCOR2), which are components of multiprotein complexes that include histone deacetylases, associate with the amino-terminal POZ domain [2,3,4]. SMRT and NCoR share an amino acid sequence (GRSIHEIPR) that is required for binding to the BCL6-POZ domain and is functionally important [5] but in contrast BCoR binding is by means of a different primary sequence (APSSWVVPG) [6]. It has been demonstrated that BCL6 promotes the proliferation of primary tonsillar B-cells [13] and prevents terminal differentiation to plasma cells in B-cell lines [12,14]
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