Binding curves for five resonances that shifted (Y-axis, in ppm) upon the addition of increasing concentrations of 79-6 (X-axis). The fitting resulted in a K_d of 138 ± 31 μM. See also Figure S5.

A,B: Selected regions of the ¹⁵N-¹H HSQC spectra of BCL6^BTB with increasing amounts of 79-6. The red spectrum was obtained in the absence of the compound. The arrows indicate shifted resonances. C: Residues whose amide NMR resonances shifted by 0.05–0.09 ppm are colored light pink, 0.10–0.14 ppm are colored medium red, and >0.15 ppm are colored dark red. The surface of the BCL6 BTB dimer is colored grey and white. See also Figure S4.

A: Structure of the 2:2 complex between the BCL6 BTB domain and the SMRT BBD peptide. The two chains of the BTB domain are shown in pink and blue and the two SMRT peptides are shown in stick representation with green carbon atoms. The SMRT 1423-1428 region is circled. B: View of the selected compounds docked to the BCL6 lateral groove pocket predicted by the CADD procedure along with the putative binding site represented by green transparent spheres. The compounds are stick representations based on the following color scheme: 28 (blue), 72 (magenta), 79 (red), 53 (yellow) and 55 (green). See also Figure S1 and Table S1.

Single cell suspensions were obtained from lymph node biopsies of patients diagnosed with DLBCL and were treated with either vehicle, 79-6 125 μM (black bars) or 79-6 250 μM (grey bars). The Y-axis represents the percent of viable cells compared to vehicle, which is represented by the line at 100%. A significant response zone is shown as a gray shadow with 75% viability as the upper limit. Whether BCL6 was detected by western blot in these samples is indicated at the bottom of the graph by the plus and minus signs. Error bars represent the SEM for replicates. See also Figure S8.

A: Reporter assays were performed to test the impact of CADD compounds on the repressor activity of a GAL4-DBD-BCL6^BTB fusion construct, compared to their effect on GAL4-DBD alone. The Y-axis shows the relative percent of repression mediated by GAL-BCL6^BTB in the presence of vehicle (V, which is set as 100%), compounds with activity (black bars and 79 as red bar) or selected inactive compounds (grey bars). Compounds were tested at 50 μM in DMSO. Experiments were performed in triplicate with compounds tested in quadruplicate. B: Reporter assay as in (A) was performed using families of compounds related to those in (A). The X-axis lists the compounds according to which parental compound they are similar to. The 79 series is shown in black and 79-6 is shown in red. Compounds were tested at 100 μM in DMSO. Error bars represent the SEM for replicates. See also Figure S2 and Table S2.

A: The crystal on the right was soaked with compound 79-6 prior to transferring it to a well with no added compound. The crystal on the left was not soaked in 79-6. B: |Fo′Fc| difference electron density contoured at 4 s (red mesh) and 2 s (grey mesh) in the lateral groove site of the BCL6^BTB dimer prior to the inclusion of the compound in the model. The stick model represents the final refined position of 79-6. The bromine atom of the compound is located in the region of highest electron density C: Compound 79-6 is shown in a space filling representation with green carbon atoms, and binds in the lateral groove of the BTB dimer. D: Details of the molecular interactions between 79-6 and the BCL6 BTB domain. The bromine atom of the indolazine ring of 79-6 is colored brown. Residues Asn-21, Arg-24, Leu-25 and Arg-28 are from one chain, and residues labeled with primes (Gly-55′, Tyr-58′ and Ser-59′) are from the other BCL6 chain. See also Figure S3 and Table S3.

A: The serum (blue curve) and tumor (red curve, inset) concentrations of 79-6 were determined after the intraperitoneal administration of 50 mg/kg to mice carrying OCI-Ly7 xenografts. Serum and tumors were harvested at several time points (X–axis) and the concentration of 79-6 was determined by HPLC-MS/MS and compared to control (time 0). B: Tumor growth plots in SU-DHL6, OCI-Ly7 and Toledo (as negative control) xenografted mice treated with vehicle (green circles) or 79-6 50 mg/kg/day (red circles) for 10 consecutive days. The Y-axis represents the percentage of tumor volume (in mm³) compared to day 1 of treatment and X-axis represents treatment day. C: Serum levels of human β₂-microglobulin at day 10 in vehicle (green bars) and 79-6 (red bars) treated SU-DHL6, OCI-Ly7 and Toledo mice. D: Representative images from SU-DHL6, OCI-Ly7 and Toledo mice tumors after treatment with vehicle or 79-6 and assayed for apoptosis by TUNEL. Scale bars represent 125 μm and 50 μm in main images and insets respectively. Error bars represent the SEM for replicates. See also Figure S7 and Table S4.

A: Reporter assay comparing the effect of 50 μM of 79-6 on the repressor activity of GAL4-BCL6^BTB, GAL4-Kaiso^BTB, GAL4-HIC1^BTB or GAL4-PLZF^BTB, compared to GAL4-DBD alone. The Y-axis shows fold repression in the presence of vehicle (black bars) compared to 79-6 (grey bars). B: Quantitative chromatin immunoprecipitation was performed in SU-DHL6 DLBCL cells to detect binding of BCL6, N-CoR and SMRT to the ATR promoter in the presence of vehicle (black bars) or 125 μM of 79-6 (grey bars). The Y-axis shows fold difference in enrichment of ATR in the various conditions. C: mRNA abundance of the BCL6 target genes ATR, TP53, CD69, p21 and CD44, and the non-target genes B2M, PCNA and HPRT was measured in SU-DLH6, SU-DHL4 (both BCL6 dependent) and Toledo (BCL6 independent) DLBCL cell lines exposed to vehicle or 50 μM of 79-6 for 8 h. The Y-axis shows 79-6 mediated fold induction of each gene normalized to RPL13A and relative to vehicle (DMSO). D: Dose-effect curves for a panel of 8 DLBCL cell lines exposed to increasing concentrations of 79-6 for 48 h. OCI-Ly7, OCI-Ly1, SU-DHL6, SU-DHL4, OCI-Ly10 and Farage are BCL6-dependent, and Toledo and OCI-Ly4 are BCL6-independent negative controls. Cell viability was determined by luminescent ATP quantization. The value between parentheses represent the drug concentration (in mM) that inhibits the growth of cell lines by 50% compared to vehicle (GI₅₀). Assays were performed in biological triplicates, with the averages of these plotted. Error bars represent the SEM for replicates. See also Figure S6.