Discovery of Pyrazolo[1,5-a]pyrimidine B-Cell Lymphoma 6 (BCL6) Binders and Optimization to High Affinity Macrocyclic Inhibitors

Abstract

Inhibition of the protein-protein interaction between B-cell lymphoma 6 (BCL6) and corepressors has been implicated as a therapeutic target in diffuse large B-cell lymphoma (DLBCL) cancers and profiling of potent and selective BCL6 inhibitors are critical to test this hypothesis. We identified a pyrazolo[1,5-a]pyrimidine series of BCL6 binders from a fragment screen in parallel with a virtual screen. Using structure-based drug design, binding affinity was increased 100000-fold. This involved displacing crystallographic water, forming new ligand-protein interactions and a macrocyclization to favor the bioactive conformation of the ligands. Optimization for slow off-rate constant kinetics was conducted as well as improving selectivity against an off-target kinase, CK2. Potency in a cellular BCL6 assay was further optimized to afford highly selective probe molecules. Only weak antiproliferative effects were observed across a number of DLBCL lines and a multiple myeloma cell line without a clear relationship to BCL6 potency. As a result, we conclude that the BCL6 hypothesis in DLBCL cancer remains unproven.

Inducible knock-out of BCL6 in lymphoma cells results in tumor stasis

“…To date the evaluation of the effects of targeting BCL6 in vitro and in vivo has been limited to the use of low affinity binding BCL6 inhibitors at high concentrations [22][23][24]32]. Recently, McCoull et al have developed highly potent inhibitors of BCL6, which, however, did not show significant anti-proliferative effects on lymphoma cells [28]. Further approaches to use BCL6 small molecule inhibitors as target binding ligand of proteolysis targeting chimeras (PROTACs) resulted in compounds that induce degradation of BCL6, albeit not to complete levels.…”

“…However, their use is limited due to the low binding affinity of most of these molecules [22][23][24]. Despite recent advances in developing BCL6 inhibitors [19,[25][26][27][28], no compound has yet reached the clinic. Furthermore, there exist controversies around the rationale and the impact of targeting BCL6 as a monotherapy due to the presence of high intra-and inter-tumor heterogeneity regarding type and number of oncogenic mutations [2,3] and the possibility of oncogene addiction switching following BCL6 targeted therapies by reactivating BCL2-family dependent anti-apoptotic pathways [29].…”

Development of a Novel B-Cell Lymphoma 6 (BCL6) PROTAC To Provide Insight into Small Molecule Targeting of BCL6

“…BCL6 was the most enriched protein identified(Figures 3D and S3A). In contrast, utilizing a PP series ligand 1315 tested at 4, 40 and 200μM (reduced compared to 12 due to lower solubility of PP series compounds) followed by treatment with the bead immobilized PP affinity probe (45) identified CK2 and CK2 binding partners in addition to BCL6, consistent with its known off-target CK2 activity(Figure 3C and 3D).…”

High throughput evaluation of macrocyclization strategies for conformer stabilization

“…Previous work has created workflows to screen macrocycles by combining these sampling methods with scoring functions such as molecular mechanics simulated annealing combined with quantum mechanical strain scoring 13 , inverse kinematics with ROSETTA 14 , implicit solvent/molecular mechanics scoring 15 , and normal-mode-based sampling with molecular mechanics scoring 16 . In this work, we implement an integrated, high-throughput method utilizing a loop-sampling-based macrocycle conformational sampling protocol along with a simple molecular mechanics strain-based scoring function.…”

“…The approach presented here is similar to that used by McCoul et al . to design macrocyclic BCL6 inhibitors 16 , however here we demonstrate an integrated workflow and validate it on diverse test sets.…”