Fragments vs BCL6, two ways

Disrupting protein-protein interactions (PPIs) tends to be challenging: interfaces are often large and flat, with few deep pockets in which small molecules can bind. Also, much like unhappy families, PPIs are usually dissimilar, meaning that HTS collections yield fewer, less attractive hits. Both of these challenges are well-addressed by fragments, and indeed last year saw the approval of venetoclax, which targets a PPI. Two new papers in J. Med. Chem. report inhibitors of another PPI.

B-Cell lymphoma 6 (BCL6) binds to other proteins to regulate gene expression. As its name suggests, it was identified in diffuse large B-cell lymphoma (DLBCL), and is thus an interesting anticancer target. Also, structures of the protein in complex with a peptide suggested that it may not be impossible to find small molecule inhibitors. Yusuke Kamada and colleagues at Takeda set out to do just this.

An SPR-based screen of 1494 fragments, each at 1 mM, identified 64 hits which confirmed in dose-response titrations. However, only seven compounds confirmed in an STD-NMR experiment. Of these, compound 1 was characterized crystallographically bound in the peptide groove.

A few tweaks to the fragment led to compound 4, with improved potency. Meanwhile, an HTS screen had identified the very weak compound 5, and crystallography showed that it bound at the same site as compound 4. Merging the two molecules led to compound 7, with mid-nanomolar biochemical potency and low micromolar activity in a cell-based assay. This is a classic case of fragment-assisted drug discovery (FADD), and a good illustration of how FBLD and HTS can be complementary.

The second paper, by William McCoull and a large team of collaborators from AstraZeneca and Pharmaron, goes somewhat further. The researchers conducted an SPR-based screen of 3500 fragments along with a virtual screen. Both found hits, and led to molecules with the same core as compound A2. Crystallography revealed that these also bind in the peptide groove, and combining elements of both molecules while tweaking the properties led to compound A5, with improved affinity.

The next step was growing compound A5 to try to make a hydrogen bond interaction with the protein. That led to compound A8, with submicromolar affinity. A crystal structure of a related compound bound to BCL6 revealed that two chemically distant portions of the molecule were in close proximity, suggesting a macrocyclization strategy similar to what we described last week. This proved highly successful, improving the affinity by more than two orders of magnitude for compound A11. NMR studies of the linear and cyclized compounds revealed that the conformation of the latter was indeed closer to that seen in the crystal structure.

The medicinal chemistry continues extensively from here. In particular, compound A11 showed some activity against the kinase CK2, but this could be engineered out. Multiple additional changes were explored, with many compounds showing low nanomolar activity in a biochemical assay and some showing high nanomolar activity in a cell-based assay. Unfortunately, none were very effective at inhibiting the proliferation of lymphoma cell lines. The authors state, “we conclude that the BCL6 hypothesis as a means of treatment for DLBCL is still unproven and we have elected not to progress this series of BCL6 inhibitors further into development.”

This makes sense, though I wouldn’t abandon all hope. For two other PPIs, BCL2 and MCL1, robust cell activity required picomolar affinity in a biochemical assay. Whether this level of potency is achievable for BCL6 remains an open question.