Fragment merging – and flipping – on the leucine zipper of MITF
Transcription factors can be
difficult drug targets, particularly those whose primary structure is a “leucine
zipper” in which two α-helices
gently coil around each other. Their three-dimensional structure provides few
pockets suitable for binding small molecules. In a new (open-access) paper in Nat.
Commun., Deborah Castelletti, Wolfgang Jahnke, and a large group of
multinational collaborators at Novartis and elsewhere present progress toward
one of these, microphthalmia-associated transcription factor (MITF), which has
been implicated in melanoma.
Most of MITF is believed to be
disordered, but the DNA-binding domain (DBD) homodimerizes as a basic
helix-loop-helix leucine zipper. Unlike related transcription factors, the
helices in MITF contain a small kink that keeps them from heterodimerizing and
also creates a small “kink pocket.”
The researchers expressed the DNA-binding domain of MITF and screened it using 19F NMR against the
LEF4000 library, which we described here. This yielded just 9 hits that
confirmed in protein-observed NMR, a hit rate the researchers note “is amongst
the lowest that we have observed across multiple FBS campaigns,” consistent with expectations for a difficult target. Two chemical series,
represented by compounds 1 and 2, were prioritized, and analogs from the
Novartis compound collection were screened to find more-potent compounds 3 and
4.
Crystallography revealed that
compounds 3 and 4 both bound in the kink pocket. Excitingly, the binding modes
are similar and overlapping, inviting fragment merging. This proved successful,
yielding a compound that bound 100-fold more tightly than either fragment. Further
optimization ultimately led to compounds 7 and 8, with low or sub-micromolar
affinity as assessed by isothermal titration calorimetry (ITC).
The bound structures of compounds
7 and 8 were determined by crystallography. Compound 7 (gray, left)
superimposes nicely onto compounds 3 (cyan) and 4 (magenta), showing successful
fragment merging. Compound 8 (green, right), however, is flipped 180 degrees compared to
compound 7, despite having similar structure and affinity. Although surprising, this is not too uncommon; we’ve written about previous flippers here, here, and
here.
The MITF homodimer is asymmetric,
with one helix kinked and the other straight. NMR experiments and
molecular dynamics show that both compounds 7 and 8 slow the interconversion
between kinked and straight forms, though it is unclear whether this has
functional implications. The compounds do not seem to affect DNA binding, and with
at best high nanomolar affinity towards MITF no cell data are reported with the
molecules.
Nonetheless, the successful
identification of ligands against a leucine zipper is exciting. The binding
pocket is small; as shown in the figure above, the best compounds already stick
out on either side of the helices. Further affinity improvements may be
difficult, though perhaps covalent approaches could help. Alternatively,
perhaps these molecules could be starting points for induced proximity strategies such as PROTACs. It will be fun to watch this story develop.