Fragments vs procaspase-6: preventing activation

The caspases are involved in a plethora of cellular processes and have been targeted by many groups using a variety of methods. They are cysteine proteases that cleave substrates immediately after an aspartic acid residue – and they pose several challenges for drug hunters. Because of their active site cysteine, they are particularly susceptible to non-specific inhibitors such as PAINs. Another difficulty is that their predilection for negatively charged substrates (such as aspartate) complicates efforts to identify cell-permeable inhibitors. To address both these issues, Jeremy Murray and colleagues at Genentech collaborated with Adam Renslo and colleagues at UCSF to avoid the active site altogether. They’ve recently published this work in ChemMedChem.

Caspases are initially translated as inactive zymogens that form dimers. These are cleaved to form a dimer of dimers, and this complex works as the active protease. The researchers wanted to see if they could find molecules that bind to the zymogen dimer interface and so block activation.

They started by performing a surface plasmon resonance (SPR) screen using 2300 fragments against both the active and inactive (procaspase) forms of the protein. Initial screening was done at a relatively low concentration of fragment (50 µM), with full dose-response assays being run on hits from either assay. This resulted in 84 hits against procaspase-6, with dissociation constants from 3 µM to 2300 µM, most of which were selective for the inactive zymogen.

Crystallography revealed that several of the fragments were in fact binding to a pocket at the dimer interface. In particular, fragments 1 and 6 bound (separately) at partially overlapping sites, suggesting the possibility of merging. This led to compound 8, with an improvement in affinity, albeit at a cost to ligand efficiency. However, modeling suggested that the bound conformation of this molecule would be strained, a situation which could be rectified by moving the position of the nitrogen atoms in the pyrimidine ring. Gratifyingly, this led to nearly a 10-fold boost in potency (compound 11), and further tweaking improved the dissociation constant to sub-micromolar (compound 12).

At the same time, the researchers noticed that the dimer interface is symmetrical, and that compound 6 binds near the center of the symmetry axis. This led them to design symmetrical compound 14, which displayed a modest improvement in potency but at a cost to ligand efficiency. Again modeling came to the rescue, this time suggesting a larger central element to yield sub-micromolar binders such as compound 16.

Whether or not targeting procaspase-6 will be useful therapeutically, this paper is a nice example of fragment merging against an unconventional binding pocket. It is also an excellent example of cooperation on multiple levels: first between biologists, biophysicists, chemists, and modelers, and second between academia and industry.