Do covalent fragments need to be larger?

A few months ago we highlighted work out of AstraZeneca detailing how to build a covalent fragment library. One of the design features was including larger molecules beyond the traditional rule of three (Ro3) criteria. A new open-access paper in J. Med. Chem. by György Keserű and collaborators at the HUN-REN- Research Centre for Natural Sciences and the Weizmann Institute of Science explores “size-dependent target engagement of covalent probes.”

 

The paper starts with a theoretical discussion of covalent inhibitors, focusing on the classic two-step mechanism in which binding of a ligand to a protein is followed by covalent bond formation. These steps are characterized by the inhibition constant (K_I) and the inactivation rate constant (k_inact). The most appropriate way to assess an irreversible covalent inhibitor is by the ratio k_inact/K_I, as we discussed last year.

 

A two-step mechanism is not the only possibility: the researchers also consider a three-step model in which binding of the ligand is followed by a second step, deprotonation of the amino acid nucleophile, before the final bond-forming step.

 

Fragments typically have lower affinities than lead-size or drug-size molecules, and thus k_inact will usually need to be higher for smaller molecules in order to see significant protein labeling. Simulations in which K_I is held constant show that at the high micromolar affinities often seen for fragments, protein modification requires either long incubation times or high reactivities. In addition to these simulations, the researchers also reanalyze publications we’ve previously covered such as this and this to argue that “reactivity contributes to labeling when the effects of other factors cancel out.”

 

Next, the researchers examine the kinase BTK and the oncology target KRAS, both of which have been successfully drugged with covalent molecules, ibrutinib and adagrasib, respectively. They trimmed back these molecules to smaller lead-like and fragment-like molecules and found that while some lead-sized molecules could still label the proteins, this was not the case for the fragment-sized molecules. From this they conclude that “fragment-sized covalent agents do not offer smooth optimization and are not ideal starting points.”

 

Two examples do not a trend make, but the researchers point to other examples in the literature. In 2020 we noted the larger size of covalent KRAS hits, and Vividion’s WRN inhibitor also started from a molecule with a molecular weight of 337 Da, while GSK’s starting point weighs in at 312 Da. The AstraZeneca library we mentioned at the start of this post yielded a hit against BFL1 that also just missed the Ro3 cutoff, coming in at 302 Da.

 

That said, there are counterexamples. Just last month we highlighted a covalent fragment hit that fits comfortably within the rule of three. Fragment-sized covalent hits can be found, but don’t expect them to be common. The alternative approach, screening lead-like compounds, will also likely require screening more compounds due to lower coverage of chemical space. Either way, libraries containing more molecules are likely to be beneficial for finding covalent starting points.