Ligand reactivity efficiency (LRE)

As covalent drug discovery continues to rise, the demand for metrics to help guide lead optimization is increasing. Last year we discussed covalent ligand efficiency (CLE). In an open-access paper just published in J. Med. Chem., Benjamin Horning, Brian Cook, and colleagues at Vividion Therapeutics describe ligand reactivity efficiency (LRE). (Benjamin presented LRE at the DDC meeting in 2024.)

 

A key challenge when developing covalent ligands is maximizing specific reactivity towards the target of interest while minimizing intrinsic reactivity towards other proteins; the two types of reactivity are not the same, as we wrote about last year. For molecules that target cysteine residues, intrinsic reactivity is usually determined by assessing reactivity against the small molecule glutathione, which is abundant in cells.

 

For lead optimization more generally, a common metric is lipophilic efficiency (LLE or LipE, see here and here), in which the logP of a molecule is subtracted from the negative log of the IC₅₀ (pIC₅₀). More lipophilic molecules have higher logP values, so maximizing LLE helps to minimize increases in lipophilicity.

 

By analogy, the researchers defined LRE to help minimize increases in intrinsic reactivity. However, distinguishing specific from intrinsic activity is not necessarily straightforward. As we previously discussed, IC₅₀ alone is an inappropriate measurement for covalent inhibitors; the incubation time before the IC₅₀ is measured is an essential variable. The most rigorous value is k_inact/K_I, and although this ratio has been historically time-consuming to determine, we described an easier method earlier this year. Yet an even simpler measurement is the TE_{50(target, 1h)}, the concentration of compound necessary to label 50% of a target after one hour, which is a function of k_inact/K_I. The researchers thus defined LRE as:

 

    LRE = pTE_{50(target, 1h)} – pTE_{50(GSH, 1h)}

 

The variable in the second term, pTE_{50(GSH, 1h)}, is calculated from the reaction rate of the ligand with glutathione; intrinsically reactive ligands have higher rates.

 

In the case of LLE, values above 5 or 6 are generally considered acceptable for advanced leads, and the same is true for LRE. For example, a molecule with TE_{50(target, 1h)} = 10 nM and a (low) GSH reactivity of 0.01 M^-1s^-1 would have an LRE = 6.3. Also analogous to LLE, one can generate plots with pTE_{50(GSH, 1h)} on the x-axis and pTE_{50(target, 1h)} on the y-axis to assess whether LRE values are improving during a lead optimization campaign.

 

In my view, LRE is superior to previously discussed CLE because it explicitly considers the time component. A one hour incubation is practical; a ligand with k_inact/K_I = 10,000 M^-1s^-1 would have TE_{50(target, 1h)} = 19 nM. Also, LRE is more intuitive for medicinal chemists than CLE due to its similarity to LLE.

 

On the minus side, the researchers note that some of the assumptions break down for ligands with high non-covalent affinity (low K_I). Also, some folks may take issue with metrics that take the logarithm of a measurement that has units.

 

The researchers note another alternative metric, the reactivity enhancement factor (REF), which I briefly discussed here. REF is simply the ratio of the specific reactivity to the intrinsic reactivity, which is conceptually simpler to me than LRE. Nonetheless, the researchers state that LRE is commonly used at Vividion, which has put several covalent drugs into the clinic, so clearly it can be useful. Whether REF, LRE, or CLE, ultimately the choice of metric is less important than the ultimate goal: maximizing specific reactivity while minimizing intrinsic reactivity.