A new tool for covalent ligands: kinact/KI made easy with dDRTC

As covalent drug discovery becomes increasingly common, researchers are becoming more rigorous in how they characterize their molecules. The simple IC₅₀ values used for reversible inhibitors are meaningless for irreversible ligands unless the incubation times are also disclosed. And as molecules become more potent during the course of optimization, the incubation time may need to be shortened. An early hit might require treatment overnight to give 50% protein modification, while a potent lead might completely modify the protein in seconds. How do you quantitatively compare these?

 

The most rigorous parameter to characterize irreversible ligands is k_inact/K_I, sometimes called covalent efficiency, which we recently discussed here and here. Unfortunately, determining k_inact/K_I is a pain: it requires running multiple dose-response studies at multiple time points, and is thus typically only done for key compounds. In a new (open-access) Nat. Commun. paper, Robert Everley and colleagues at Frontier Medicines (including yours truly) provide a shortcut.

 

The new method relies on the fact that, especially for low-affinity fragments, much of the data collected in a conventional dose-response time course (DRTC) is redundant, providing little additional value. For example, if a compound at one concentration gives virtually no modification after 8 hours, it also won’t modify after 1 hour. The trick is to collect just the most informative data in a “diagonal” dose-response time course, or dDRTC.

 

I won’t go into the mathematics and full implementation details since the paper is open-access, but suffice it to say that dDRTC lowers the number of required data points by a factor of eight, thus saving both time and reagents – including precious protein.

 

The paper appropriately notes limitations, such as the fact that for compounds with better affinities (K_I < 50 µM), the values derived from dDRTC can underestimate the true k_inact/K_I. However, this situation is uncommon for fragments, and indeed the potencies for even some clinical compounds such as sotorasib and VVD-133214 are largely driven by (specific) k_inact rather than K_I. The paper shows good agreement for k_inact/K_I values determined using dDRTC with those determined using the conventional approach for compounds having k_inact/K_I from 1 to 2000 M^-1s^-1.

 

Perhaps most relevant for this blog, dDRTC is a practical solution for collecting important data. The next time you’re running a covalent program, give it a try!