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Mel reviewed an important article on maximal affinity of ligands to kick off our sequence of posts on ligand efficiency. There are a number of reasons that this upper limit for potency might be observed and it's worth having a bit of think about them.
One interpretation of the upper limit is that it represents a validation of the molecular complexity concept. If a ligand makes many interactions with the protein they are less likely to be of ideal geometry. Hydrogen bonds between the binding partners and water are more likely to be of near-ideal geometry. Another factor that can impose limits on affinity is the finite size of a binding site. Once the site has been filled, increasing the size of the ligand does not lead to further increases in affinity because all the binding potential of the protein has already been exploited.
However, there is another reason that an upper limit for affinity might be observed and it has nothing to do with molecular complexity or fully exploited binding sites. Measuring very strong binding is not as easy as you might think it would be. In a conventional enzyme assay, you normally assume that the concentration of the ligand is much greater than that of the enzyme. This works well if you’ve got 10nM enzyme in the asaay and a micromolar ligand. However, things will get trickier if you’re trying to characterise a 10pM inhibitor since you’ll observe 50% inhibition of the enzyme for a 5nM concentration of the inhibitor. And you’ll see something very similar for a 1pM inhibitor…
This behaviour is well known and is called tight-binding inhibition. If you want to characterise very potent inhibitors you need reduce the concentration of the enzyme and be a bit more careful with the math. However, not everybody does this and I suspect that this may be one reason there appears to be an upper limit for affinity.
Kuntz et al, The maximal affinity of ligands. PNAS 1999, 96, 9997-10002. Link to free article.
Williams & Morrison, The kinetics of reversible tight-binding inhibition. Methods Enyzmol. 1979, 63, 437-467 DOI