Tuesday, 14 April 2009

The upper limits of binding: Part 2

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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.

Literature cited

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


Joerg Kurt Wegner said...

Nice summary for 'tight binding', thanks for sharing.

Dan Erlanson said...

Great series of posts – thanks. This is a fascinating topic, and the “practical” limit of mismeasuring tight binding seems likely to be common.

One possibility that I haven’t seen proposed is what I’ll call the “good-enough” hypothesis: once you get to low nanomolar or high picomolar affinity, is there any reason to get tighter? Most biological interactions do just fine with nanomolar, micromolar, and even millimolar binding affinities. Indeed, given the importance of reversibility in biology, one could argue that tight binding affinities (biotin aside) have been actively selected against.

Similarly, once chemists develop low nanomolar molecules they usually (and appropriately) stop worrying about improving affinity further and focus on pharmaceutical properties.

It is certainly possible to design higher affinity molecules. In addition to the famous “Clicked” acetylcholinesterase inhibitors, there is a neat older paper from Alan Kaplan and Paul Bartlett (Biochemistry 1991 8165) describing a femtomolar carboxypeptidase inhibitor. In this case it was indeed challenging to obtain quantitative data. I remember a talk Alan gave on this where he showed a kinetics time course with the x-axis measured in days or weeks; there was a discontinuity that reflected the effects of a large earthquake on the assay!