Many interesting proteins have
flat, featureless surfaces, lacking the deep pockets in which small molecules
usually bind. But structures can be deceptive: crevasses can open unexpectedly,
revealing “cryptic sites” for ligands. Or not – just because a site is
available does not mean it is ligandable (able to bind to ligands with high
affinity). A new (open access) paper in Drug Disc. Today by Sandor Vajda and collaborators
at Boston University and Stony Brook University asks “which cryptic sites are
feasible for drug targets?” (Sandor presented some of this at FBLD 2024 last
month.)
To get started, the researchers
turned to the aptly named CryptoSite, a previously published list of 93 proteins
where unexpected pockets had been found. Each protein has at least two published
crystal structures, one in the apo form and one with a ligand bound to the (no
longer) cryptic pocket. Cryptic sites form primarily through two mechanisms. In
the first, amino acid side chains move aside, opening a pocket. In the second,
larger motions occur in protein loops or secondary structural elements, such as
alpha helices, creating pockets.
Of the 18 cases for which cryptic
sites formed primarily through the movement of side chains, ten had published affinities
for the ligands, and all of these were weak, with the best being low micromolar.
In contrast, of the 27 cryptic sites created by loop movements for which
affinity information was available, all but two were nanomolar binders. From
this evidence, the researchers suggest that cryptic sites formed only by the
motion of side chains are not sufficient to support high affinity ligands. Why?
The researchers note that side
chain motions occur very rapidly, on a timescale of 10-11 to 10-10
seconds, much faster than ligand binding, which at its fastest is 10-8
seconds. Thus, “a fast-moving side chain that spends a substantial fraction of
time in the pocket interacting with the other residues competes with ligands
for binding and, hence, acts as a competitive inhibitor.” This intuitive
picture is supported in the paper by mathematical simulations.
In contrast, loop movements occur
on 10-9 to 10-6 second timescales, while the movements of
secondary structure elements are even slower. Thus, a ligand could bind while
the cryptic site is open, and, like a wrench in a machine, keep it open.
This finding is important. As
the researchers point out, the molecular dynamics calculations frequently used
to find cryptic pockets are typically run at short timescales likely to miss
loop movements. Other computational methods used to assess ligandability may also
suffer; the researchers note that their program FTMap, which we’ve written
about here and here, overestimates the ligandability of cryptic sites created
by side chain movements.
Of course, just because a cryptic
site is created by loop movements does not mean it is ligandable, as we
discussed for interleukin-1β. And the researchers acknowledge that covalent inhibitors
might be able to take advantage of less traditionally ligandable sites, cryptic
or otherwise. Certainly this has been the case for KRAS. I’m confident that
many more examples will be forthcoming.
Posts
6 comments:
Hi Dan, the article isn’t actually open access so my comments are based on your post and the authors may have addressed my points. My understanding is that it generally costs energy to open a cryptic site and this energy penalty is effectively a tax on ligand binding which needs to be taken account of when assessing ligandability. Invoking timescales for conformational interconversion to explain affinity looks flaky from the physicochemical perspective and may even be straying into the realm of Maxwell’s Demon.
Hi Pete, sorry about the open-access, it seemed to be when I downloaded it last week.
Your tax metaphor is interesting, but how would you account for the observed differences between side-chain motions vs loop motions, blood demon arts aside?
No worries, Dan, and I've been caught out like this on more than one occasion (I think some journals make their articles accessible for a few days after publication before placing them behind the paywall). I consider a causal relationship between timescale for motion and affinity to be improbable because it would lack a physical basis (an alternative explanation is that the timescale for motion serves as as a 'latent' indicator variable that encodes whether side-chains or loops are moving). On an unrelated note, best wishes for a good result in the election tomorrow.
Dear Peter,
Probably I am too illiterate in the topic, but why does it lack a physical basis? If one simplify things and considers collision theory, the timeframe the interaction-competent conformation exists should be related to the probability of a productive collision. Of course, the relationship and the model are not as simple. However, one might expect an effect on the association rate constants, and at least on the observable association rates. By no means it excludes energy penalpties and thermodynamics.
I think the discussion on kinetics in the paper does muddy the waters. They say they start from the observation that side-chains change conformation with rate constants in 10e-10s range or faster.
However, the argument they then go on to make is a thermodynamic one and comes down to "if the side chain spends 1000x more in the open state than the closed state, the observed affinity will be lower than if it were 50:50". But this is pretty much Peter's point anyway - if it's 1000x more in the closed state that suggests that the closed state is more stable (by -RT ln(1000)), and that's the energy tax Peter mentions.
I think the whole discussion around kinetics is unhelpful here, it's just a bunch of chained equilibria, the actual implication is that all the high-affinity ligands (where the protein has undergone significant motion) are paying a lower "tax" than the simple side-chain rearrangement. This is still very interesting though - why are large movements cheaper? My guess is that it's some kind of selection bias in how we classify these things - that for some reason we don't "see" the high-cost larger rearrangements (or we ignore the low-cost side-chain movements?). If it *isn't* a selection bias, then we need to invoke something along the lines of how we're looking at evolved mechanisms which carefully balance the open/closed states, and that these always involve multiple residues??
Many scientist involved in drug discovery tend to forget that the binding constant is a ratio, i.e., Kb = kon/koff, and thus the thermodynamics and kinetics are inherently connected. The off rate control residence time, is determined by the atomic interactions in the ligand-bound state, and is the prime focus in drug discovery. The on-rate, while also depends on long-range forces such as electrostatics, is primarily determined by the availability of receptor conformation available for productive binding. Papers in the field discuss ether the thermodynamics or the kinetics of binding, and I had difficulty finding papers that connect the two. Please let me know if you can suggest some.
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