Which cryptic sites are ligandable, and why?
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.