An early example of fragment linking was reported by Abbott
researchers in 1997: two fragments that bound to the matrix metalloproteinase
stromelysin were linked together to give a molecule that bound about 14-fold
more tightly than the product of the affinities of the two fragments.
Thermodynamic analyses were conducted to explore the roles of entropy and
enthalpy, but these were complicated by the fact that one of the fragments
contained an acidic phenol that was removed in the course of linking. In a new paper published in Bioorg. Med. Chem.
Lett., Eric Toone and colleagues at Duke University
have re-examined this system.
The researchers dissected several of the originally reported
linked molecules into component fragments and examined their thermodynamics of
binding using isothermal titration calorimetry. All of the experiments produced
similar results; a particularly illustrative example is shown in the figure, in
which a single bond in compound 1 was conceptually broken to yield component
fragments 5 and 8.
As the researchers note, weirdly, the “favorable additivity
in ligand binding – that is a free energy of binding greater than the sum of
those for the constituent ligand fragments – is enthalpic in origin,” not entropic. It is not clear why this is the
case, but what is clear is that the
results are completely different from those obtained by Claudio Luchinat and
colleagues on another matrix metalloproteinase. In that report, the enhanced
affinity of the linked molecule was entirely entropic in origin, as might be
expected. So what’s going on here?
One clue is provided by Fesik and colleagues in their
original analysis of their stromelysin inhibitors. They noted that, when
fragment 8 (acetohydroxamic acid) was added to the protein, biphenyl ligands
similar to fragment 5 bound considerably more tightly than when fragment 8 was
not present. In other words, the ligands displayed cooperative binding even
when they were not covalently linked, probably due to non-covalent interactions between the two bound ligands or possibly to changes in protein
structure and dynamics.
It is easy to assume that two ligands bind independently to
two sites on a rigid protein, when in fact proteins are anything but rigid, and
the addition of one ligand to a protein can dramatically change its properties.
Thermodynamics measures changes in the entire system, not just the ligands, and
if the protein changes upon ligand binding things can quickly get complicated.
As Fesik and coworkers noted:
The observed
cooperativity between the two ligands is a factor that should be considered
when optimizing compounds for binding to nearby sites, since a portion of the
binding energy is due to the cooperativity rather than interactions between the
ligands and the protein.
All of which is to say that we remain woefully ignorant of
the forces driving ligand binding, let alone fragment linking. But assessing
how much better (or worse) a linked molecule binds than its component fragments
can still be a useful exercise to guide optimization, even if the thermodynamic
origins of the effects are unclear.
1 comment:
I'd be thinking about conformational strain (which is likely to be 'seen' as an enthalpic effect) as a rationale for these results. Be warned that I've not actually looked at any crystal structures and not really read the paper properly.
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