The binding studies and the four structures suggest that a flexible amine linkage on the library fragment side of the tether (as in the MA2 and DA analogues) negates binding of the library fragment irregardless of whether an amine or oxime linkage is present on the uracil side of the tether. Conversely, introducing an amine linkage on the uracil side of the tether (i.e. MA1) enhances binding only when an oxime linkage is present on the library side, and cannot rescue the binding deficit brought about by an amine linkage on the library side of the tether. These intriguing positional effects of the oxime and amine linkages provide unambiguous evidence that the tether is not a passive medium for presenting the binding fragments.
The enzyme-ligand system used here and the extensive thermodynamic and structural data provide a favorable opportunity to dissect the energetic contributions of the linker in a fragment-based ligand. First, the linkers do not interact directly with the target, and thus, any observed differences in binding affinities cannot be trivially attributed to such interactions. Importantly, the linkers derived from the various combinations of amine and oxime linkages are each capable of presenting the binding fragments to their respective sites (), requiring that any observed differences in the binding affinities must be related to the conformational preferences of the given linker, its internal flexibility or linker strain. In addition, these linkers are solvent exposed in the bound state which makes it unlikely that observed differences in binding affinity might be derived from differences in solvation of the various linker forms upon binding. However, despite solvent exposure of the linkers in the free and bound states, it should be noted that the amine linkages are protonated under the conditions of the binding reactions based on the pKa
= 9.8 for N
-methyl-benzyl amine 17
, whereas the oxime linkages are neutral. The electrostatic differences between these linkages might give rise to differential effects on binding, but this possibility is unlikely because (i
) the binding affinities of the individual fragments are similar regardless of whether an amine or oxime linkage is employed (see Results), and (ii
) there is no correlation between binding affinity of the linked fragments and whether an amine or oxime linkage is present on the uracil side of the linker (Figs. and ). Taken together, these results indicate that the linker effects arise from how well the linkers present the two fragments to their sites and not electrostatic effects. Finally, the effects of introducing flexible or rigid linkages on each side of the linker can be explored in a combinatorial fashion. This aspect is particularly informative because position dependent effects of the two linkage types, and their energetic communication across the tether, provide an opportunity to deconvolute connectivity effects and understand how linkers with greater internal flexibility influence binding of each individual fragment.
Figure 4 Free energy changes (ΔΔG) arising from switching between flexible amine and rigid oxime linkages that connect the uracil and benzoic acid (30) binding fragments. Difference free energies are in kcal/mol relative to the DA (27) compound. (more ...)
The differences in binding free energies between DA, DO, MA2 and MA1 can be most reasonably attributed to two effects: linker strain and favorable freezing of bond rotations when the two amine linkages (1NH
) are switched to rigid oximes (1ox
). Using this framework of linker strain and/or entropy freezing, the free energy diagram shown in can be rationalized. Using DA as the reference state, compound MA2 is generated by the single switch 1NH
, resulting in a −0.6 kcal/mol enhancement in binding as compared to DA. This 1NH
switch does not result in binding of fragment 30
to its site, but does freeze rotation of a single bond on the uracil side of tether, negating a potentially unfavorable rotational entropy decrease of the 1NH
linkage upon DA binding. The energetic effect of the 1NH
switch is similar to estimates of −TΔSrot
in the range 0.4 to 1 kcal/mol for freezing of single rotatable bonds 18-20
. The next compound, DO, is generated by switching the second amine linkage to an oxime (2NH
), which results in binding of 30
to its site (). Thus, freezing rotations of the linker at a critical position (2NH
) has allowed realization of the free energy benefit of docking fragment 30
in its binding site (ΔΔG(2NH
) = −1.2 kcal/mol). Finally, the highest affinity ligand MA1 is generated from DA by the single switch 2NH
, which also results in binding of 30
to its site, but with a free energy change that is −2.0 kcal/mol more negative than DO. One potential basis for the enhanced binding of MA1 is that the 1NH
linkage relieves the linker strain present in DO (). However, even though DO and MA1 position the uracil and 30
fragments in an indistinguishable way, it cannot be excluded that part of the enhanced binding affinity of MA1 arises from better positioning of the binding fragments (). In summary, MA1 binds most tightly because the 1NH
linkage reduces linker strain leading to optimal positioning of the binding fragments, and the rigid 2ox
linkage lowers the single bond rotational entropy at the position where it can have the largest effect on the binding of fragment 30
These intriguing positional effects of the oxime and amine linkages suggest that binding of a loosely interacting fragment such as 30 will be more highly sensitive to small alterations in linker flexibility than a tighter interacting substrate fragment such as uracil. The substrate fragment has sufficient interactions with its site to remain bound even in the presence of linker strain. In contrast, the binding energy for the library fragment may not be sufficient to overcome a suboptimal linker that has excessive flexibility or strain. In this regard, the uracil fragment serves as a molecular anchor for docking fragment 30 in its site, and the flexibility and strain properties of the linker dictate how much of the binding energy of 30 can be realized.
The ligands explored here are remarkably ordinary in their molecular properties and are also drug like, suggesting that the findings may be general to fragment-based ligand design. The most sobering conclusion is that it is impossible to predict, even with high-resolution structures in hand, how a linker will affect binding of two fragments. Thus, hidden strain and other energetic penalties can only be discovered by iterative optimization and binding measurements. Many fragment based ligands are suboptimal binders because they do not even reach the expectation of additive binding energies of the fragment pieces, let alone the additional expected entropic benefit arising from binding a single tethered molecule as opposed to two fragments see (see examples in references)4, 21
. Just one notable example is the widely used immunosuppressant drug FK506 which binds about 3 kcal/mol less tightly than expected from simply summing the binding free energies of its component fragments 22
. Although this binding deficit with FK506 cannot necessarily be attributed to the linker without further experimentation, it is clear that even extremely useful fragment based drugs have not reached the theoretical potencies that would be expected, exemplifying the potential for further improvement.
It has been noted that some high-affinity enzyme inhibitors that were not discovered by fragment tethering cannot be parsed into their component fragments that recapitulate the binding modes of the parent linked compound 23
. This is strong evidence for nonadditive (cooperative) binding effects in the linked compound that must be in place before one or both fragments can bind to their sites. The significant implication is that such high affinity ligands would be missed in a fragment-based discovery approach because neither fragment has sufficient binding energy for its site to overcome the large rotational and translational entropy losses that occur upon binding of a small molecule to a protein 24, 25
. In contrast, substrate fragment tethering has the potential to detect such weak binding fragments because screening is performed while library fragments are already linked to the substrate fragment anchor. Thus, an expanded region of chemical space is explored as compared to screening individual fragments. Nevertheless, the current results also demonstrate how the internal entropy or strain of a tether can also be sufficient to completely negate binding of a tethered fragment that is capable of providing significant binding energy if it were presented properly.