Taking Advantage Of Structural Understanding Of Allosteric Binding Sites

The sections above have discussed methods for screening allosteric modulators using wild-fype receptors. However, one of the attributes of allosteric binding sites is that they are topographically distinct from orthosteric sites. The distinction between allosteric and orthosteric sites is obvious for some GPCRs, such as for mGluRs and the GABAb receptor, where the orthosteric site is formed by the large N-terminal domain and allosteric sites have been identified in the transmembrane bundle [3]. For other GPCRs, such as musca-rinic acetylcholine receptors, allosteric binding sites at the top of the transmembrane domain and in the extracellular loops (ECLs) [39] are much closer to the orthosteric binding site, which is found slightly deeper in the transmembrane bundle.

These structural differences have enabled the use of truncated, chimeric, or mutated GPCRs to aid in the identification of allosteric ligands. The mGluRs are an excellent example in this regard. Positive [40, 41] . negative -36] . and neutral [42] allosteric modulators have been identified for receptors within this family and have been shown to interact with multiple binding sites in the transmembrane domain [24, 40, 43]. DFB is a positive allosteric modulator of mGluR5, which shows no intrinsic allosteric agonism. However, in a mGluR5 construct in which the N[[erminal glutamate-binding domain was removed, DFB was shown to act as a full agonist [43]. In a similar fashion, the negative allosteric modulator, MPEP, appeared as an inverse agonist at the N-terminally truncated receptor. Such effects of allosteric modulators have also been demonstrated in N-terminal truncates of GABAb and calcium-sensing receptors [44, 45]. The ability of the transmembrane domain of mGluRs to activate cellular effector systems can be exploited at a screening level; it is easy to imagine that it might be considered attractive to express an ^terminally truncated Family C receptor and perform a screen to detect agonists, rather than run a more complex screen for positive modulators at the wild- type receptor.

A similar example of exploiting the structural basis of allosteric binding sites comes from the muscarinic M[ receptor. The orthosteric binding site across muscarinic acetylcholine receptors shows a high degree of conservation, and as such, subtype- selective agonists for this family have proven hard to identify [46]. Recently, several agonists have been discovered that display unprecedented levels of selectivity for the muscarinic M[ subtype [47-49]. AC-42 exemplifies this class of molecule, and studies with chimeric M[-M5 muscarinic receptors suggested that the N-terminus/TMl and ECL3/TM7 regions are key to the selective activity of AC-42 at the M1 receptor [47]. This region is clearly distinct from the orthosteric binding site, which is formed by residues in TM3 and TM6 [ 50] . One of the key residues in the orthosteric binding site is Tyr381 within TM6. Mutation of this residue to Ala greatly reduces the affinity and potency of orthosteric agonists, such as acetylcholine and carbachol [47, 51]. However, this mutation leaves the activity of agonists, such as AC-42, relatively unaffected [47] and even potentiates the activity of others [49] [ Thus, activity at the Tyr381Ala mutation is a useful marker of atypical agonism, which may prove to be allosteric and/or selective. The examples given above are somewhat specific to the receptors involved. However, the increasing understanding of the structural basis of allosteric regulation will potentially increase the number of surrogate assays that can be performed to identify allosteric ligands.

0 0

Post a comment