Ligands targeting PAR2 receptors: state of the art

The PAR2 receptor is known to be involved in several physiological and pathological processes and different ligands targeting this receptor have been synthesized and proposed since its discovery and cloning in 1994;⁷ however, no PAR2 modulators have yet entered the clinical trials.¹

The first molecules proposed were based on the structure of the tethered ligand shown in the previous paragraph. Hence, in 1996 different short peptides were synthesized and, among these, only the sequence SLIGRL-NH217 resulted more potent than the native sequence SLIGKV-NH2.¹⁸ Unfortunately, this peptide could be used only as a tool for biological investigations due to its poor potency (it is active at micromolar concentration). Later, in 2004 Hollenberg and colleagues synthesized a library of more potent and stable peptides modified at the N-terminus,¹⁹ among these the 2-furoyl-LIGRLO-NH2 ligand 1 (Figure 4), showing the best potency in the series, became a pioneering candidate in the search of PAR2 binders.

Figure 4. Structure of the sequence 2-furoyl-LIGRLO-NH2 (1) and other PAR2 agonists (I). Adapted from Ref. 20.

Going further on this direction, other research groups proposed new short peptide sequences of type I as PAR2 agonists (Figure 5) with EC50 in the 0.1-0.8 mM range (measured by the concentration

72 of intracellular calcium released in various cell lines), confirming that a terminal heterocyclic ring is beneficial for the agonist activity.^21,20 These results taken together evidenced that agonist activity is positively affected by a C-terminal amide group and a heterocyclic ring linked at the N-terminus, thus opening the way to the development of peptidomimetic PAR2 ligands.

Therefore, the research moved toward the synthesis of small molecules and peptidomimetics having better stability and pharmacokinetic properties. In 2010, Barry et al. described an important SAR study²² that resulted in the discovery of the novel agonist GB110 (2, Figure 5, EC50 0.28 μM, iCa²⁺ release) and the antagonist GB88 (3, Figure 5, IC50 2 μM, inhibition of iCa²⁺ release). As for the previously described ligands, GB110 presents an N-terminal heterocycle and two apolar amino acid residues close to this ring, suggesting that these residues are important for the binding to the receptor. On the contrary, GB88 has a bulky residue at the C-terminus, although the rest of the structure is very similar to GB110, once more suggesting that this last portion is crucial for switching agonist/antagonist activity towards PAR2.

Relying on these studies, in 2016 Yau et al.²⁰ tried to reduce size, polar surface and bond mobility with the aim to synthesize a potent, rule-of-five compliant compound with higher potency and better stability than the previously described ones. During this study, they found the novel potent agonist AY77 (4, Figure 5, EC50 33 nM, iCa²⁺ release) and proposed a possible binding mode, where AY77 is supposed to form multiple hydrogen bonds and hydrophobic interactions with PAR2 through its terminal amide group (with Asp228 and Tyr156) and the isoxazole ring (with Tyr82) (Figure 5). The authors hypothesized that isoxazole, cyclohexylalanine and cyclohexylglycine portions should occupy three distinct pockets in the PAR2 binding site; in particular, cyclohexylglycine seems to develop specific hydrophobic interactions with Tyr326 and Leu307.

Figure 5. Structures of the PAR2 agonists 2 and 4, and the antagonist 3. The predicted binding mode for 4 in a PAR2 homology model derived from nociceptin/orphanin FQ receptor (PDB code 4EA3)²⁰ is shown at the bottom right.

One year later, the same research group synthesized the biased ligand AY117 (5, Figure 6),²³ which showed antagonist activity in calcium release induced by trypsin and 2-furoyl-LIGRLO-NH2 (IC50 2.2 and 0.7 M), but it also showed selective PAR2 agonist activity in inhibiting cAMP stimulation and in activating ERK1/2 phosphorylation. In fact, in this work the authors described the switching from agonist to antagonist properties for several low micro- to submicromolar PAR2 ligands in the iCa²⁺

73 release assays; they demonstrated that the presence of a primary amide or H-bond acceptor at the C-terminus was crucial for the agonist activity and that the switch to antagonist activity depended on the presence of H-bond acceptor units at the N-terminus.

Figure 6. Structure of the antagonist AY117 (5) and examples of compounds with antagonist/agonist activity.²³

In the field of non-peptidic small molecular ligands, only a few candidates appeared in the literature up to now. In 2008, Gardell et al. discovered the first non-peptidic small molecular PAR2 agonists AC-55541 and AC-264613 by means of a high-throughput functional screening (6 and 7, Figure 7); these compounds were able to activate PAR2 signalling in cellular proliferation, phosphatidylinositol hydrolysis and calcium mobilization assays, with potencies ranging from 200 to 1000 nM for AC-55541 (6) and 30 to 100 nM for AC-264613 (7).²⁴ Interestingly, these compounds share a completely non-peptidic scaffold, a N'-acetyl-hydrazone central core, on which two hydrophobic portions (the aromatic rings) and the H-bond donor groups (the phthalazin-1(2H)-one for AC-55541 and at the -lactam for AC-264613) are grafted.

Figure 7. On the left, structures of the antagonists AC-55541 (6), AC-264613 (7), AZ8838 (8) and AZ3451 (9). On the right, the crystal structure (PDB: 5NDD) of PAR2 in complex with AZ8838. Adapted from Ref. 25.

In 2017, Cheng et al. first reported the crystal structure of PAR2 in complex with two antagonists, AZ8838 and AZ3451 (8 and 9, Figure 7).²⁵ According to these studies, compound AZ8838 (8) binds

74 in a fully occluded pocket near the extracellular surface and exhibits slow binding kinetics, whilst compound AZ3451 (9) binds to a remote allosteric site outside the helical bundle; hence, the authors concluded that AZ3451 is not an orthosteric antagonist, as its binding to PAR2 is able to prevent the structural rearrangements required for receptor activation and signalling. This important study allowed to expand considerably the knowledge about PAR2 and prompted the development of new PAR2 ligands.

Along this line, Gmeiner et al. recently published a SAR study concerning a series of peptidomimetic PAR2 agonists.²⁶ The authors planned to replace the cyclohexylglycine moiety within the previously described ligand 4 AY77 with benzamide-homologues to achieve a better fitting in the lipophilic pocket with respect to compound AY77, synthesizing a panel of compounds of type II (Figure 8). However, the three most potent molecules in the Gq activation (EC50 0.59-0.64 M) are not enough potent in the -arrestin recruitment (EC50 6.0-8.9 M).

Figure 8. Left, design of novel nonpeptidic PAR2 agonists. Right, overlay of the best poses for AY77 (orange) and type II ligands (green) in the PAR2 model (grey). Adapted from Ref. 26.

In this research context is inserted the project I carried out during my six-month secondment in the Prof. Dr. Gmeiner group at the University of Erlangen-Nürnberg. Considering the remarkable potential of PAR2 targeting ligands as both therapeutic agents and/or investigating probes, it is not surprising that great efforts are being addressed to the development of new PAR2 modulators;

indeed, although several ligands have been reported so far,²⁷ no drugs addressing PAR2 receptors have been approved yet.

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