The discovery of cyclic γ-AApeptides as the promising ligands targeting EP2.

In our previous work, linear OBOC library was established based on γ-AA peptides, from which inhibitors towards Aβ aggregation and STAT3-DNA interaction were found through screening 17 . Thereafter, the cyclic γ-AApeptide libraries with amplified conformational rigidity were designed and screened against numerous targets to discover novel enzyme inhibitors and receptor antagonists 20 , 24 – 27 . The parallel synthesis of the encoded sequence and the targeting compound was achieved on the TentaGel beads with each bead displaying a cyclic γ-AApeptide on the surface. Each γ-AA building block ( Figure 2B ) used in this library has one chiral side chain originally, and the other side chain was protected with Alloc protecting group which was replaced by eight different carboxylic acid or acyl chlorides later 19 , leading to the remarkable diversity of this One-bead Two-compounds library. To successfully prepare the designed library, the TentaGel microbeads were first soaked with water thoroughly ( Figure S1 ). After gently rinsing the outer surface of beads with DCM/ether to remove the water, Boc protecting group was introduced. Then the methionine amino acid was introduced to the inner surface promoting the coding-sequences release for MS analysis upon CNBr treatment. The coding sequences consist of eight Dde-protected amino acid and each chiral side chain of γ-AA building block added to the outer surface was represented by one coding amino acid. Then those beads were split to 8 portions followed by another Dde-protected amino acid added while the other achiral side chain of γ-AA building block were attached to the beads. Subsequent repeats of the previous procedures of splitting and pooling led to the desired library. In the end, the sequences on the beads were cyclized utilizing the thiol-ether bridging moiety. The combination of different positive/negative charged, and hydrophobic side chains made the library with a diversity of more than 300,000 sequences. The detailed synthesis protocol is included the Supporting Information . The quality of designed OBTC library was confirmed by randomly picking 10 beads and cleaving decoding sequence for analysis by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) MS. Seven out of ten coding sequences could be analyzed illustrating the high quality of the library synthesis. The library beads were firstly equilibrated in Tris buffer overnight followed by incubation with protein target. Then His-tag antibody Fluor 488 was treated to the beads which could emit strong green fluorescence when the cyclic peptides showed great binding with PTGER2 protein ( Figure 3 ). Eighteen beads were picked up under microscope and the corresponding coding sequences were released by CNBr cleavage and analyzed by MS/MS of MALDI. The exact structure of the eighteen cyclic γ-AApeptides were then figured out and target cyclic peptides with fluorescein isothiocyanate (FITC) labelled were resynthesized. The binding affinity between PTGER2 and FITC labelled cyclic γ- AA peptides were measured through fluorescence polarization (FP) assay 28 , 29 . Briefly, a constant amount of 50 nM FITC-labeled cyclic peptide was incubated with a serial dilution of the PTGER2 protein in 96 well plate. The K d values were calculated using the following equation and the L st is the concentration of the AApeptide while the x is the concentration of the protein. Among all the found structures, several hits can moderately bind to target protein and two hits showed excellent binding affinity ( Figure 4A , C ). The measured K d for FITC-labelled EP2–6 was 0.016 μM and FITC-labelled EP2–14 was as low as 0.013 μM ( Figure 4B , D ). Therefore, the EP2–6 and EP2–14 were selected as the most promising EP2 ligands, and the binding specificity was further confirmed by Confocal Laser Scanning Microscopy. After incubation of FITC-labelled EP2–14 with cultured mammalian C6 cell line, the green florescence could be observed sporadically on the surface of cells that do not overexpress EP2 ( Figure 5A – C ), whereas evenly diffused fluorescence was noticed upon incubation of EP2–14 with the EP2-overexpressing cell line, which may imply that EP2–14 binding specifically on the EP2. Although further demonstration is needed, the preliminary data suggests that EP2–14 maybe promising for cell-based studies to interrogate the function of EP2. The PGE2 signaling has been known as a significant anti-inflammatory target since 2011 and scientists have been dedicated to optimizing inhibitor structures or developing new generation of drugs in the past decades 30 . Selective inhibition of EP receptor subtype appears to be valuable drug candidates in treating many pathophysiological states, for instance, alleviating inflammatory processes in arthritis, endometriosis, and different status epilepticus models. 31 Moreover, PGE2-associated tumorigenesis, proliferation, and metastasis in colorectal cancers 32 as well as brain inflammation in neurodegenerative diseases could all be benefit from selective EP2 inhibition 33 . However, the EP2 selectivity has always been a difficult point compared with the other three subtypes. Consequently, the γ- AA peptide based macrocyclic peptidomimetic screening provides us with the new scaffold which could potentially display excellent binding ability toward EP2. The diversity of combinatorial library could be remarkably enhanced by increasing more side chains during the synthesis while the screening of one single target remains simple. In addition, the unique properties of cyclic γ-AApeptide endowed the ligand with strengthened conformational rigidity and proteolytic resistance which contribute to the investigation of more potentially bio-active ligands. The two active hits identified here, EP2–6 and EP2–14, showed similar binding affinity with some previous reported antagonist such as PF-04418948 and TG4–155, demonstrating their potential for further development and future applications. Other than EP2–6 and EP2–14, those hits with moderate binding affinity could also exhibit some pharmacological activities via acting on the PGE2/EP2 signaling, which should also be evaluated in the future. In summary, the parallel synthesized One-bead Two-compound peptidomimetic library is a systematic, efficient, and easily adjusted strategy for developing bio-active binding molecules persistently.

As bioactive lipids commonly found in various tissues and cells, prostanoids are known to bind to their receptors on the cell surface and modulate inflammation and immune responses. Two prostaglandin-endoperoxide synthases, cyclooxygenase 1 (COX-1) and cyclooxygenase 2 (COX-2), are responsible for the conversion of prostanoids from arachidonic acids that are liberated from the cell membrane. It is currently known that different prostanoids ( Figure 1 ), including prostaglandin D2 (PGD2), prostaglandin E2 (PGE2), prostaglandin F2α (PGF2α), prostacyclin and prostaglandin I2 (PGI2), and thromboxane A2 (TXA2), are generated from the temporary intermediate PGH2. 1 The important roles of prostaglandins in different pathological and physiological processes such as inflammation and protection of gastric mucosa 2 are achieved through acting on their cognate G protein–coupled receptors 3 . Non-steroidal anti-inflammatory drugs (NSAIDs), also known as classical cyclooxygenase (COX) inhibitors, are therapeutically used for their analgesic, anti-inflammatory, and antipyretic properties. Aspirin, the first discovered NSAID, has venerable therapeutic history of more than 100 years, 4 followed by the breakthrough development of more COX inhibitors such as ibuprofen, naproxen, celecoxib, indomethacin, and diclofenac 5 . However, classic NSAIDs non-selectively inhibit both constitutively expressed COX-1 and inducible COX-2, causing severe side effects including gastric ulcerations and platelet function disorder 6 . COX-2 selective inhibition has been proved possessing analgesic and anti-inflammatory effects but also coming with significant cardiovascular side effects with prolonged-use. Therefore, identifying new therapeutic strategies targeting the downstream receptors of COX-2 with improved drug sensitivity 7 and minimized adverse effects has attracted much attention. Among the five bioactive PGs generated by cyclooxygenases, the lipid signaling molecule prostaglandin E2 (PGE2), as a main inflammatory mediator, could increase pro-inflammatory factors such as cytokines inducing pain, fever, tumor, and anaphylactic reaction, 8 through binding with four G protein-coupled receptors (EP1, EP2, EP3, EP4). It has been reported that the EP2 (encoded by PTGER2) abnormal over-expression in breast, colon, liver, and prostate cancer is greatly associated with chronic inflammation, immunoregulation 9 , angiogenesis, metastasis, and multidrug resistance in cancer development 10 . Moreover, the deletion of EP2 receptor in AD mouse model also shows reduction of oxidative damage and amyloid-β burden. 11 Therefore, targeting the EP2 receptor directly could be more rational and promotional to find novel therapeutic agents. Natural agonist of EP2 such as derivatives of pyridyl-sulfonamide PF-04217329 was developed by Pfizer in 2011 12 for the treatment of glaucoma. A series of thiophene carboxylate compounds were developed as positive allosteric modulators for EP2 receptor by Emory University. Thereafter, numerous small-molecule ligands 13 including antagonists PF-04418948 and TG4-155 have been reported in the last decades 14 , 15 , providing more chemical tools on modulation of PGE2 signaling through the EP2 receptor. However, due to their limitations in pharmacokinetics and pharmacodynamics, there is still a need to identify novel ligands for the therapeutic potentials of targeting EP2. To this end, we planned to use the affinity-based combinatorial chemistry screening to uncover compounds with high binding affinity toward the EP2 receptor from a macrocyclic peptidomimetic library. In the combinatorial library, compounds can be screened concurrently meanwhile each compound has the equal possibility of target screening 16 . We have previously demonstrated the feasibility of making γ-AApeptide-based combinatorial library for biological applications ( Figure 2 ) 17 – 21 . γ-AApeptides have tremendous chemical diversity, preeminent resistance to proteolytic degradation and multifarious possibility of structure modification, which makes the combinatorial libraries of γ-AApeptides a promising strategy to discover ideal candidates as molecular probes or therapeutic agents. 22 Moreover, the macrocyclic library based on γ-AApeptides would exhibit enhanced conformational rigidity and constraints compared with the linear counterparts which is more beneficial in the identification of bioactive ligands 23 . We believe that with the highly efficient screening methods, ligands which possess significant affinity towards PTGER2 could be elected. Screening the combinatorial macrocyclic γ-AApeptide library entailed incubation of the library with the target the extracellular domain of the protein EP2, isolation individual elected “hit” beads for analysis, and then determination of the macrocyclic structure. Finally, the putative hits were confirmed for their binding affinity toward the extracellular domain of EP2.