Design of Y2 receptor selective and proteolytically stable PYY3-36

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Design of Y2 receptor selective and proteolytically stable PYY3-36 analogues Søren Østergaard, Jacob Kofoed, Johan F. Paulsson, Kim Grimstrup Madsen, Rasmus Jorgensen, and Birgitte S. Wulff J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01046 • Publication Date (Web): 06 Nov 2018 Downloaded from http://pubs.acs.org on November 7, 2018

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Journal of Medicinal Chemistry

Design of Y2 receptor selective and proteolytically stable PYY3-36 analogues Søren Østergaard*a, Jacob Kofoeda, Johan F. Paulssonb, Kim Grimstrup Madsena, Rasmus Jorgensenb, Birgitte S. Wulffb. a) Global Research Technologies, b) Global Drug Discovery, Novo Nordisk A/S, Novo Nordisk Park, 2760 Måløv, Denmark

KEYWORDS: PYY3-36, selective Y2 agonist, NPY, obesity, diabetes.

ABSTRACT

In recent years peptide YY (PYY) has attracted attention within the area of diabetes and obesity due to its involvement in food intake regulation and glucose homeostasis. It is well known that PYY1-36 is rapidly cleaved by dipeptidyl peptidase-4 to the more Y2 receptor selective analogue PYY3-36 which is further cleaved to the inactive analogue PYY3-34. In order to improve selectivity and proteolytic stability of the C-terminus we synthesized several analogues incorporating N-methyl amino acids or -homo amino acids and other non-natural amino acids. These were tested against all four NPY receptors and highly potent and Y2 receptor selective analogues were identified by combining a tryptophan residue in position 30 with either N-methyl

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or -homo arginine in position 35. We also identified an analogue with a MeGln34 substitution that surprisingly displayed high affinity towards all four receptors. In addition these analogues displayed improved stability towards C-terminal proteolysis compared to native PYY3-36.

Introduction Peptide YY (PYY) belongs to the NPY family of peptides that also includes neuropeptide Y (NPY) and pancreatic polypeptide (PP). All three peptides mediate their effect via four receptors Y1, Y2, Y4 and Y5 receptors. PYY1-36, which is co-released with GLP-1 from L-cells upon food stimuli, is rapidly cleaved to PYY3-36 by dipeptidyl peptidase-4 (DPP-4 EC 3.4.14.5) cleavage1. In recent years PYY3-36 has become of interest within the area of obesity and diabetes2. After DPP-4 cleavage of PYY1-36, PYY3-36 maintains high potency on the Y2 receptor, a loss of potency >300-fold is observed for the Y1 receptor and less for the Y4 receptor (approximately 15-fold) and Y5 receptor (approximately 6-fold). However, this improved selectivity displayed by PYY3-36 versus PYY1-36 may not be optimal if long-acting analogues (e.g., once-weekly) are to be administered. Instead it would be important to significantly increase the selectivity to the Y2 receptor by lowering or completely eliminating the Y1 receptor activity, but also Y4 and Y5 receptor activity. A higher preference for the Y2 receptor would minimize the risk of undesired effects such as increasing appetite from Y1 and Y5 receptor activation3 and additional inhibition of gall bladder emptying and enzyme secretion from pancreas from Y4 receptor activation4. Furthermore, for Y4 receptor also to minimize potential off target effects on the repro axis as described by Sainsbury et al5 , or in prostate where Y4 receptor mRNA has been demonstrated in humans6.


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NPY has in particular been studied in detail with regards to the structure activity relationship and the contribution of the individual amino acids concerning binding and potency towards the NPY family of receptors. Truncation analogues7 , an Ala scan8 and a D-amino acid scan9 all reveal that the C-terminal residues in NPY peptides from position 29 to position 36 are highly critical for maintaining binding and potency on all receptors. For most optimal Y1 receptor activation and binding, an intact N-terminus needs to be present either as full length NPY or in truncated analogues where the first 2-4 residues of the N-terminal part are linked to the C-terminal part of e.g., NPY25-36 by a small spacer and in some examples constrained by a chemical linkage, e.g., disulphide or lactam bridge10-13. The PYY interaction with the Y2 receptor is less effected by Nterminal truncations and requires only the intact C-terminal part from approximately residues 25 to 36. N-terminally-truncated versions of PYY or NPY display good Y2 receptor potency with expected decreases in potency and binding affinity with increasing N-terminal truncation11, 14, 15. With the truncations it is likely that the alpha-helix becomes more and more disordered as has been shown for NPY16. However, this loss of affinity towards the Y2 receptor in very truncated PYY analogues can be reconstituted by amino acid substitutions and/or introducing conformational restrictions such as lactamization or disulphide bridges10, 11, 13, 17-20. The in vivo stability of PYY3-36 has recently been addressed and it was reported that PYY3-36 is rapidly cleaved to PYY3-34 as observed in humans by antibodies directed against PYY3-34 21, 22 or by liquid chromatography mass spectrometry in mini-pigs and monkeys23. Similar observations has been reported for NPY1-36 and analogues24. Since the C-terminus is critical for receptor activation it is important to stabilize this part in the design of long-acting analogues (e.g., onceweekly administration). Along these lines it was shown with the glucagon like peptide-1 (GLP-1) analogue liraglutide that a palmitic acid connected to a -glutamyl residue and attached to lysine


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in position 26 partially stabilises against DPP-4 degradation and allows for once-daily dosing25. In the GLP-1 analogue semaglutide, the DPP-4 cleavage site is protected by replacing the Ala8 with Aib8. By combining the improved proteolytic stability with a protractor with stronger albumin binding, a GLP-1 receptor agonist was developed, which is suitable for once-weekly dosing26. Peptide backbone modification represents a feasible way to stabilise against proteolytic cleavage of the scissile peptide bond while keeping the side chains intact. PYY analogues have been reported where the peptide bond has been modified either as a reduced peptide bond19 or as an ester bond27. A reduced peptide bond will stabilise against proteolysis whereas the stability of the ester bond is likely less favourable making the latter approach unsuitable for peptide drug stabilisation. While many truncated analogues may display improved selectivity compared to PYY3-36 the pI of these shortened Y2 receptor agonists is very high (approximately pI 12) and this imposes a potential risk of injection site reactions as reported earlier with truncated NPY analogues 28. With the aim to identify proteolytically-stable and Y2 receptor selective analogues that can be the basis for the development of long-acting Y2 receptor selective analogues for the treatment of obesity, we generated a structure-activity relationship around the C-terminal part of PYY3-36. We decided to use the PYY3-36 scaffold for the design of PYY3-36 analogues with particular attention to the very C-terminal portion of PYY3-36 since this is the essential part docking into the deep receptor binding pocket of the Y2 receptor29-31. Furthermore, we wanted to address the rapid proteolytic degradation of the C-terminus that has been observed in vivo. N-methyl and -homo amino acid scans were employed in order to evaluate whether any of these modifications is tolerated with respect to Y2 receptor affinity and also to determine whether any of these changes might also affect selectivity against Y1, Y4 and Y5 receptors. Analogues with other substitutions,


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combinations with aforementioned substitutions, as well as peptides earlier reported in the literature, to serve as reference comparators. A subset of the analogues was evaluated to test whether some of these modifications improved the in vitro proteolytic stability in pig plasma. Results In total we synthesised 39 peptide analogues of the NPY family of peptides with focus on the PYY3-36 backbone. An overview of the peptide sequences is listed in Table 1 and the structural details concerning the modifications are described in Figure. 1. We measured the binding affinity on all four NPY receptors Y1, Y2, Y4 and Y5 (Table 2) and the relative selectivity is shown in Table 4. We also addressed the potency of selected high affinity Y2 receptor analogues in receptor signalling as shown in Table 3, and in addition, we measured the binding affinity against the mouse NPY receptors for the most selective analogues (Table 5). The proteolytic stability of a subset of analogues was determined in an in vitro plasma stability assay using pig plasma (Table 6). Discussion and Conclusions N-methyl scan of the C-terminal region from Val31 to Tyr36 The N-methyl scan from positions 36 to 31 was done by standard solid-phase peptide synthesis using commercially available Fmoc-N-methyl amino acids (for synthesis details see experimental and supporting information). Among the set of seven N-methylated PYY analogues we observed that only positions 35 and 34, [MeArg35]PYY3-36 (4) and [MeGln34]PYY3-36, (5) retained good Y2 receptor affinity and potency (Table 2 and 3). The most inactive analogue was the PYY3-36 methylamide (1) which lost more than 2500-fold affinity on the Y2 receptor compared to PYY3-36. This underlines that the very deep part of the binding pocket has little


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space to accommodate even a relatively small change such as addition of a methyl group to the C-terminal amide nitrogen. The same dramatic loss of affinity was observed for the other three receptors as well (Table 2). Another C-terminal modified analogue (alcohol) the PYY3-36-ol (2) also lost affinity for all the receptors albeit the changes were less dramatic as in peptide (1). The loss in affinity by the removal of a carbonyl group may be due to the loss of favourable hydrogen bond interactions to the carbonyl group and not by steric hindrance, which is likely the effect of peptide (1). A significant loss in affinity for all four NPY receptors was also seen for the three analogues [MeThr32]PYY3-36, [MeArg33]PYY3-36 and [MeVal31]PYY3-36, peptides (6-8) suggesting an important role for these three amide bonds in the receptor recognition for all four receptors. The analogue [MeTyr36]PYY3-36 (3) reduced both the Y2 receptor affinity and potency approximately 15-fold and 25-fold, respectively, but interestingly the peptide bond 35-36 can be replaced by an ester bond and still maintain full Y2 receptor potency27. The analogue [MeArg35]PYY3-36 (4) was the only N-methyl amino acid substitution that retained good Y2 affinity while also substantially lowering the affinity on all the other three receptors Y1, Y4 and Y5 (Table 2). The potency of peptide (4) corresponds to the binding affinities showing a larger reduction in potency for the Y1, Y4 and Y5 receptors as compared to Y2 receptor (Table 3). If the peptide bond 34-35 is replaced by an ester bond, similar reductions in potencies towards the receptors are observed27 further substantiating the critical involvement of the amide nitrogen in the interaction with the Y1, Y4 and Y5 receptors and less towards the Y2 receptor. [MeGln34]PYY3-36 as a novel promiscuous ligand The analogue [MeGln34]PYY3-36 (5), displayed good Y2 receptor affinity (1.3 nM) compared to PYY3-36 (0.35 nM), but interestingly the selectivity of peptide (5) for the Y2 receptor went dramatically down with the affinity for Y1 receptor being increased by a 100-fold, giving an


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affinity comparable to that of NPY1-36 and an increased affinity by 70-fold for the Y4 receptor and keeping the Y5 receptor affinity similar to PYY3-36 (see Table 4). The potency of [MeGln34]PYY3-36 (5) was also tested against all four receptors and it follows the same profile as for the binding data. The Y1 receptor potency of peptide (5) is identical to NPY1-36 which also is 0.20 nM and thus approximately 80-fold improved compared to PYY3-36. Also a high increase in potency is observed for the Y4 receptor going from >30 nM for PYY3-36 to 0.50 nM for the peptide (5). The fact that [MeGln34]PYY3-36 (5) binds with so high affinity to all four receptors makes this analogue to our knowledge the most promiscuous PYY analogue identified so far. We included the analogue [Pro34]PYY3-36 (30) as a control since this residue is known to increase affinity of PYY3-36 towards Y1 receptor while the affinity is strongly reduced for Y2 receptor32. Since PP1-36 contains a Pro34, the Y4 receptor affinity of peptide (30) is also increased compared to PYY3-36, while a 3-fold increase in affinity for the Y1 receptor was observed. The Y2 receptor affinity of peptide (30) is, however, significantly reduced 4000-fold compared to PYY3-36, while the Y5 receptor affinity is also reduced by approximately a 5-fold. Altering the peptide bond torsion angles in [MeGln34]PYY3-36 (5) increased Y1 receptor affinity more than introducing MeAla34 as in the analogue [MeAla34]PYY3-36 (33) (Table 2); thus, significantly higher affinity can be obtained for the Y1 receptor when keeping the side chain of Gln34 but altering peptide bond torsion angles imposed by the N-methyl group. In contrast, the side chain of Gln34 is not important for Y4 receptor interaction as both analogues [MeGln34]PYY3-36 (5) and [MeAla34]PYY3-36 (33) display similar Y4 receptor affinity of 0.16 nM. Native PP1-36 has a proline in position 34, but surprisingly the analogues [MeAla34]PYY3-36 (33) and [MeGln34]PYY3-36 (5) both display approximately 10-fold better affinity towards Y4 receptor compared to the analogue [Pro34]PYY3-36 (30), suggesting that the increase in Y4 receptor affinity


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may be due to a binding mode imposed by the N-methyl peptide amide bond 33-34, that is more favourable than that of a proline residue. With respect to Y2 receptor binding of analogue [MeGln34]PYY3-36 (5) we observed only a small reduction in affinity approximately 3-fold, whereas the analogues [Pro34]PYY3-36 (30) and [MeAla34]PYY3-36 (33) dramatically reduced the affinity approximately by 5000-fold and 1000-fold, respectively. This again illustrates that the glutamine side chain is important for Y2 receptor recognition, which is in agreement with the observations done for [Ala34]NPY1-36 and [Ala34]PYY3-368,

33.

We also synthesised two PP

analogues [MeGln34]PP1-36 (31) and [Gln34]PP1-36 (32). While the affinity towards the Y2 receptor was increased (800-fold) as expected for [Gln34]PP1-36 (32) compared to PP1-36, the affinity was similar for the [MeGln34]PP1-36 (31) analogue indicating that the N-methyl group has a modest effect on the Y2 receptor binding mode which is in close agreement with what was observed for the PYY3-36 backbone. However, while the Y1 receptor affinity of PP1-36 and [Gln34]PP1-36 (32) are the same, a 10-fold increase in Y1 receptor affinity was observed for the [MeGln34]PP1-36 (31) and is comparable to observations with the analogue [MeGln34]PYY3-36 (5) and again demonstrates the importance of the N-methyl group and the likely involvement of cis/trans isomeri of peptide bond 33-34 for the Y1 receptor binding mode. Both [Gln34]PP1-36 (32) and [MeGln34]PP1-36 (31) display high affinity (100 pM and 10 pM, respectively) towards the Y4 receptor and with [MeGln34]PP1-36 (31) actually being identical to PP1-36. The dramatic gain in Y4 receptor affinity seen with [MeGln34]PYY3-36 (5) was not observed with the analogue [MeGln34]PP1-36 (31) likely reflecting that the PP1-36 per se has a high Y4 receptor affinity being the natural ligand with little room for further improvement. -homo amino acid scan of the C-terminal region from Asn29 to Tyr36


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We also studied the impact of - to -amino acid residue substitution by replacing the -amino acid with that of the corresponding -homo amino acid covering the region from Asn29 to Tyr36. As opposed to N-methylation, which lacks the hydrogen bond-donating properties of the amide nitrogen and thus are also helix-breakers, -peptides are able to mimic the structure properties of -helices34. We decided to replace a few more residues upstream from residue Thr32 which are part of the alpha-helix in PYY3-36. Among the -homo amino acid analogues peptides (9-16) we tested, it was observed that only the -peptide analogues [-homoArg35]PYY3-36 (10) and [homoAsn29]PYY3-36 (16) were tolerated with respect to maintaining good Y2 receptor affinity and potency. We observed that [-homoArg35]PYY3-36 (10) was more selective than the analogue [MeArg35]PYY3-36 (4) with a 160-fold reduction in affinity of the Y1 receptor compared to only a 10-fold reduction in Y1 receptor affinity for the analogue [MeArg35]PYY3-36. The Y2 receptor affinity for the analogue [-homoArg35]PYY3-36 (10) was on the other hand slightly reduced (approximately 2-fold) compared to analogue [MeArg35]PYY3-36 (4) . With respect to Y2 receptor potency (Table 3) the [-homoArg35]PYY3-36 lost 10-fold compared to PYY3-36. For the [-homoAsn29]PYY3-36 analogue similar Y2 receptor affinity and relative selectivity against the Y1,Y4 and Y5 receptors as [MeArg35]PYY3-36 (4) were observed (Table 2). However, this substitution is expected to be less protective against C-terminal degradation compared to MeArg35 or -homo Arg35 since -homo Asn29 is farther away from the proteolytically-labile peptide bond 34-35. Analogues with Arg modification in position 35 In our studies, position 35 was the only hot spot where changes could be made while maintaining good Y2 receptor affinity and increased selectivity relative to all other three NPY receptors and


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at the same time potentially increase stability versus proteolytic degradation. We therefore decided to expand the repertoire of arginine-derived analogues with substitutions only in position 35 in search for an analogue that may be even more Y2 receptor potent and selective. Not surprisingly most of these analogues displayed very poor affinity towards all the NPY receptors. It was observed that when inserting one methylene group in arginine as in [homoArg35]PYY3-36 (18) a 100-fold reduction in Y2 affinity was observed. Removing one or two methylene groups of the arginine side chain as represented in analogues [norArg35]PYY3-36 (19) and [dinorArg35]PYY3-36 (20) also dramatically reduced Y2 receptor affinity approximately 60 and 600-fold respectively, again demonstrating that there is not much flexibility and space to accommodate minor structural rearrangements in this particular position, which is in agreement with models of the PYY analogues interacting with the Y2 receptor29-31. The overall modelled structure of the Y2 receptor reported by Xu et al31 is supported by a recent Y1 receptor structure bound to two antagonists by Yang el al35, and reveals a deep cavity densely surrounding the very C-terminal part of NPY/PYY3-36. However, further structural details with NPY or PYY bound to their receptors are needed to fully understand the endogenous agonist binding mode, since the reported Y1 receptor structure is in complex with a small molecule antagonist and thus the conformation of the receptor (inactive) is expected to differ as compared to the (active-like) model suggested by Xu et al. Since minor changes like removing or inserting one methylene group in an amino acid side chain may have little influence on proteolytic stability36, the above mentioned analogues peptides (18-20) which only differ in the distance between the guanidinium group and the C-alpha carbon may not be expected to be good candidates in stabilising against proteolytic cleavage. As opposed to changing the side chain of arginine, modification of the peptide bond as in an N-methyl amino acid or changing side chain relative to peptide bond as in


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-homo amino acids, D-amino acids or substituted glycines (peptoid) is expected to have a more stabilizing effect versus proteolytic degradation as the important recognition area for proteases are the peptide amide bond or the orientation of the side chain close to the scissile bond. Two peptide analogues [-D-homoArg35]PYY3-36 (21) and

[-L-dihomoArg35]PYY3-36 (22) both

displayed very low affinity on all four receptors whereas another -peptide analogue [-LArg35]PYY3-36 (17) displayed identical Y2 receptor affinity (2.5 nM) as the analogue [-LhomoArg35]PYY3-36 (10) but with less selectivity for Y2 receptor versus Y5 receptor. We also included an analogue [GuaP35]PYY3-36 (23), which is a cyclized version of an arginine (Figure 1) mimicking peptide bond properties of a proline residue and thus, presenting the guanidine group in a more constrained orientation. This analogue was inactive on notably the Y2 receptor but also much reduced affinity for the Y1 and Y5 receptors. However, the substitution was much more tolerable for the Y4 receptor and displayed an affinity towards the Y4 receptor comparable to PYY3-36. The introduction of a residue imposing the structural constrains of that of a proline residue is not necessarily limited to position 34 in the context of PYY3-36 when it comes to Y4 receptor interaction. Three other analogues were tested [Ntyr36]PYY3-36 (24), [Narg35]PYY3-36 (25) and [Ngln34]PYY3-36 (26) in which the side chain of tyrosine, arginine and glutamine, respectively, was placed on the amide nitrogen and thus represent a hybrid of a peptide and a peptoid37, 38. Again this type of modification resulted in inactive analogues on all NPY receptors. Combining substitutions In addition to making backbone modifications in various analogues, another way to improve Y2 receptor selectivity against the Y1, Y4 and Y5 receptors is to empirically search for amino acid substitutions by synthesizing and screening a large number of analogues with various


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substitutions. By combining substitutions even greater selectivity may be expected by an additive or even synergistic effect of the individual contributions. We have previously performed a single amino acid scan of PYY3-36 (deep mutational analysis) in which each residue one by one from position 3 to 36 was replaced with proteinogenic as well as non-proteinogenic L-amino acids (unpublished data). Among the residues that increased selectivity and maintained Y2 receptor affinity, we identified tryptophan in position 30, a residue also previously reported in truncated PYY analogues19. The analogue [Trp30]PYY3-36 (27) had similar affinity towards Y2 receptor as PYY3-36, but displayed a 10-fold decrease of the Y1 receptor affinity compared to PYY3-36 and approximately 4-fold decrease in Y5 receptor affinity. No significant improvement in selectivity was observed against the Y4 receptor. The potency of [Trp30]PYY3-36 (27) follows that of the binding data with equal Y2 receptor potency as PYY3-36 but increased selectivity towards the Y1 receptor of approximately 50-fold, however with no improvement in Y5 receptor selectivity. By combining Trp30 with either MeArg35 or -homoArg35 we obtained two analogues [Trp30,MeArg35]PYY3-36 (28) and [Trp30,-hArg35]PYY3-36 (29) that showed even greater selectivity than the analogues with one substitution while maintaining good Y2 receptor affinity and potency. For the analogue [Trp30,MeArg35]PYY3-36 (28) adding tryptophan increased the affinity and potency approximately 2-fold and 4-fold, respectively, for the Y2 receptor as compared to the single substitution in analogue [MeArg35]PYY3-36 (4), but in addition further decreased both Y1 and Y5 receptor affinity 5-fold. For the analogue [Trp30,-hArg35]PYY3-36 (29) modest improvement in selectivity and Y2 receptor affinity was observed for the combination of Trp30 and -hArg35 (Table 4). While the affinity of peptide 28 is approximately 3-fold better than peptide 29, the fold selectivity versus the other NPY receptors are within the same range (Table 4).


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Comparison to other Y2 receptor selective NPY and PYY analogues In order to put our analogues in perspective we also included analogues previously described in the literature for having improved Y2 receptor selectivity. Among the first Y2 receptor selective analogues published were the truncated NPY analogues reported back in the early 90s39. Most of these NPY analogues are truncated from residues 5-24 and many are also constrained by a lactam bridge. Although peptides 35-37 were improved with respect to Y2 receptor selectivity against the Y1 or the Y5 receptor, the Y2 receptor affinity was lower compared to peptide 28 and 29 with peptide 37 being the best with Y2 receptor binding of 2.0 nM and potency of 7.6 nM. This could be slightly improved to 1.3 nM (binding) and 2.0 nM (potency) if Ile28,31 in NPY was replaced with Leu28 and Val31 in PYY as represented by peptide 38, but the achieved selectivity versus the other receptors was lower than for peptides 28 and 29. A more recent truncated analogue Abz-PYY25-3614 (34) displayed very increased selectivity notably against the Y1 and Y5 receptor with a ratio of Y1/Y2 and Y5/Y2 of approximately 2500 and 650. However, the Y2 receptor affinity was approximately 5-fold lower than that of peptide 28 and furthermore the fold selectivity versus the Y4 receptor was approximately 6-fold less. The analogue [4fluoroPhe36]PYY3-36 (39) has been shown by Pedersen et al

40

to display very good selectivity

versus Y1 and Y4 receptors. Indeed we could confirm the improved selectivity of 39 compared to PYY3-36 with decreased affinity towards Y1 and Y4 receptors, but only to find that the Y5 receptor affinity and potency was in fact increased approximately 5-fold and 2-fold, respectively compared to native PYY3-36 thereby having lower selectivity for the Y2 receptor versus Y5 receptor. In vitro binding for the mouse receptors


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The four analogues [MeArg35]PYY3-36 (4), [hArg35]PYY3-36 (10), [Trp30,MeArg35]PYY3-36 (28) and [Trp30,-hArg35]PYY3-36 (29) were also tested against the mouse NPY receptors (Table 5). The two most potent and selective analogues [Trp30,MeArg35]PYY3-36 (28) and [Trp30,hArg35]PYY3-36 (29) showed an affinity for mouse Y2 receptor of 0.5 nM and 1 nM, respectively, which is similar to PYY3-36 (0.4 nM). Indeed we could not observe any binding (>10000 nM) for the two analogues towards the Y1 and Y4 receptors, whereas the Y5 receptor affinity was reduced compared to PYY3-36 approximately 25-fold and 80-fold thereby increasing the selectivity versus the Y5 receptor by 1200 and 2000 fold, respectively, confirming that the analogues 28 and 29 maintained to be very Y2 receptor selective concerning the mouse NPY receptors. Proteolytic stability of selected PYY analogues The very C-terminal part of PYY is prone to proteolytic degradation in vitro and in vivo and removal of the last two residues 35-36 will lead to the inactive PYY3-34. It is therefore essential to protect the peptide against proteolytic degradation if long-acting analogues are to be designed. We tested 11 analogues for in vitro proteolytic stability in plasma from mini-pigs and the results are listed in Table 6. Plasma from mini-pigs was used in order to bridge to in vitro and in vivo observations reported by Olsen et al23 and Torang et al21. We included GLP-1 as a positive control throughout to ensure consistency in the assays. While GLP-1(7-37) is rapidly cleaved by DPP-4 to GLP-1(9-37) with an in vitro half-life of approximately 46 minutes, the cleavage of PYY3-36 to PYY3-34 is slightly slower with an in vitro half-life of approximately 200 min. A Cterminal methylamide PYY3-36 analogue (1) did not protect against C-terminal cleavage, while the

half-lives

of

the

analogues

[MeTyr36]PYY3-36

(3),

[MeArg35]PYY3-36

(4)

and

[MeGln34]PYY3-36 (5) were increased by approximately 2-, 3- and 4-fold, respectively. The [MeArg33]PYY3-36 (6) did not show any increase in half-life compared to native PYY3-36 and


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most likely peptide bond modifications farther upstream will not improve stability any further. With respect to the -homo amino acid series peptides (10-16) we included analogues substituted with -homo amino acid from position 36 to 33. The same pattern was observed and the most optimal protection was observed for positions 35 and 34 as in the two analogues [homoArg35]PYY3-36 (10) and [-homoGln34]PYY3-36 (11) with a 3-fold and 6-fold increase in plasma half-lives, respectively. Again position 34 seems to offer the best protection against cleavage. We also included the two highly selective analogues [Trp30, MeArg35]PYY3-36 (28) and [Trp30,-homoArg35]PYY3-36 (29) to verify that the Leu30 to Trp30 substitution did not shorten the plasma half-life compared to [MeArg35]PYY3-36 (4) and [-homoArg35]PYY3-36 (10) and indeed these two analogues (28) and (29) also display similar extended half-life (T½ = 980 min and 570 min, respectively) as analogues (4) and (10). Torang et al also identified the PYY3-34 metabolite in pigs21 as well as in humans22 and we believe the improved in vitro stability that is observed in mini-pig plasma for analogues 4,5,10,11,28,29 can be correlated to improved in vitro stability in human plasma. By performing systematic N-methyl and -homo amino acid scans in the C-terminal end of PYY3-36 and by combining this with structural information from an amino acid scan we have been able to co-evolve PYY3-36 analogues with both increased Y2 receptor selectivity and proteolytic stability. We believe that the two PYY3-36 analogues [Trp30,MeArg35]PYY3-36 (28) and [Trp30,-hArg35]PYY3-36 (29), as compared to a minor subset of Y2 receptor selective and truncated analogues reported earlier (34-39, except 38), qualifies as some of the most Y2 receptor potent and selective PYY3-36 analogues against Y1, Y4 and Y5 receptors identified so far. In addition, the analogues (28) and (29) also display improved in vitro stability versus proteolysis thereby ensuring the needed stability for designing analogues with sufficient long half-life.


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Indeed we have found that attaching a fatty acid to the N-terminal region of native PYY3-36 halflives in the range of 10-20 hours were observed in mini-pigs41, whereas introducing MeArg35 or -homo-Arg35, but keeping the same albumin protractor, increases the half-lives to 60-90 hours42, 43

illustrating the importance of stabilising the C-terminus in addition to attachment of protractor

moiety alone. In addition, we also identified an analogue [MeGln34]PYY3-36 (5) that we believe represents the most promiscuous PYY3-36 analogue identified to date displaying low nanomolar affinity and potency towards all four NPY receptors. We hope these data may also help to guide to more precise computer models of the structural requirements for the interaction of NPY, PYY and PP with the NPY receptors. Experimental Section The set of N-methylated and beta-homo peptide analogues were synthesised by standard solid phase peptide synthesis (SPPS) using Prelude or Prelude X equipment (Gyros Protein Technologies) and commercially available Fmoc-N-methyl-amino acids or Fmoc--homo-amino acids. While these amino acids, with the exception of Fmoc-beta-arginine, couple normally under standard SPPS conditions, the coupling onto a N-methyl substituted amino acid or Nsubstituted glycine unit (peptoid monomer) is notoriously slow and extended coupling time, or double coupling and/or heating (using Prelude X) was needed in order to obtained full coupling yield. With respect to Fmoc--Arg, which was used only in position 35, this residue had to be in situ coupled three times followed by a capping step and this still resulted in lower crude yield (approximately 30%) than expected. Likely this particular residue may lactamize more rapidly than normally observed for the corresponding Fmoc-arginine. PYY3-36 with an alcohol as the Cterminal was synthesised using a 2-Chloro-trityl resin and the coupling of the first amino acid was accomplished using the tyrosine alcohol Fmoc-O-t-butyl-L-tyrosinol (8 equivalents) as a 0,3


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M using 3% DPEA in DCM/DMF (4:1) for 2 hours followed by a capping step with 25% MeOH for 15 min. Synthesis of PYY3-36 methyl amid (1) was done using the linker from Estep44. In particular for this linker a longer coupling time of the tyrosine (>2 hours) was needed in order to obtain acceptable yield followed by capping with acetic anhydride in order to avoid deletion peptides. The lactamization of the cyclic peptides was done by using Lys(Mtt) and Fmoc-Glu(2O-PhiPr)-OH protection group strategy with the N-terminal of the cyclized analogues either Bocprotected or acetylated. Removal of Mtt and 2-phenylisopropyl group was removed using 1% TFA, 5% TIPS in HFIP/DCM (3:1) for 15-20 min (3 x 1 min + 12-17 min). After deprotection the resin was washed DCM then with 1M DPEA followed by DMF. Activation of the carboxylic group of glutamic acid was done by addition of 4 equivalents PyAOP and 8 equivalents DPEA and the allowed to couple for 5-19 hours. All other peptides were synthesised by standard solid phase methods using 25% piperidine to remove the Fmoc group and coupling was done by using 0,3 M Fmoc-amino acid in 0,3 M Oxymapure in DMF added in 6-8 eq excess and activated by 68 eq DIC (3M solution in DMF) and 3-4 eq collidine (3M solution in DMF). Coupling time was typically 30-60 min at room temperature or 10-15 min using heating (Prelude X). For cleavage, purification by reverse HPLC and characterization by LC-MS see supporting information. Peptides were analysed by RP-UPLC with an average purity found to be 96.0% [91.0%-99.9%]. We decided to run very flat gradients of ACN in our analytical setup in order to detect as much impurity as possible, since we observed that if we run a standard gradient often used e.g., 5-60% ACN or 5-90% ACN all peptides were >95% in purity. For the PYY peptides below 95% [91.0%-94.7%] (1,3,9,12,19,21,22,26,31,33,36,38) the main impurities were deamidation of Asn18 and Asn29 analogues or oxidation of methionine in the PP analogues. For PYY analogues 9,21,22 and 38 impurities could not be detected by MS and this is due to a slight tailing of the


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peak and thus it is difficult to exactly define the peak or the amount of impurity was too low to be detected. All of the PYY analogues with purity below 95% were also of low activity against the NPY receptors and the very small amount of impurities has no influence of the interpretation of the in vitro results. We have previously found that deamidation in Asn18 leads to equipotent analogues whereas deamidation products in Asn29 lead to completely inactive (nonbinding) analogues (unpublished results). Likewise with regards to the very small amount of oxidized methionine in the PP analogue this has also no effect on the conclusions. For the purity, MS and specific analytical conditions of each peptide see supporting information Table 1 and 2. Binding assay NPY receptor scintillation proximity assays (SPA) were performed using cell membrane preparations from cell lines expressing one of the human Y1, Y2, Y4 or Y5 receptors (n = 3 per analogue). Each receptor binding assay is described in detail in the supporting information. In short, wheat germ agglutinin SPA beads (PerkinElmer, Waltham, MA, USA) were reconstituted in SPA binding buffer and mixed with membrane preparation to obtain 0.5 mg/well SPA beads. Serial diluted PYY analogues in binding buffer were added to give a final assay concentration of PYY analogues ranging from 10 μM to 10 pM and 1 μM to 1 pM for reference analogues. Fifty thousand counts per minute per well of human [125I]-PYY1-36 (Chemistry and Isotope Lab, Novo Nordisk A/S) for Y1, Y2 and Y5 receptors or human [125I]-PP (Cat # NEX3150, PerkinElmer) for Y4 receptor was added corresponding to a concentration of 100 pM radio ligand. Plates were sealed and incubated at 25°C for 2 hours and centrifuged prior to reading of light emission in a TopCount NXT (PerkinElmer). Specific binding was calculated by performing saturation experiments with increasing concentrations of radio ligand for total binding and for non-specific binding in the presence of 1 µM cold ligand. Equilibrium binding constant Kd corresponding to


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the radio ligand concentration needed to achieve a half-maximum binding at equilibrium were determined, and the values were; Y1 Kd = 556 pM, Y2 Kd = 275 pM, Y4 Kd = 111 pM and Y5, Kd = 345 pM. The pKi values of the tested analogues were calculated by nonlinear regression analysis of sigmoidal dose-response curves using the Cheng-Prusoff equation in GraphPad Prism v 7.03 (Graph Pad software, La Jolla, CA, USA). Mouse Y receptors were transiently expressed in HEK-293 cells and membrane preparation and binding assays were performed as described for the human counterparts. pIC50 values were calculated by nonlinear regression analysis of sigmoidal dose-response curves. Potency assay Reduction of cyclic adenosine monophosphate (cAMP) through Gi-coupled Y receptor activation was measured in a FLIPR Tetra System (Molecular Devices, Sunnyvale, CA, USA) using human embryonic kidney (HEK) 293 cells stably expressing one of the human Y1, Y2, Y4 or Y5 receptors and a cAMP sensitive calcium channel (Codex Bio-solution, Gaithersburg, MD, USA) (n = 3 per analogue; except analogue 3 where n = 2). The calcium channel has a selective binding site for cAMP resulting in cellular calcium influx in presence of cAMP which is detected by a calcium sensitive dye. In 384 well format, 14000 cells/well, cells were pre-treated for 2 hours with 25 µl/well calcium dye buffer containing: 1 vial Calcium 5 dye (Molecular Devices) dissolved in 100 ml Hank's balanced salt solution (HBSS) (Gibco®, Life Technologies, Carlsbad, CA, USA) containing 20 mM Hepes, 1.5 mM probenecid (Sigma-Aldrich, St. Louis, MO, USA), 250 µM phosphodiesterase (PDE) inhibitor 4-(3-Butoxy-4-methoxybenzyl)imidazolidin-2-one (Sigma-Aldrich) and 0.129 mM CaCl2 at pH 7.40. When assayed, 1 µl isoproterenol (SigmaAldrich, 0.05 µM final assay concentration) to stimulate cAMP and 1 µl PYY analogue was added followed by fluorescence signal measurement (Ex540/Em590). Final assay concentrations


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for PYY analogues ranged from 0.03 nM to 30 nM for Y2 receptor assays and 1 nM to 1000 nM for Y1, Y4 and Y5 receptor assays. Reference analogues were serial diluted ranging from 0.03 nM to 30 nM in all assays. Fluorescence intensity at time point 360 seconds was used for calculations of pEC50 values. For all in vitro binding and potency assays, dose response measurements were performed in technical duplicates and data presented represents a mean of 3 independently performed assays. Mean pKi/pIC50/pEC50 with the 95% confidence interval are stated in the tables as well as the mean Ki/IC50/EC50 values which were calculated by 10^-(pKi/pIC50/pEC50) respectively and presented in nM. In vitro metabolism The in vitro plasma stability studies (n = 3 per analogue) were conducted in heparinized plasma from mini-pigs (BioreclamationIVT, Westbury, NY). Prior to the in vitro studies, four volumes of plasma were diluted with one volume of 0.1 M PBS-buffer (pH 7.4). Diluted plasma from mini-pigs and the PYY-analogues (and native GLP-1 as positive control) in 0.1 M PBS-buffer (pH 7.4) was pre-incubated at 37°C for 30 min. The reaction was started by adding the prewarmed solution of PYY-analogues to plasma and mixture was kept at 37°C. The final concentration of PYY-analogue (and positive control) was 1 uM. The reaction was stopped at selected time points (5, 15, 30, 60, 90, 120, 150, 180, 210, 240, 270 and 300 min) by transferring one volume of plasma to three volumes of methanol (containing 20 nM of internal standard). The mixture was centrifuged for 6000g for 20 minutes at 4°C. After centrifugation, one volume of supernatant was diluted with one volume of Milli-Q water. The mixture was analysed by LCMS. Calibration curves were prepared in cold heparinized plasma from mini-pigs. The


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calibration curves covered a range from 20 nM to 2000 nM. Quality control samples were included. The deviation between nominal and calculated concentration in the calibrators and quality control samples was below 20%. The in vitro samples were analysed by LC-MS on a MaXis QTOF mass spectrometer (Bruker, Bremen, Germany) to which an Aquity UPLC (Waters, Milford, MA) was connected. The mass spectrometer was equipped with an electrospray interface, which was operated in positive ionisation mode. Analysis was conducted in full scan mode in the range from m/z 500 to 1850. Analytical separation was conducted on an Aeris Peptide XB-C18 column (50x2.1 mm, 3.6 um, Phenomenex, CA) using gradient elution. Mobile phase A consisted of Milli-Q water with 0.1% formic acid and mobile phase B consisted of acetonitrile with 0.1% formic acid. The flow was 0.3 ml/min and the column was operated at 60°C. The percent of PYY-analogue remaining in the sample (%-remaining) was calculated by normalizing the concentration of the PYY-analogues at the selected time point to the concentration of the same PYY-analogue in the 15 minute sample. Calculation of half-lives was conducted in Prism 7 (Version 7.03; GraphPad software, La Jolla, CA, USA) assuming first order degradation kinetics. The plateau where set to constant (0). Ancillary Information Corresponding author *email: [email protected] Author contribution All authors have given approval to final version of the manuscript.


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SO designed and synthesized the peptides, interpretation of in vitro results and wrote the manuscript. BSW gave input to design, interpretation of in vitro results and assisted in writing and reviewing. JCKF designed and synthesized peptides and assisted in reviewing, JFPL performed in vitro analysis, interpretation of in vitro results and assisted in writing and reviewing, KGUM performed the in vitro plasma stability and assisted in writing and reviewing. RSJR gave input to design, interpretation of in vitro results and assisted in reviewing. Author Notes The authors declare the following competing interest: All authors are full time employees of Novo Nordisk A/S and most hold a minor share portions as part of their employment. Novo Nordisk A/S has currently two PYY3-36 analogues in clinical trial phase I. Acknowledgments We thank Yvonne Broby Madsen, Lene Stoltze, Martina Mørkenborg, Anne Meincke and Masja Blicher Hansen for excellent technical assistance and Hugo Gutierrez de Teran, University Uppsala for comments. Abbreviations ACN, acetonitril; cAMP, cyclic adenosine monophosphate ; DIC, N,N´-diisopropylcarbodiimide; DPEA, diisopropylethylamine; DPP-4, dipeptyl peptidase 4, FA,

formic acid; Fmoc,

fluorenylmethyloxycarbonyl; GLP-1, glucagon like peptide-1, HBSS, Hank's balanced salt solution;

HEK,

human

embryonic

kidney;

HEPES,

4-(2-hydroxyethyl)1-

piperazineethanesulfonic acid; HFIP, hexafluoroisopropanol; LC-MS, liquid-chromatography mass spectrometry; Mtt, 4-methyltrityl; NMP, N-methylpyrrolidone; NPY, neuropeptide Y; PDE, phosphodiesterase; pI, isolelctric point; PP, pancreatic polypeptide; PYY, peptide YY;


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PyAOP,

7-Azabenzotriazol-1-yloxy)trispyrrolidinophosphonium

hexafluorophosphate;

RP-

UPLC, reverse phase ultra-performance liquid chromatography; SPA, scintillation proximity assay; SPPS, solid phase peptide synthesis; TIPS, triisopropylsilane; Y1, Y2, Y4, Y5, NPY receptor subtype 1, 2, 4 and 5 respectively; Supporting information Solid phase peptide synthesis protocol, tables with MS, purity and UPLC methods, methods for performing binding and potency assays, illustrative graphs with binding curves and graph with plasma stability included in Supporting Information.


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and/or Y4 Receptor Agonists. U.S. Patent 2011/0275559 A1, Nov. 10, 2011.


29


42.

Page 30 of 43

Ostergaard, S.; Conde Frieboes, K. W.; Wieczorek, B.; Kaalby Thomsen, J.; Wulff, B. S.;

Jessen, C. Selective PYY Compounds and Uses Thereof. U.S. Patent 2015/0152150 A1, Jun 4, 2015. 43.

Ostergaard, S.; Conde Frieboes, K. W.; Wieczorek, B.; Thomsen, J. K.; Wulff, B. S.

hPYY(1-36) Having a Beta-Homoarginine Substitution at Position 35. U.S. Patent 2016/0263197 A1, Sep. 15, 2016. 44.

Estep, K. G.; Neipp, C. E.; Stephens Stramiello, L. M.; Adam, M. D.; Allen, M. P.;

Robinson, S.; Roskamp, E. J. Indole resin: a versatile new support for the solid-phase synthesis of organic molecules. The Journal of Organic Chemistry 1998, 63, 5300-5301.


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Tables Table 1. List of PYY, NPY and PP analogues synthesized and tested against the NPY receptors. No.

Peptide

Sequence (modification in bold)

NPY1-36

YPSKPDNPGEDAPAEDLARYYASLRHYINLITRQRY

PP1-36

APLEPVYPGDNATPEQMAQYAADLRRYINMLTRPRY

PYY1-36

YPIKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY

PYY3-36

IKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY

1

PYY3-36 (methylamide)

IKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY-CONH-CH3

2

PYY3-36 (ol)

IKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY-CH2-OH

3

[MeTyr36]PYY3-36

IKPEAPGEDASPEELNRYYASLRHYLNLVTRQR(MeTyr)

4

[MeArg35]PYY3-36

IKPEAPGEDASPEELNRYYASLRHYLNLVTRQ(MeArg)Y

5

[MeGln34]PYY3-36

IKPEAPGEDASPEELNRYYASLRHYLNLVTR(MeGln)RY

6

[MeArg33]PYY3-36

IKPEAPGEDASPEELNRYYASLRHYLNLVT(MeArg)QRY

7

[MeThr32]PYY3-36

IKPEAPGEDASPEELNRYYASLRHYLNLV(MeThr)RQRY

8

[MeVal31]PYY3-36

IKPEAPGEDASPEELNRYYASLRHYLNL(MeVal)TRQRY

9

[-homoTyr36]PYY3-36

IKPEAPGEDASPEELNRYYASLRHYLNLVTRQR(-homoTyr)

10

[-homoArg35]PYY3-36

IKPEAPGEDASPEELNRYYASLRHYLNLVTRQ(-homoArg)Y

11

[-homoGln34]PYY3-36

IKPEAPGEDASPEELNRYYASLRHYLNLVTR(-homoGln)RY

12

[-homoArg33]PYY3-36

IKPEAPGEDASPEELNRYYASLRHYLNLVT(-homoArg)QRY


31


13

[-homoThr32]PYY3-36

IKPEAPGEDASPEELNRYYASLRHYLNLV(-homoThr)RQRY

14

[-homoVal31]PYY3-36

IKPEAPGEDASPEELNRYYASLRHYLNL(-homoVal)TRQRY

15

[-homoLeu30]PYY3-36

IKPEAPGEDASPEELNRYYASLRHYLN(-homoLeu)VTRQRY

16

[-homoAsn29]PYY3-36

IKPEAPGEDASPEELNRYYASLRHYL(-homoAsn)LVTRQRY

17

[35-arg35]PYY3-36

IKPEAPGEDASPEELNRYYASLRHYLNLVTRQ(-Arg)Y

18

[homoArg35]PYY3-36

IKPEAPGEDASPEELNRYYASLRHYLNLVTRQ(homoArg)Y

19

[norArg35]PYY3-36

IKPEAPGEDASPEELNRYYASLRHYLNLVTRQ(norArg)Y

20

[dinorArg35]PYY3-36

IKPEAPGEDASPEELNRYYASLRHYLNLVTRQ(dinorArg)Y

21

[35--D-homoArg35]PYY3-36

IKPEAPGEDASPEELNRYYASLRHYLNLVTRQ(-D-homoArg)Y

22

[35--L-dihomoArg35]PYY3-36

IKPEAPGEDASPEELNRYYASLRHYLNLVTRQ(-dihomoArg)Y

23

[GuaP35]PYY3-36

IKPEAPGEDASPEELNRYYASLRHYLNLVTRQ(GuaP)Y

24

[Ntyr36]PYY3-36

IKPEAPGEDASPEELNRYYASLRHYLNLVTRQR(Ntyr)

25

[Narg35]PYY3-36

IKPEAPGEDASPEELNRYYASLRHYLNLVTRQ(Narg)Y

26

[Ngln34]PYY3-36

IKPEAPGEDASPEELNRYYASLRHYLNLVTR(Ngln)RY

27

[Trp30]PYY3-36

IKPEAPGEDASPEELNRYYASLRHYLNWVTRQRY

28

[Trp30, MeArg35]PYY3-36

IKPEAPGEDASPEELNRYYASLRHYLNWVTRQ(MeArg)RY

29

[Trp30,-homoArg35]PYY3-36

IKPEAPGEDASPEELNRYYASLRHYLNWVTRQ(-homoArg)Y

30

[Pro34]PYY3-36

IKPEAPGEDASPEELNRYYASLRHYLNLVTRPRY

31

[MeGln34]PP1-36

APLEPVYPGDNATPEQMAQYAADLRRYINMLTR(MeGln)RY

Page 32 of 43


32

Page 33 of 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


32

[Gln34]PP1-36

APLEPVYPGDNATPEQMAQYAADLRRYINMLTRQRY

33

[MeAla34]PYY3-36

IKPEAPGEDASPEELNRYYASLRHYLNLVTR(MeAla)RY

34

Abz-PYY25-36

(2-Amino-benzoyl)-RHYLNLVTRQRY

35

[Ahx5-24]NPY1-36

YPSK-(6-amino-hexanoyl)-RHYINLITRQRY

36

[cyclo(Ac,Glu28,Lys32]NPY25-36

Ac-RHY[E*NLIK*]RQRY [* cyclolactam]

37

[Cyclo(Glu2,Lys30),Ahx5-24]NPY1-36

Y[E*SK-(6-amino-hexanoyl)-RHYINK*]ITRQRY [*cyclolactam]

38

[Cyclo(Glu2,Lys30),Ahx5-24]PYY1-36

Y[E*IK-(6-amino-hexanoyl]-RHYLNK*]VTRQRY [*cyclolactam]

39

[36p-fluoroPhe]PYY3-36

IKPEAPGEDASPEELNRYYASLRHYLNLVTRQR(p-fluoro-Phe)


33

Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Page 34 of 43

Table 2. In vitro binding of peptide analogues against human Y1, Y2 Y4 and Y5 receptors as measured by SPA binding (n=3). pKi is calculated as the mean of the 3 independent experiments, and 95% confidence interval (CI), given as [lower; upper]. Mean Ki is calculated from mean pKi.

Peptide PYY3-36 PP1-36 PYY1-36 NPY1-36 PYY3-36 methylamide PYY3-36 (ol) [MeTyr36]PYY3-36 [MeArg35]PYY3-36 [MeGln34]PYY3-36 [MeArg33]PYY3-36 [MeThr32]PYY3-36 [MeVal31]PYY3-36 [-homoTyr36]PYY3-36 [-homoArg35]PYY3-36 [-homoGln34]PYY3-36 [-homoArg33]PYY3-36 [-homoThr32]PYY3-36 [-homoVal31]PYY3-36 [-homoLeu30]PYY3-36 [-homoAsn29]PYY3-36 [-Arg35]PYY3-36 [homoArg35]PYY3-36 [norArg35]PYY3-36 [dinorArg35]PYY3-36 [-D-homoArg35]PYY3-36 [-L-dihomoArg35]PYY3-36 [GuaP35]PYY3-36 [Ntyr36]PYY3-36 [Narg35]PYY3-36 [Ngln34]PYY3-36 [Trp30]PYY3-36 [Trp30,MeArg35]PYY3-36 [Trp30,-homoArg35]PYY3-36 [Pro34]PYY3-36 [MeGln34]PP1-36 [Gln34]PP1-36

No.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

Human Y1 receptor

Human Y2 receptors

Human Y4 receptor

Human Y5 receptor

Ki [nM] 40 50 0.13 0.25 >10000 1600 4000 500 0.40 2500 >10000 5000 4000 6300 160 200 6300 >10000 >10000 630 >10000 1000 1300 10000 >10000 >10000 2000 >10000 >10000 5000 400 2500 >10000 13 0.79 40

Ki [nM] 0.40 5000 0.25 0.79 1000 40 6.3 1.3 1.3 200 2000 630 79 2.5 2.5 250 160 50 50 0.79 2.5 40 25 250 1600 160 7900 1300 1000 2500 0.25 0.63 2.0 1600 5.0 6.3

Ki [nM] 13 0.010 0.79 2.5 2500 1300 1000 630 0.16 320 1600 100 2500 4000 100 130 2000 2500 130 790 6300 63 50 1000 5000 5000 6.3 630 500 200 16 790 2500 1.6 0.010 0.10

Ki [nM] 3.2 10 0.50 0.40 1300 160 100 130 1.6 200 3200 790 630 790 25 400 32 40 100 79 100 1000 200 1000 5000 1300 250 1300 1000 50 16 400 1300 16 0.63 1.3

pKi [95% CI] 7.4 [7.5; 7.2] 7.3 [7.6; 7.0] 9.9 [9.9; 9.8] 9.6 [9.8; 9.5] 5.0 5.8 [6.0; 5.7] 5.4 [5.8; 5.1] 6.3 [6.3; 6.2] 9.4 [9.6; 9.3] 5.6 [5.8; 5.3] 5.0 5.3 [5.6; 4.9] 5.4 [5.6; 5.2] 5.2 [5.6; 4.8] 6.8 [7.0; 6.6] 6.7 [7.0; 6.4] 5.2 [5.6; 4.8] 5.0 5.0 6.2 [6.8; 5.7] 5.0 6.0 [6.2; 5.8] 5.9 [5.9; 5.8] 5.0 [5.0; 5.0] 5.0 5.0 5.7 [6.1; 5.3] 5.0 5.0 [5.1; 5.0] 5.3 [5.5; 5.1] 6.4 [6.7; 6.2] 5.6 [5.9; 5.3] 5.0 [5.0; 5.0] 7.9 [8.1; 7.7] 9.1 [9.3; 8.9] 7.4 [8.0; 6.9]

pKi [95% CI] 9.4 [9.5; 9.3] 5.3 [6.8; 3.9] 9.6 [10; 9.1] 9.1 [9.4; 8.7] 6.0 [6.1; 5.9] 7.4 [7.6; 7.2] 8.2 [8.4; 7.9] 8.9 [9.3; 8.6] 8.9 [9.1; 8.8] 6.7 [6.9; 6.5] 5.7 [6.0; 5.5] 6.2 [6.4; 6.0] 7.1 [7.3; 7.0] 8.6 [8.9; 8.3] 8.6 [8.7; 8.4] 6.6 [6.7; 6.4] 6.8 [7.0; 6.7] 7.3 [7.8; 6.9] 7.3 [7.8; 6.7] 9.1 [9.4; 8.7] 8.6 [8.9; 8.2] 7.4 [7.7; 7.1] 7.6 [7.8; 7.3] 6.6 [6.8; 6.4] 5.8 [5.9; 5.7] 6.8 [7.0; 6.7] 5.1 [5.3; 4.8] 5.9 [6.2; 5.6] 6.0 [6.2; 5.9] 5.6 [5.8; 5.4] 9.6 [10; 9.3] 9.2 [9.4; 9.0] 8.7 [9.1; 8.3] 5.8 [5.9; 5.7] 8.3 [8.6; 8.0] 8.2 [8.4; 7.9]

pKi [95% CI] 7.9 [8.3; 7.6] 11 [11; 11] 9.1 [9.2; 8.9] 8.6 [8.9; 8.3] 5.6 [5.9; 5.3] 5.9 [6.0; 5.8] 6.0 [6.4; 5.6] 6.2 [6.3; 6.2] 9.8 [10; 9.5] 6.5 [6.7; 6.2] 5.8 [5.9; 5.7] 7.0 [7.3; 6.7] 5.6 [5.7; 5.4] 5.4 [5.6; 5.3] 7.0 [7.3; 6.8] 6.9 [7.1; 6.7] 5.7 [6.1; 5.3] 5.6 [5.8; 5.5] 6.9 [7.3; 6.5] 6.1 [6.2; 5.9] 5.2 [5.4; 5.1] 7.2 [7.3; 7.1] 7.3 [7.3; 7.2] 6.0 [6.2; 5.9] 5.3 [5.5; 5.2] 5.3 [5.4; 5.1] 8.2 [8.7; 7.8] 6.2 [6.5; 5.8] 6.3 [6.3; 6.2] 6.7 [6.9; 6.5] 7.8 [8.9; 6.8] 6.1 [6.2; 6.0] 5.6 [5.8; 5.3] 8.8 [9.3; 8.4] 11 [11; 10] 10 [11; 10]

pKi [95% CI] 8.5 [8.9; 8.1] 8.0 [8.3; 7.7] 9.3 [9.4; 9.3] 9.4 [9.7; 9.1] 5.9 [6.6; 5.3] 6.8 [7.2; 6.5] 7.0 [7.5; 6.5] 6.9 [7.1; 6.6] 8.8 [9.2; 8.3] 6.7 [6.8; 6.6] 5.5 [5.6; 5.4] 6.1 [6.6; 5.7] 6.2 [6.6; 5.7] 6.1 [6.2; 6.1] 7.6 [8.3; 6.8] 6.4 [6.7; 6.1] 7.5 [8.0; 7.0] 7.4 [7.6; 7.1] 7.0 [7.2; 6.8] 7.1 [7.5; 6.7] 7.0 [7.5; 6.5] 6.0 [6.3; 5.7] 6.7 [7.1; 6.4] 6.0 [6.4; 5.6] 5.3 [5.5; 5.2] 5.9 [6.0; 5.8] 6.6 [7.4; 5.9] 5.9 [6.4; 5.4] 6.0 [6.1; 6.0] 7.3 [7.9; 6.7] 7.8 [8.1; 7.6] 6.4 [6.8; 5.9] 5.9 [6.3; 5.6] 7.8 [8.0; 7.6] 9.2 [9.4; 9.0] 8.9 [9.1; 8.6]


34

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[MeAla34]PYY3-36 [Abz]PYY25-36 [Ahx5-24]NPY [cyclo(Ac,Glu28,Lys32)]NPY25-36 [cyclo(Glu2,Lys30),Ahx5-24]NPY1-36 [cyclo(Glu2,Lys30),Ahx5-24]PYY1-36 [p-fluoroPhe36]PYY3-36

33 34 35 36 37 38 39

4.0 7900 400 >10000 500 1300 630

8.4 [8.9; 7.8] 5.1 [5.3; 4.9] 6.4 [6.8; 6.1] 30 0.10 0.20 >1000* 320 0.20 >1000 130 320 >1000 790 >1000 >1000 0.20 16 >1000 >1000 >1000 79

Human Y2 receptor

pEC50 [95% CI] 7.8 [8.2; 7.4] 7.5 10.0 [10; 9.7] 9.7 [10; 9.1] 1000 500 >1000 >1000 0.16 0.20 >1000 >1000 >1000 >1000

pEC50 [95% CI] 7.5 9.7 [9.9; 9.6] 8.4 [8.7; 8.2] 8.0 [8.9; 7.1] 10000 10000 >10000

pKi [95% CI] 10 [11; 9.9] 6.1 [6.2; 5.9]

Design of Y2 receptor selective and proteolytically stable PYY3-36

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