Development of Novel Melanocortin Receptor Agonists Based on the

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Development of novel melanocortin receptor agonists based on the cyclic peptide framework of sunflower trypsin inhibitor-1 Thomas Durek, Philipp M. Cromm, Andrew M. White, Christina I Schroeder, Quentin Kaas, Joachim Weidmann, Abdullah Ahmad Fuaad, Olivier Cheneval, Peta J. Harvey, Norelle L. Daly, Yang Zhou, Anita Dellsén, Torben Österlund, Niklas Larsson, Laurent Knerr, Udo Bauer, Horst Kessler, Minying Cai, Victor J. Hruby, Alleyn T. Plowright, and David J Craik J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00170 • Publication Date (Web): 31 Mar 2018 Downloaded from http://pubs.acs.org on March 31, 2018

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Journal of Medicinal Chemistry

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Development of novel melanocortin receptor agonists based on the cyclic peptide framework of sunflower trypsin inhibitor-1

Thomas Dureka#, Philipp M. Cromm#a,b,d, Andrew M. Whitea, Christina I. Schroedera, Quentin Kaasa , Joachim Weidmanna, Abdullah Ahmad Fuaada, Olivier Chenevala, Peta J. Harveya, Norelle L. Dalya,e, Yang Zhouc, Anita Dellsénf, Torben Österlundgh, Niklas Larssong, Laurent Knerri, Udo Baueri, Horst Kesslerb, Minying Caic, Victor J. Hrubyc, Alleyn T. Plowrightij and David J. Craik*a a

Institute for Molecular Bioscience, The University of Queensland, Brisbane, Qld, 4072, Australia.

b

Institute for Advanced Study and Center of Integrated Protein Science, Department Chemie, Technische Universität München, Lichtenbergstrasse 4, 85747 Garching, Germany.

c

Department of Chemistry and Biochemistry, University of Arizona, Tuscon, AZ 85721, USA.

d

Current address: Department of Molecular, Cellular & Developmental Biology, Yale University, New Haven, CT 06511, USA. e

Current address: AITHM, Centre for Biodiscovery and Molecular Development of Therapeutics, James Cook University, Cairns, QLD 4870, Australia.

f

Mechanistic Biology & Profiling, Discovery Sciences, IMED Biotech Unit, AstraZeneca, Gothenburg, 43183 Sweden.

g

Discovery Biology, Discovery Sciences, IMED Biotech Unit, AstraZeneca, Gothenburg, 43183 Sweden. h

Drug Safety and Metabolism, IMED Biotech Unit, AstraZeneca, Gothenburg, 43183 Sweden.

i

Medicinal Chemistry, Cardiovascular and Metabolic Diseases, IMED Biotech Unit, AstraZeneca, Gothenburg, 43183 Sweden.

j

Current address: Integrated Drug Discovery, Sanofi-Aventis Deutschland GmbH, Industriepark Hochst, 65926 Frankfurt am Main, Germany

#Equally contributing first authors *To whom correspondence should be addressed: Professor David J. Craik, Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia, Tel.: (+61) 7 3346 2019; Fax: (+61) 7 3346 2101; E-mail: [email protected] *Running title: Novel melanocortin agonists based on the SFTI-1 scaffold Keywords: cyclic peptides, drug design, N-methylation, structure 1 ACS Paragon Plus Environment

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Abstract Ultra-stable cyclic peptide frameworks offer great potential for drug design due to their improved bioavailability compared to their linear analogues. Using the sunflower trypsin inhibitor-1 (SFTI-1) peptide scaffold in combination with systematic N-methylation of the grafted pharmacophore led to the identification of novel subtype selective melanocortin receptor (MCR) agonists. Multiple bicyclic peptides were synthesized and tested towards their activity at MC1R and MC3–5R. Double N-methylated compound 18 showed a pKi of 8.73±0.08 (Ki = 1.92±0.34 nM) and a pEC50 of 9.13±0.04 (EC50 = 0.75±0.08 nM) at the human MC1R and was over 100 times more selective for MCR1. NMR structural analysis of 18 emphasized the role of peptide bond N-methylation in shaping the conformation of the grafted pharmacophore. More broadly, this study highlights the potential of cyclic peptide scaffolds for epitope grafting in combination with N-methylation to introduce receptor subtype selectivity in the context of peptide-based drug discovery.

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Introduction The mammalian melanocortin (MC) system is an evolutionary ancient regulatory system implicated in a wide range of important physiological functions, including food intake, energy metabolism, skin pigmentation, sexual function and inflammation.1 These physiological effects are mediated by five MC receptor subtypes (MC1–5R), which are members of the rhodopsin-like G-protein coupled receptor (GPCRs) family. MCRs are activated by several endogenous peptide ligands, including adrenocorticotropic hormone (ACTH), α/β/γ-melanocyte stimulating hormone (MSH) and agouti-related protein (AGRP).2 They constitute drug targets for obesity and inflammatory diseases. However, it is still unclear which specific isoform or combination of MCRs needs to be targeted in each particular disease state.3-4 Furthermore, the design of subtypespecific molecules is challenging because of the high degree of sequence and structure similarity among MCRs.5 The natural hormones themselves are unsuitable as drugs or probes because of their low selectivity as well as short half-life in vivo.6 Organisms achieve selective activation of MCR isoforms by distinct tissue distribution of the MCRs as well as by the spatial release of the native hormones. The MSH peptides and ACTH share the tetrapeptide His-Phe-Arg-Trp (HFRW) pharmacophore, which is the shortest peptide active on MCRs.6 In recent years, numerous modifications to this small sequence have been made to explore the role of individual residues and to obtain molecules with increased potency and selectivity.5 One strategy that has proven particularly successful is to conformationally constrain the tetrapeptide pharmacophore (typically through sidechain-to-sidechain cyclization), thereby reducing conformational flexibility and minimizing entropic losses upon receptor binding. This approach has resulted in highly potent (IC50/EC50


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~pM) MCR ligands, including melanotan (MT)-I and –II,7 RM4938 and bremelanotan (Pt-141).9 However, despite their high potency, the MCR subtype selectivity of these molecules is modest.

Peptides are widely regarded as excellent drug leads because of their high potency and specificity for pharmacological targets, but typically they are under-represented as drugs because of their shortcomings of poor oral bioavailability and susceptibility to proteolysis in the body.10 Recent advances in peptide design and synthesis have helped to ameliorate these shortcomings and to better exploit the enormous potential of peptides as drugs.10 One particularly successful approach involves backbone cyclized peptides, which have a number of advantages over their linear counterparts, including well-defined conformations and high resistance to proteolysis.11 Cyclosporin is a prime example of a naturally occurring cyclic peptide that has been approved as a drug,12 and a number of other synthetic cyclic peptides are in advanced stages of clinical trials.13 However, these examples typically comprise small highly constrained peptides that lack disulfide bonds, and hence are missing an opportunity for further stabilization. Recent approaches of applying cyclization to larger disulfide-rich peptides, include the artificial cyclization of bioactive conotoxins14 and the ‘grafting’ of bioactive linear peptides into stable, naturally-occurring cyclic disulfide-rich peptide frameworks.15 For example, we recently demonstrated that incorporation of the HFRW pharmacophore into the 29-amino acid residue kalata B1 cyclotide resulted in a potent MC4R-selective agonist.16

Sunflower trypsin inhibitor-1 (SFTI-1) is a small (14 residue) naturally-occurring disulfidecontaining cyclic peptide. As its name suggests, it was originally derived from the seeds of sunflowers and has attracted attention as a stable peptide template.17 This macrocyclic peptide is



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an exceptionally potent trypsin inhibitor (Ki = 2 pM)18 and features N-to-C backbone cyclization as well as a single disulfide bond. This architecture gives rise to a rigid bicyclic structure in which one of the two loops between Cys residues adopts a β-turn conformation while the other forms a ω-turn (Figure 1).19 The former loop harbours the biosynthetic cyclization site (Asp-Gly) and is hereafter referred to as cyclization loop, whereas the latter contains the lysine P1 (Lys3 in Table 1) residue critical for anti-trypsin activity referred to as the binding loop.

Homology modelling and functional data have suggested that MCR ligands bind using a β-turn conformation into a deep acidic pocket situated between the seven MCR transmembrane helices.20 This proposed binding mode suggests that the SFTI-1 scaffold is ideal for displaying MCR-targeting pharmacophores. To further constrain the conformational freedom of the grafted pharmacophore, we systematically introduced N-methylation of specific peptide bonds and evaluated their pharmacological and structural impact on MCR subtype selectivity. Our results show that by grafting the MC pharmacophore into SFTI-1 in combination with N-methylation of selected peptide bonds, this stable cyclic trypsin inhibitor scaffold can be converted into highly potent and selective GPCR agonists.


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Results Design and chemical synthesis The MSH-derived HFRW tetrapeptide has been shown to be the minimal core sequence for activating MCRs6, 21 and therefore was chosen as the key epitope for the grafting approach. The bicyclic SFTI-1 framework (1) comprises two structurally different loops, referred to as the binding and cyclisation loops, and we envisioned incorporation of the HFRW pharmacophore into either loop in the various designed peptides. Figure 1 gives an overview of the design strategy. For grafts in the binding loop, the region comprising Lys3–Pro6 was chosen as the insertion site for the HFRW motif to optimally place the pharmacophore in the anticipated turn conformation. Using a similar line of thought, Phe10–Gly13 was chosen for grafts in the cyclization loop. In the latter designs, Lys3 was substituted by Ala to abolish native trypsin inhibitory activity.22

Extensive structure-activity relationship (SAR) studies over recent years have established that inversion of the configuration of L-Phe in the HFRW motif (HfRW) generally results in more potent MCR analogs. To determine the role of the Phe residue configuration in the context of the SFTI-1 framework, both diastereoisomers were synthesized (2,3 and 11,12). Pharmacological analysis revealed that the D-Phe-containing diastereoisomers were at least an order of magnitude more potent than the L-Phe analogs (see below and Table 1). Hence, all further SAR studies were carried out using the D-Phe containing pharmacophore. An N-methyl scan of the HfRW epitope was conducted to modify the conformational space with the aim to improve potency and to gain subtype selectivity. A similar strategy was recently used to tune subtype specificity and potency of MT-II.23 N-Methylation of the D-Phe residue was spared because in previous studies it



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resulted in a complete loss of activity for every MCR.23 The N-methylation pattern as well as the grafted sequences are summarized in Figure 2 and Table 1. In total, 19 SFTI-1 analogs were prepared by automated Fmoc solid-phase peptide synthesis. The yields of the cyclic and Nmethylated products after two rounds of RP-HPLC purification were acceptable at 5–20% (based on initial resin loading). In line with previous observations,24-25 acylation of the secondary amine of N-methylated residues was found to be slow and inefficient, which necessitated double couplings and extended coupling times (2 h) to prevent accumulation of significant amino acid deletion side-products.

Pharmacological characterization The grafted analogs were tested for binding affinities at MC1R and MC3–5R in competition binding assays using 100 pM radiolabeled [125I]-NDP-αMSH (Figure 3A). Activity at MC2R was not tested because this receptor subtype is structurally and pharmacologically different to the other MCRs (for example, unlike all other subtypes, MC2R is not activated by α-MSH and it’s ACTH-signaling is strictly dependent on the interaction with MC2R accessory protein (MRAP)).26-27 Functional activation of MC1R, 3R and 4R was evaluated via cAMP assays (Figure 3B). Because most analogs showed only weak competitive binding to MC5R, receptor activation was not evaluated further. In the cAMP assays, receptor expression was induced via doxycycline-controlled transcriptional activation. For hMC4R, two doxycycline concentrations were used (low (L) = 0.1 ng/mL and high (H) = 10 ng/mL) to better characterize ligand behavior at low and high receptor expression levels, respectively. The higher doxycyclin concentration leads to excess of receptors resulting in apparent enhancement of potencies, in this case approximately 10-fold compared to the lower doxycyclin condition.


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The unmodified SFTI-1 framework (1) and the positive controls MT-II, RM493 and bremelanotide were included for comparison. The pharmacological data are summarized in Table 1 and reveal a range of potencies and selectivities for the various MCRs. As expected, the wildtype SFTI-1 framework did not compete with NDP-αMSH in binding to any of the MCRs tested and failed to induce any cAMP response at concentrations up to 100 µM. Insertion of the HFRW pharmacophore, or the improved HfRW sequence, into the binding (2, 3) or cyclization (11, 12) loops resulted in molecules with modest agonist activities, primarily at hMC1R (EC50’s: nM to µM). As noted above, the D-Phe carrying compounds 3 and 12 showed substantially stronger activity in competition and functional assays than the corresponding molecules harboring L-Phe (2, 11).

N-methylation of selected peptide bonds within the HfRW-binding loop grafts (compounds 4– 10) produced mixed results. Single N-methylation of either His or Trp led to molecules with noticeably reduced activity at MC1R, 3R and 4R. In contrast, N-methylation of Arg (compound 5) resulted in partly reduced activity at MC1R, whereas activity at MC3R was slightly increased and substantially increased by at least one order of magnitude against MC4R, respectively. Double N-methylation of either His/Arg (7) or His/Trp (8) yielded molecules with reduced activity at all MCRs, whereas N-methylation of both Arg and Trp (9) resulted in a selective MC1R agonist. Activity against MC3R and 4R was significantly reduced, but essentially unchanged for MC1R when compared to the non-N-methylated parent compound 3. The triple Nmethylated analog 10 only showed weak micromolar activity (EC50/Ki) at MC1R and was inactive at MC3/4R.



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Surprisingly, the N-methyl scan of the cyclization loop graft (12–19) produced markedly different outcomes. Single N-methylation of His (13) resulted in loss in activity by at least an order of magnitude across all MCRs relative to the non-N-methylated parent molecule 12. In contrast, N-methylation of either Arg (14) or Trp (15) was well tolerated, particularly by MC1R. Compound 14 has at least 200-fold selectivity for MC1R over MC3R and 4R with low nanomolar Ki (pKi = 8.24±0.06) and sub-nanomolar agonist activity at MC1R (pEC50 = 9.28±0.05). N-methylation of Trp (15) on the other hand produced a potent pan-specific agonist active on MC1R, 3R and 4R. Interestingly, of all the molecules synthesized, this analog also displayed by far the strongest competitive binding to MC5R with a pIC50 of 7.23±0.01. The beneficial effects of Arg/Trp N-methylation as well as the deleterious effects of NMeHis were partially recapitulated in the dual N-methylated cyclization loop grafts (compounds 16–18). All of these molecules exhibit low nM potency in binding and functional assays for MC1R with varying degrees of selectivity over MC3R and 4R. Compound 18 containing the NMeArg-NMeTrp dipeptide motif, for example, is the most potent molecule identified in this study (pKi = 8.73±0.08, pEC50 = 9.13±0.04) and has at least 100-fold selectivity over MC3R and 4R in binding and functional assays. Introduction of an additional methyl group at the NMeHis in compound 19 yielded an agonist with slightly reduced activity at MC1R (pKi = 8.02±0.16, pEC50 = 9.03±0.06), but with at least 250-fold improved selectivity over MC3R and 4R. Compounds 5, 12, 14 and 18 were tested for residual trypsin inhibitory activity. As expected, all compounds exhibited greatly reduced anti-trypsin activity when compared to wild-type SFTI-1 (Ki = 0.014 ± 0.001 nM, n=3). Activity of compound 5 was reduced by more than 4 orders of magnitude (Ki = 310 ± 14 nM, n=3) while 12, 14, 18 only showed partial inhibition at 5 µM (Supplementary Figure S2). The in vitro stability of compounds 5, 14 and 18 was assessed in


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human serum after 12h incubation at 37˚C. Compounds 5 and 18 retained the excellent stability of the parent SFTI scaffold (~70% recovery after 12h), while compound 14 was slightly less stable (35% recovery).

Structural investigations by NMR All compounds were structurally evaluated by 1D and 2D homo- and heteronuclear NMR spectroscopy to verify compound identity and to examine the structural consequences of grafting into the SFTI-1 framework and of backbone N-methylation. The parent SFTI-1 scaffold adopts a highly rigid and well-defined 3D structure characterized by two short β-strands connected via a type I β-turn (10FPDG13, 1) and an extended ω-turn (2TKSIPPI8).19 The three proline residues of SFTI-1 adopt well defined conformations: the Ile5-Pro6 peptide bond is in a cis conformation, whereas Pro6-Pro7 and Phe10-Pro11 are in trans conformations.19 We expected that inserting the HFRW pharmacophore into either binding or cyclization loops would result in changes to the backbone conformation of SFTI-1, which would then be further affected by N-methylation. NMR analysis was carried out in water at 298 K and pH 3–4. The NMR spectra were recorded at low pH as the amide protons in peptides of the SFTI-1 size typically exchange quickly at physiological pH and can limit the information available from the spectra. Although Nmethylation generally increased the RP-HPLC retention time of the synthesized molecules, all analogs were water-soluble to at least 1 mM concentration. Under the studied conditions several of the peptides displayed multiple conformations, which slowly interconvert on the NMR time scale. The NMR fingerprint region of compound 2, for example, indicates three conformers that coexist in a ratio of approximately 60:30:10, while under the same conditions compounds 11 and 18 showed two isomers in a ~80:20 ratio. Evidence for chemical exchange is based on



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observations that: a) the isomer ratios were affected by temperature; b) NMR exchange peaks appeared at high temperatures (>50 ºC) and long EXSY mixing times (1 sec); and c) isomers that could be separated by RP-HPLC resulted in the original two isomer profile when re-injected. Collectively, these observations suggest a conformational exchange process that is slow on the NMR and HPLC time scales and rule out the possibility that the observed species are co-eluting side products derived from chemical synthesis. Most grafts in the cyclization loop (11–19) had a single conformation, whereas grafts in the binding loop (2–10) tended to adopt multiple conformations, complicating NMR analysis.

Three-dimensional structures of key grafted SFTI-1 analogs Three-dimensional structures were determined for a selection of analogs to provide insights into SAR. Structures of the binding loop graft 5 and the cyclisation loop grafts 12, 16 and 18 were determined using CYANA and torsion angle dynamics. For analogs 5, 12 and 18, one major conformation was observed for each peptide, and 20 out of 50 structures with the best MolProbity scores were chosen to represent the ensemble (Figure 4, Table 2). For analog 16, 20 out of the 50 calculated structures adopted one conformation, whereas two distinct (but equally populated) conformations were present across the remaining 30 structures. The differences in conformers occurred across residues 10–14 in the grafted loop, as highlighted by the differing dihedral angles across this section of the peptide. MolProbity scores and statistics are given in Table 2 and dihedral angles across the grafted pharmacophore residues for analogs 5, 12, 16 and 18 are reported in Table 3.


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Compound 5 was the only binding loop graft adopting a well-defined conformation. The conformation of the unchanged cyclization loop in 5 was identical to that observed in SFTI-1 (1) and involved a type-I β-turn that is stabilized by a hydrogen bond between Phe10 (NH) and Arg14 (CO) as evidenced by H/D exchange and temperature coefficient NMR experiments. The two prolyl peptide bonds (Trp6-Pro7 and Phe10-Pro11) both adopted well-defined trans conformations showing strong Phe/Trp Hα – Pro Hδ NOEs. The pharmacophore-embedded binding loop conformation is different from SFTI-1 and, based on the measured Φ and Ψ backbone dihedral angles, does not adopt a classical turn motif. The D-Phe4-NMeArg5 peptide bond is predominantly in a trans configuration, but a minor alternative conformation was identified around NMeArg5, indicating local conformational heterogeneity, probably caused by cis/trans isomerization.

Compound 12, featuring a non-N-methylated HfWR pharmacophore in the cyclization loop, adopts a single conformation in solution. The minor structural change in the binding loop [Thr2Ala] results in a conformation that is virtually identical to wild-type SFTI-1 and includes the preserved configurations of the Ile5-Pro6 (cis) and the Pro6-Pro7 (trans) peptide bonds, as evidenced by strong NOEs between Ile5 Hα – Pro6 Hα and Pro6 Hα – Pro7 Hδ, respectively. According to the average Φ and Ψ backbone dihedral angles in the 20 NMR models (see Table 3) and amide temperature coefficients, the HfRW pharmacophore adopts a type II’ β-turn that is stabilized by a hydrogen bond between His10 (NH) and Arg14 (CO). This turn type frequently features a Gly residue in position 2 (i+1), which is occupied by a D-Phe in 12.



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Analog 16, displays the double N-methylation of the pharmacophore residues NMeHis10 and NMe

Arg12, and the conformation of its binding loop is highly similar to SFTI-1 and 12. By

contrast, the conformation of the HfRW pharmacophore-bearing loop is different from 12 in that: a) it lacks internal hydrogen bonding as suggested by NMR H/D exchange and temperature coefficient experiments, b) the Φ and Ψ backbone dihedral angles do not fall into any standard turn category, and c) despite the presence of two N-methylations, all peptide bonds in this loop adopt a trans conformation (experimentally supported by the absence of defined (i) Hα – (i+1) Hα NOEs).

The structure of 18, one of the most potent MC1R agonists identified in this study carrying both NMe

Arg12 and NMeTrp13, recapitulated several features observed in 12 and 16. The conformation

of the unmodified binding loop is similar to that of SFTI-1, although some structural flexibility around Ala3 was observed in some of the calculated structures. As in 16, the HfRW loop adopts a non-standard turn conformation and lacks internal stabilizing hydrogen bonding. In contrast to 12 and 16, dual N-methylation of the adjacent Arg12 and Trp13 induces a dominant cis conformation of the Arg12-Trp13 peptide bond as evidenced by a strong Arg12 Hα - Trp13 Hα NOE. Discussion and Conclusions In this study we used the SFTI-1 cyclic peptide scaffold to design selective agonists for MC receptors. The most potent analog (compound 18) showed high agonist potency at MC1R and high selectivity over the other MCRs tested, highlighting the potential of the SFTI-1 scaffold for targeting specific GPCRs. Previously, engineering of this Bowman-Birk type protease inhibitor focused on analogs with altered protease specificity and led to potent and selective inhibitors of 13 ACS Paragon Plus Environment

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medically important serine proteases (cathepsin G, KLK4, 5, 7 and 14).28-30 Recently, the Tam group reported the design of an orally-active analgesic SFTI-1 analog antagonizing the bradykinin B1 receptor.31 Here, we extend the applicability of SFTI-1 scaffold by demonstrating its suitability to target GPCRs and to incorporate N-methylated amino acids. Peptide N-methylation has been a favored tool for medicinal chemists to help overcome the traditional drawbacks of peptides.32-33 Specifically, N-methylation has been shown to improve selectivity and biological activity of the modified scaffold23, 34 and has helped to enhance pharmacokinetic properties by boosting metabolic stability and improving bioavailability.35-36 Furthermore, N-methylation increases the lipophilicity and thereby potentially the membrane permeability of a peptide scaffold and, in some cases, can even lead to oral bioavailability such as found for cyclosporine.12

In the present study the major effect of N-methylation of various peptide bonds within the SFTI1 scaffold is the introduction of conformational constraints. These constraints enhance the population of single conformers that are only transiently present in their non-N-methylated analogs and thereby induce biological activity and especially subtype selectivity. A similar strategy has been recently applied to modulate MCR subtype selectivity, mode of action and potency of MT-II23 and SHU9119.37

Incorporation of the unmodified HFRW tetrapeptide into the cyclization loop of the SFTI-1 framework (11) produces an agonist with modest MCR activity, while grafting the same sequence into the binding loop does not (2). In case of the latter, and in line with previous observations, significant activity could only be established by inversion of the configuration of



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Phe4 (3). The subsequent N-methylation scan of the binding loop grafts generally produced modest results: compared to compound 3 the activity of most analogs was significantly reduced or unchanged. However, the most interesting compound (5) with a single NMeArg5 showed significantly improved agonist activity at MC4R, while activity at MC1R was unchanged. The modest pharmacological data for 2-10 are reflected in the NMR structural studies of these compounds, revealing pronounced conformational heterogeneity in most binding loop grafts (except 5). The molecular basis for this heterogeneity has not been established, but is probably caused by slow (ms-sec timescale) cis/trans isomerization particularly around N-methylated peptide bonds and/or Xaa-Pro bonds. Collectively these data suggest that N-methylation of 3 did not produce a stable conformation that could more favorably interact with MCRs (with the exception of 5 and overall unaffected, modest MC1R agonism in general).

By contrast, N-methylation of the cyclization loop grafts produced several molecules with improved potency and altered selectivity compared to the non-N-methylated parent molecule 12. In particular, N-methylation of Arg12 or Trp13 increased potency at MC1R by at least one order of magnitude together with slight gains in activity at MC3/4R. Further improvements were observed in the double Arg/Trp N-methylated analog 18, suggesting that the beneficial effects are additive. In contrast, N-methylation of His10 generally was not well tolerated, unless combined with N-methylation of Trp13 (17).

From a mechanistic perspective, all analogs acted as full agonists, typically resulting in 100±10 % MCR activation and competition binding when compared to α-MSH. This finding suggests that the effects of N-methylation of the SFTI-HFRW motif are subtle and do not cause major


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mechanistic changes in these compounds, contrasting with N-methylated variants of MT-II (agonist-partial agonist switch) or SHU9119 (agonist-antagonist switch).23, 37 Future structural analysis of the SFTI-1 analogs in complex with MCRs will facilitate this process and allow a greater understanding of the effects of the mutations on potency and selectivity.

Overall, pharmacological analysis and N-methyl scanning revealed several new HfRW-grafted SFTI-1 molecules acting as potent and selective MC1R agonists. The in vitro potencies and selectivities achieved for MC1R in 18 or 19 compare favorably to a recently described tetra Nmethylated analog of MT-II23 as well as a rationally designed γ-MSH derivative.38 For comparison, the latter compound has a pIC50 of 7.6 and a pEC50 of 8.3 at hMC1R and modest (16-fold) selectivity over hMC4R.38 Whereas several of our analogs had varying degrees of activity at MC3R and MC4R, only one had appreciable binding to MC5R (15). The initial selectivity profiles observed in a largely non-optimized molecular framework suggests plenty of scope for further pharmacological optimization. In this context, the bicyclic structure of the grafted SFTI-1 molecules offers the unique opportunity to co-incorporate other functionalities, such as tissue targeting epitopes, molecular tracers, or MCR-unrelated bioactivities. The feasibility of this approach is supported by our NMR studies, suggesting that both binding and cyclization loop can be grafted independently without significantly affecting the conformation of the other unmodified loop. This successful example of grafting a function into SFTI-1 adds to a growing body of evidence that naturally-occurring cyclic peptide frameworks can accommodate a diverse range of bioactivities. With further optimization, this group of peptides could provide new peptide-based leads with the potential to treat MCR-related disorders.



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Experimental Section Peptide synthesis All peptides were synthesized via automated Fmoc-SPPS on a 2-chlorotrityl resin using 2-(6chloro-1-H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU) as coupling reagent. To form the head-to-tail cyclized backbone, the fully sidechain protected peptides were cleaved from the highly acid labile solid support and cyclized in solution using 2(7-Aza-1-H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU).39 Following side-chain deprotection (TFA:H2O:TIPS (95:2.5:2.5), 2,5h, RT) and purification by RP-HPLC, the two cysteine residues of the cyclic peptides were oxidized to disulfides in 50% acetonitrile (ACN)/H2O, containing 20 eq. iodine. The desired product was isolated following another round of RP-HPLC purification and lyophilized. N-Methylated residues were introduced by standard coupling using suitably sidechain protected N-Fmoc-N-methyl amino acid building blocks (Fmoc-NMeArg(Pbf), Fmoc-NMeTrp(Boc), FmocNMe

His(Trt), Chempep, USA). Following Fmoc deprotection (30% piperidine/DMF, 2 x 30min),

acylation of the secondary amine with the next Fmoc amino acid was performed twice with HATU and coupling times were extended to 2 h. The resin was capped by acetylation with acetic anhydride/DIEA/DMF and the synthesis continued using standard protocols. Peptides were purified by RP-HPLC on a Shimadzu Prominence system at room temperature using Phenomenex Jupiter C18 columns (5 µm, 300 Å, 250 x 21.2 mm or 250 x 10 mm) with gradients of 0.05% aqueous TFA (buffer A) to 90% acetonitrile and 0.045% TFA (buffer B) over 60 min and a flow rate of 8 mL/min. The eluent was monitored at 214 nm and 280 nm. The final products were characterized by electrospray ionization mass (ESI-MS), 1D and 2D 1H NMR and


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analytical RP-HPLC on a Phenomenex Jupiter C18 column (5 µm, 300 Å, 150 x 2 mm, see Methods and Supplementary Figure S1). All compounds had a purity of at least 95%. MS data for all peptides were acquired on an Applied Biosystems 4700 proteomics analyzer in positive reflectron mode using α-cyano-4-hydroxycinnamic acid (CHCA) as matrix. Calculated molecular weight values refer to most abundant isotope compositions. 1: Mfound: 1512.68 ± 0.08 Da, Mcalc: 1512.74 Da; 2: Mfound: 1713.91 ± 0.10 Da, Mcalc: 1713.78 Da; 3: Mfound: 1713.84 ± 0.10 Da, Mcalc: 1713.78 Da; 4: Mfound: 1727.72 ± 0.10 Da, Mcalc: 1727.79 Da; 5: Mfound: 1727.81 ± 0.10 Da, Mcalc: 1727.79 Da; 6: Mfound: 1727.78 ± 0.10 Da, Mcalc: 1727.79 Da; 7: Mfound: 1741.90 ± 0.10 Da, Mcalc: 1741.81 Da; 8: Mfound: 1741.88 ± 0.10 Da, Mcalc: 1741.81 Da; 9: Mfound: 1741.78 ± 0.10 Da, Mcalc: 1741.81 Da; 10: Mfound: 1755.88 ± 0.10 Da, Mcalc: 1755.83 Da; 11: Mfound: 1665.78 ± 0.10 Da, Mcalc: 1665.82 Da; 12: Mfound: 1665.81 ± 0.10 Da, Mcalc: 1665.82 Da; 13: Mfound: 1679.79 ± 0.10 Da, Mcalc: 1679.84 Da; 14: Mfound: 1679.80 ± 0.10 Da, Mcalc: 1679.84 Da; 15: Mfound: 1679.89 ± 0.10 Da, Mcalc: 1679.84 Da; 16: Mfound: 1693.91± 0.10 Da, Mcalc: 1693.85 Da; 17: Mfound: 1693.87 ± 0.10 Da, Mcalc: 1693.85 Da; 18: Mfound: 1693.79 ± 0.10 Da, Mcalc: 1693.85 Da; 19: Mfound: 1707.78 ± 0.10 Da, Mcalc: 1707.87 Da. NMR Peptides were dissolved at a concentration of ~1 mM in 10% D2O and 90% H2O. Spectra were recorded at 298 K on a Bruker AVANCE 600 MHz NMR spectrometer equipped with a cryogenically cooled probe. Two-dimensional spectra recorded included TOCSY, NOESY and 1

H-13C HSQC. The mixing times for the TOCSY and NOESY spectra were 80 ms and 200 ms

respectively. The processed spectra were analyzed using the program CcpNmr Analysis.40-41 For 5, 12, 16 and 18 additional experiments including 1H-15N-HSQC, ECOSY and TOCSY spectra recorded at various temperatures (280–305 K) were acquired and three-dimensional structures 18 ACS Paragon Plus Environment


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were calculated from the recorded two-dimensional spectra using CYANA42 as described previously. Structures were analyzed using MolProbity43 and PROMOTIF44 and represented using MOLMOL45 and PyMol. Chemical shifts for 5, 12, 16 and 18 are reported in Supplementary Tables 1-4 and have been deposited in the BMRB database (5: 30382; 12: 30380; 16: 30383; 18: 30381). Structural coordinates have been deposited in the PDB (5: 6BVX; 12: 6BVU; 16: 6BVY; 18: 6BVW).

DNA constructs, cell culturing, transfections and generation of stable cell lines cDNAs encoding human MC1 (Genbank accession number NM_002386), MC3 (NM_019888), and MC4 (NM_005912) and MC5 (NM_005913) were cloned into the pcDNA4/TO vector (ThermoFisher). In the constructs, a consensus Kozak sequence (GCCACC) was incorporated immediately before the start ATG. DNA was amplified, isolated and sequenced using standard techniques.

T-Rex™-293 cells (ThermoFisher) were cultured in DMEM medium, supplemented with 10% fetal bovine serum and 5 µg/mL blasticidin at 37 °C in a humidified atmosphere with 10% CO2. Lipofectamine 2000 (ThermoFisher) was used for transfections according to the manufacturer’s recommendations. Resistant polyclonal populations were generated by selection with Zeocin (0.3 mg/mL, ThermoFisher) for about two weeks after transfection. Individual clones were isolated using flow cytometry, subsequently expanded, tested for responsiveness to [Nle4,d-Phe7]-αMelanocyte Stimulating Hormone (NDP-α-MSH) and cryopreserved. To induce receptor expression, cells were treated with 10 (high) or 0.1 (low) ng/ml doxycyclin for 24 h before assay.


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Melanocortin receptor radioligand binding and cAMP assays. A 384-well format scintillation proximity assay (SPA) was used to identify compounds that show competitive binding to the human melanocortin receptor 1,3,4 or 5 ligand binding domain (LBD). Membranes expressing the melanocortin receptor bind to the wheat germ agglutinin (WGA) coated polyvinyltoluene (PVT) SPA beads. The inhibition of the scintillation signal by displacement of [125I]-NDP-α-MSH by test compounds were detected by a 1450 MicroBeta Trilux scintillation counter (PerkinElmer).

Binding assays were performed in a 384 format PS-microplate (Greiner 781095). Membranes were prepared from HEK293-6E suspension cells transiently expressing human MC1R, MC3R, MC4R or MC5R. The assays were done as follows: 20 µL WGA coated PVT SPA bead (Perkin Elmer RPNQ0001) resuspended in assay buffer (25mM Hepes pH 7, 1.5 mM CaCl2, 1 mM MgSO4, 0.2 % BSA (Sigma A3059), 1 mM 1,10-phenanthroline monohydrate (Sigma P9375)) at a final concentration of 0.5 µg/µL (MC1R and MC4R) or 2 µg/µL (MC3R and MC5R) was mixed with 0.5 µL of compounds at various concentrations or DMSO. 20 µL membrane homogenate to a final concentration of 5 µg/µL (MC1R), 50 µg/µL (MC3R) or 10 µg/µL (MC4R, MC5R) were added together with 10 µL [125I]-NDP-α-MSH (2200 Ci/mmol specific activity, PerkinElmer NEX352) to a final concentration of 0.1 nM. Plates were incubated for 16– 23 hours in the dark at room temperature before reading on a 1450 MicroBeta Trilux. Measurements of cAMP production were performed using LANCE® cAMP kits (PerkinElmer) in a total assay volume of 12 µL/well. In brief, ligands were dispensed into small-volume 384well plates (Greiner, 784075) before addition of 1000 cells/well in buffer (Hank´s balanced salt solution with Ca2+ and Mg2+ (HBSS) (Thermo Fisher), 20 mM HEPES pH 7.4 (Thermo Fisher),



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0.1% BSA (Sigma)). cAMP production was stimulated for 15 min (MC4R high dox condition) or 45 min (MC1R, MC3R, and MC4R low dox condition) at 37°C in the presence of 1 mM IBMX (Sigma) and 250 µM Alexa Fluor® 647-anti cAMP antibody (LANCE cAMP kit). The reaction was stopped with detection buffer containing 10 µM Biotin-cAMP and 15 µM LANCE EuW8044 labelled streptavidin (PerkinElmer LANCE cAMP kit). cAMP production was detected by homogenous time resolved fluorescence (HTRF) (λex = 340 nm, λem = 665 and 615 nm) using a Pherastar (BMG Labtech).

Compounds were tested in ten-point concentration response (½ log serial dilution) with both 100 and 1 µM as final start concentrations. Raw data output, counts per minutes for binding assay and time-resolved fluorescence signals for cAMP assays, was analyzed in Screener software (Genedata AG). Potency in functional agonist assays (pEC50) and competition binding assays (pIC50) were calculated using a four parameter logistic fit, y = A+((B-A)/(1+((C/x)D))), where A is curve bottom, B is curve top, C is EC50 or IC50, D is slope (Hill coefficient) and x is concentration of test compound. EC50 and IC50 refer to half maximal effective concentrations of test compounds in receptor activation or radioligand-binding inhibition assays, respectively. pEC50=log(1/EC50) and pIC50=log(1/IC50) values were reported as mean ± SE with the number of experiments n≥3. Ki values were calculated from the IC50 value using the equation Ki= IC50/1+[L]/Kd). Kd values used for [125I]-NDP-α-MSH: MC1R 0.13 nM, MC3R and MC4R 0.6 nM. Ki values were not calculated for MC5R. Table 1 shows the corresponding pKi values (=log(1/Ki).


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PDB ID Codes: Structural coordinates have been deposited in the PDB (5: 6BVX; 12: 6BVU; 16: 6BVY; 18: 6BVW). Authors will release the atomic coordinates and experimental data upon article publication. Corresponding Author Information: email: [email protected] Present Author addresses: d

Department of Molecular, Cellular & Developmental Biology, Yale University, New Haven, CT

06511, USA. e

Queensland Tropical Health Alliance, School of Pharmacy and Molecular Sciences and Centre

for Biodiscovery and Molecular Development of Therapeutics, James Cook University, Cairns, QLD 4870, Australia. j

Integrated Drug Discovery, Sanofi-Aventis Deutschland GmbH, Industriepark Hochst, D-65926

Frankfurt am Main, Germany

Author Contributions: T.D., P.M.C., J.W., A.A.F. and O.C. designed and performed the chemical synthesis of SFTI analogs. Y.Z., A.D., T.Ö., N.L., L.K. and M.C. performed pharmacological analysis. T.D., A.M.W., C.I.S., P.J.H., N.L.D. and D.J.C. performed NMR spectroscopy and structure calculations. T.D., P.M.C., Q.K., L.K., U.B., H.K., V.J.H., A.T.P. and D.J.C. performed data analysis. T.D., P.M.C. and D.J.C. wrote the manuscript with input from all authors. T.D., U.B., H.K., M.C., V.J.H., A.T.P. and D.J.C. coordinated the work and secured funding. T.D and P.M.C. contributed equally.



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Acknowledgments This work was funded by an Australian Research Council (ARC) Australian Laureate Fellowship (FL150100146) to D.J.C., an ARC Discovery Project grant (DP150100443 awarded to D.J.C. and T.D.) and a National Health and Medical Research Council project grant (APP1084604 awarded to D.J.C, T.D., Q.K. and C.I.S). P.M.C. is thankful for a German Academic Exchange Service (DAAD) scholarship and C.I.S. is an ARC Future Fellow (FT160100055). This work was also supported by the Deutsche Forschungs-gemeinschaft to H.K. Koselleck grant KE147/42-1.

Abbreviations used: AGRP, agouti related protein; ACTH, adrenocorticotropic hormone; MCR, melanocortin receptor; MRAP, MC2R accessory protein; MSH, melanocyte stimulating hormone.

Competing financial interests statement: The authors declare no competing financial interests.

Supporting Information: NMR chemical shifts for compounds 5, 12, 16, 18. NMR spectra of all compounds. Trypsin inhibition activity and human serum stability data. Molecular formula strings and associated pharmacological data in CSV file format.


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Figure Legends: Figure 1. Schematic representation of the SFTI-1 grafting strategy. The structure of wild-type SFTI-1 is shown in the left panel (PDB ID: 1JBL)19 and the grafted analogs in which the MCR pharmacophore has been inserted into the cyclization or binding loops are schematically shown in middle and right panels, respectively.

Figure 2. Structural variations of the HFRW pharmacophore. A systematic N-methyl scan is performed on the grafted pharmacophore resulting in three single-, three double- and one triple N-methylated SFTI-1 derivatives per loop.

Figure 3. Pharmacological characterization of grafted SFTI-1 molecules (compounds 1, 2, 3, 11, 18, 19 and Bremelanotide) at the human MCR1 expressed in HEK293 cells. Competitive radioligand binding assay using 100 pM [125I]-NDP-α-MSH (A) and functional cAMP production assay (B). The cAMP response was normalized to the maximum effect stimulated by NDP-α-MSH. Symbols represent the same compounds in panels A and B. Values shown are means ± SEM of three to five independent experiments.

Figure 4. Comparison of backbone conformations observed in SFTI-1 (PDB ID: 1JBL),19 5, 12, 16, and 18. For each analog, the peptide backbone and disulfide crosslinks of the calculated 20 lowest energy NMR structures are shown. The N-methyl groups are shown as light-blue spheres. Disulfide bridges are in yellow sticks, nitrogen atoms in blue, and oxygen in red.



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Figure 1


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Figure 2



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Figure 3


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Figure 4


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Table 1. Systematic N-methyl scan of the SFTI-HFRW framework #

Sequenceb pKi

hMC1R pEC50

pKi

hMC3R pEC50

pKi

hMC4R pEC50 (L)c

pEC50 (H)c

hMC5R pIC50

c[CTKSIPPICFPDGR] NA NA NA NA NA NA NA NA 1 a c[CTHFRWPICFPDGR]

Development of Novel Melanocortin Receptor Agonists Based on the

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