Development of Novel CXC Chemokine Receptor ... - ACS Publications

Subscriber access provided by NEW YORK UNIV

Article

Development of Novel CXC Chemokine Receptor 7 (CXCR7) Ligands: Selectivity Switch from CXCR4 Antagonists with a Cyclic Pentapeptide Scaffold Shinya Oishi, Tomoko Kuroyanagi, Tatsuhiko Kubo, Nicolas Montpas, Yasushi Yoshikawa, Ryosuke Misu, Yuka Kobayashi, Hiroaki Ohno, Nikolaus Heveker, Toshio Furuya, and Nobutaka Fujii J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00216 • Publication Date (Web): 04 Jun 2015 Downloaded from http://pubs.acs.org on June 11, 2015

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 30

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

Journal of Medicinal Chemistry

Development of Novel CXC Chemokine Receptor 7 (CXCR7) Ligands: Selectivity Switch from CXCR4 Antagonists with a Cyclic Pentapeptide Scaffold Shinya Oishi,*,† Tomoko Kuroyanagi,† Tatsuhiko Kubo,† Nicolas Montpas,‡,§ Yasushi Yoshikawa,|| Ryosuke Misu,† Yuka Kobayashi,† Hiroaki Ohno,† Nikolaus Heveker,‡,§ Toshio Furuya,|| and Nobutaka Fujii*,† †

Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan, ‡

§

Département de Biochimie, Université de Montréal, Montréal H3T 1J4, Canada,

Research Centre, Sainte-Justine Hospital, University of Montreal, Montréal H3T 1C5, Canada, and ||

Drug Discovery Department, Research & Development Division, PharmaDesign Inc., 2-19-8 Hatchobori, Chuo-ku, Tokyo 104-0032, Japan e-mail: [email protected] (S.O.); [email protected] (N.F.)

RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required according to the journal that you are submitting your paper to) *To whom correspondence should be addressed: Shinya Oishi, Ph.D. and Nobutaka Fujii, Ph.D. Tel.: +81-75-753-4551;

Fax:

+81-75-753-4570;

e-mail:

[email protected]

[email protected] (N.F.)

ACS Paragon Plus 1 Environment

(S.O.);


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

Page 2 of 30

Abstract: The CXC chemokine receptor 7 (CXCR7)/ACKR3 is a chemokine receptor that recognizes stromal cell-derived factor-1 (SDF-1)/CXCL12 and interferon-inducible T-cell α chemoattractant (ITAC)/CXCL11. Here, we report the development of novel CXCR7-selective ligands with a cyclic pentapeptide scaffold through an SAR study of CXC chemokine receptor 4 (CXCR4)-selective antagonist FC131 [cyclo(-D-Tyr-L-Arg-L-Arg-L-Nal-Gly-), Nal = L-2-naphthylalanine]. Substitution of Gly with L-Pro switched the receptor preference of the peptides from CXCR4 to CXCR7. The SAR study led to the identification of a potent CXCR7 ligand, FC313 [cyclo(-D-Tyr-L-Arg-L-MeArg-L-NalL-Pro-)], which recruits β-arrestin to CXCR7. Investigations via receptor mutagenesis and molecular

modeling experiments suggest a possible binding mode of the cyclic pentapeptide CXCR7 agonist.

Introduction CXC chemokine receptor 7 (CXCR7, also recently renamed as atypical chemokine receptor 3 (ACKR3)) is a receptor of stromal cell-derived factor 1 (SDF-1)/CXCL12.1,2 CXCL12 binding to CXCR7 leads to extracellular signal-regulated kinase 1/2 phosphorylation through β-arrestin recruitment to CXCR7 without activation of G-protein dependent signaling.3,4 When CXCR7 forms a heterodimer with CXC chemokine receptor 4 (CXCR4), which is another receptor of CXCL12, in the same cells, the heterodimer regulates G-protein-mediated signaling.5,6 CXCR7 is involved in a number of physiological and pathological processes, including embryonic development,5 tumor progression,7-9 directional cell migration,10,11 and immune functions.12 More importantly, distinct CXCR4 and CXCR7 functions cooperatively regulate a number of biological processes including tissue migration,13,14 interneuron migration,15,16 and therapeutic homing of progenitor cells.17 A recent report also revealed that binding of opioid peptides to CXCR7 regulates the glucocorticoid oscillation.18 Several allosteric modulators of CXCR7 were originally reported to be CXCR4 antagonists.19 AMD3100/plerixafor, which is a bicyclam CXCR4 antagonist used in the clinic for the mobilization of hematopoietic stem cells, activates β-arrestin recruitment to CXCR7 as an allosteric agonist.3 An ACS Paragon Plus 2 Environment

Page 3 of 30

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


inverse agonist of CXCR4 works as an agonist of the β-arrestin pathway of CXCR7.20 In addition, a CXCL12-derived 17-mer peptides with a modification at the N-terminus is an allosteric modulators of CXCR7 that activates CXCR7 signaling.21 These observations imply that CXCR7 and CXCR4 share at least a part of the receptor surfaces to recognize similar scaffolds and/or pharmacophore substructures. FC131 (1a) is a potent CXCR4-selective antagonist with a cyclic pentapeptide scaffold (Figure 1) that was developed via a molecular size reduction study of an antimicrobial peptide, polyphemusin II.22 Although a number of the derivatives of peptide 1a with potent CXCR4 inhibitory activity have been reported,22-29 no CXCR7 binding ligands have been obtained among the various derivatives.24,25 Recently, it was demonstrated in patents that a series of cyclic hexapeptides induced CXCR7-mediated β-arrestin recruitment.30,31 Of interest, these CXCR7 ligands contain a substructure motif (Arg-Arg-Nal) of 1a. This indicates that both CXCR4 and CXCR7 may possibly recognize an identical combination of functional groups in two distinct arrangements. The other component amino acids such as Gly-D-Tyr substructure in 1a may also subserve the distinct receptor selectivity of the cyclic peptides. On the basis of this finding, we speculated that novel CXCR7 ligands with a cyclic pentapeptide scaffold can be developed via a selectivity switch from 1a. Reported herein are the design and SARs of novel CXCR7 ligands, in which peptide 1a-derived functional groups were displayed within the context of a conformationally constrained, cyclic pentapeptide platform. The possible binding modes of novel CXCR7 ligands by computational docking studies using a homology model of CXCR7 are also discussed.

Results and Discussion SAR Study for CXCR7-selective Ligands. In our previous SAR study of the derivatives of peptide 1a for CXCR4 antagonists,23 the use of chiral or conformationally constrained amino acids at the Gly5 position altered the CXCR4 antagonistic activity (Table 1). For example, L-Ala-substituted peptide 1b showed significantly lower activity for CXCR4, whereas D-Ala-substituted peptide 1c reproduced the potent bioactivity of 1a. In contrast, modification with cyclic amino acids such as L/D-Pro and L/DACS Paragon Plus 3 Environment


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

Page 4 of 30

pipecolic acid (Pic) at this position led to loss of activity towards CXCR4. These derivatives of 1a contain an identical combination of the pharmacophore elements (phenol of Tyr, two guanidine groups of Arg and naphthalene of Nal), but have diverse spatial dispositions of these elements. NMR and simulated annealing molecular dynamics analyses demonstrated different conformational preferences between 1b and 1c, which could be determined by possible 1,3-pseudo allylic strain between the L-Nal4 carbonyl oxygen and the side chain methyl group of L-Ala5.23 Initially, a series of these derivatives of 1a were evaluated for CXCR7 binding activity (Table 1). The receptor binding of the peptides to CXCR7 was measured based on the inhibitory activity against radiolabeled SDF-1 binding to CXCR7. Potent CXCR4-binding peptides 1a,c,d,j with an achiral or Damino acid at the Gly5 position of 1a showed no binding to CXCR7 at 30 µM. Peptide 1b with L-Ala5 alone bound to both CXCR7 and CXCR4 with moderate affinity [IC50(CXCR7): 18 µM; IC50(CXCR4): 23 µM], whereas peptides 1f,h with a cyclic D-amino acid (D-Pro or D-Pic) neither bound to CXCR7 nor CXCR4. Interestingly, peptides 1e,g,i, which contain an N-alkylated L-amino acid (L-Pro, L-Pic and L-MeAla, respectively) at the Gly5 position of 1a, exhibited CXCR7-selective binding [IC50, 1e: 1.5

µM; 1g: 13 µM; 1i: 15 µM]. The limited φ angle flexibility of these amino acids appears to provide favorable global conformations of the cyclic peptides for the interaction with CXCR7. The SARs of peptides 1a-j with modifications at position 5 suggested that the local conformational restriction by peptide backbone modification would determine the distinct binding preference towards CXCR7 and CXCR4. Thus, to further investigate the conformational effects, the epimers (2a-d) and Nmethylated analogs (3a-d) of 1e were included in the SAR study (Table 2). With the exception of peptide 2b (D-Arg2 substitution), which showed slightly decreased CXCR7 binding [IC50(2b): 4.6 µM], the epimers of 1e did not show measurable affinity for CXCR7 (IC50: >30 uM). Among the peptides 3ad in N-methylamino acid scanning, the L-MeArg3 modification resulted in a 1.5-fold increase in bioactivity [IC50(3c): 0.80 µM]. The other N-methylated peptides (3a,b,d) exhibited lower CXCR7 binding inhibition compared with the parent 1e. A series of peptides with modification by enantiomeric


Page 5 of 30

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


N-methylamino acids (4a-c) had a similar SAR, but slightly lower affinity in comparison with peptides 3a-c, whereas 4d exhibited no activity. No peptides among these modifications (2-4) inhibited SDF-1 binding to CXCR4 at 30 µM, indicating that L-Pro5 could be a key residue to prevent the interaction with CXCR4 for high CXCR7 selectivity. Next, we investigated the comparative significance of the side chain functional groups in peptide 3c for CXCR7 binding by an alanine-scanning experiment, in which each component amino acid (except for L-Pro) in 3c was substituted with Ala (Table 3). We expected that a series of peptides 5a-d would exhibit similar conformations to the parent peptide 3c, because of the identical combinations of amino acid chiralities. No or significantly less potent CXCR7 binding was observed in the Ala-substituted peptides 5a-d, suggesting that all the side-chain functional groups in 3c contributed to the interaction with CXCR7. D-Ala1- or L-Ala4-substituted peptides 5a,d showed no CXCR7 binding inhibition at 30 µM. In contrast, L-Ala2-substituted peptide 5b and L-MeAla3-substituted peptide 5c were 20- and 13fold less potent than 3c, respectively. These results suggest that two aromatic groups (a phenol of DTyr1 and a naphthalene of L-Nal4) are indispensable for the bioactivity via possible hydrophobic interactions, while two guanidino groups of L-Arg2 and L-MeArg3 could be further optimized for design of more potent derivatives. Of note, the indispensable functional groups is different from those of 1a, which requires a guanidino group of L-Arg3 and a naphthalene group of L-Nal4 for the interaction with CXCR4.23 CXCR7-mediated β -Arrestin Recruitment Activity of Potent CXCR7 Ligands. The potent peptides (1e and 3c) were evaluated for agonistic activity in the CXCR7-arrestin pathway (Table 4). The ability to induce the CXCR7-mediated β-arrestin recruitment was assessed by a bioluminescence resonance energy transfer (BRET)-based assay using CXCR7-yellow fluorescent protein (YFP) and βarrestin2-Renilla luciferase (RLuc) constructs.3,20,32 Peptides 1e and 3c efficiently induced CXCR7mediated β-arrestin recruitment with an EC50 of 0.074 and 0.095 µM, respectively,33 which corresponded to approximately half the potency of the known cyclic hexapeptide CXCR7 ligand 6



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

Page 6 of 30

[cyclo(-L-Ile-D-Arg-L-Arg-L-Nal-D-Pro-L-Thr-)].30,31 This suggested that cyclic pentapeptide CXCR7 ligands function as CXCR7 agonists such as SDF-1 and peptide 7 [(TC14012) H-Arg-Arg-Nal-Cys-TyrCit-Lys-D-Cit-Pro-Tyr-Arg-Cit-Cys-Arg-NH2 (S-S bridged)]. During the course of this investigation, we found that peptide 1a also showed weak activation of the CXCR7-arrestin pathway [EC50(1a): 2.4 µM]; although inhibition of SDF-1 binding to CXCR7 was not observed at 30 µM. To determine the key residue(s) on CXCR7 for receptor activation, the recruitment of β-arrestin by potent peptides (1e and 3c) was evaluated in three CXCR7 mutants (D179N, S198R, and D275N) (Table 5) as a preliminary study. D179 and D275 in CXCR7 are in the transmembrane domain 4 (TM4) and in TM6, respectively, which are both involved in peptide 7-mediated activation of the CXCR7-βarrestin pathway.32 CXCR7:S198 is located behind the conserved Cys residue (CXCR7:Cys196) in extracellular loop 2 (ECL2). SDF-1 showed comparable bioactivities for D179N and S198R mutants when compared with CXCR7/WT, whereas SDF-1 less efficiently activated the CXCR7/D275N-βarrestin pathway, probably owing to the D275N mutation itself. The activities of peptides 1e and 3c for CXCR7/WT-mediated β-arrestin recruitment were less potent compared with that of SDF-1. Peptides 1e and

3c

were

significantly less

potent

for the

D179N mutant

compared

with

SDF-1

[EC50(CXCR7/D179N), 1e: 3.5 µM; 3c: 2.0 µM], suggesting that CXCR7:D179 would be the potential interface residue for binding of 1e and 3c. In S198R and D275N mutants, the relative bioactivities among SDF-1, peptides 1e and 3c were similar to CXCR7/WT [EC50(CXCR7/S198R), 1e: 0.36 µM; 3c: 0.36 µM; EC50(CXCR7/D275N), 1e: 1.5 µM; 3c: 0.65 µM]. This was in contrast with the bioactivity of peptide 7 towards CXCR7, which was abolished by either the S198R or D275N mutation.32 These results indicate that S198 and D275 are not key residues involved in the direct interaction with peptides 1e and 3c. As such, peptides 1e and 3c bound to CXCR7 through a possible direct interaction with D179, which corresponds to the interaction of CXCR4:D171 with L-Arg3 of 1a via an electrostatic interaction.26-29,34,35 Binding Mode of Potent CXCR7 Ligands. We and others have reported the possible binding modes ACS Paragon Plus 6 Environment

Page 7 of 30

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


of 1a and the derivatives for CXCR4 by molecular modeling studies,26-29,34-36 in which an X-ray crystal structure of a complex of CXCR4 and a cyclic peptide antagonist CVX1537 were employed for calculations. On the basis of the model of the CXCR4-1a complex,34 the possible binding modes of potent CXCR7 ligands (1e and 3c) were predicted by molecular modeling studies. Initially, the CXCR7 homology models were obtained using the structure of the CXCR4-1a complex.34,38 CXCR7 ligand structures were manually generated with reference to that of 1a in the CXCR4-1a model.34 The CXCR7ligand complex structures were optimized by energy minimization. The overall binding pose of most potent 3c with CXCR7 was similar to that of 1a with CXCR4 (Figure 2). The side chain orientations of D-Tyr1, L-Arg2, L-MeArg3, and L-Nal4 in 3c were similar to that of 1a. Several interactions were conserved between CXCR4-1a and CXCR7-3c complexes; however, a number of different interactions are present. Fewer hydrogen bonds between the L-Arg2 side chain in 3c and CXCR7 (S103 side chain and C196 backbone carbonyl group) exist when compared with those in the CXCR4-1a model (Figure 2B), in which two Asp residues (D97 and D187 in CXCR4) are involved.34 The interaction between the carbonyl oxygen of L-Arg2 in 3c and the backbone NH of CXCR7:S198 is conserved (Figure 2C). L-MeArg3 in 3c also has similar interactions with H121, S125, and D179 in CXCR7, which are conserved in the CXCR4-1a model (H113, T117, and D171 in CXCR4). A binding pocket for MeArg3 in 3c was found to be larger, because of the small-sized side chain of CXCR7:S198, which is equivalent to CXCR4:R188. Nal4 in 3c formed a nonpolar interaction with the side-chain alkyl group of CXCR7:E213 in the ligand binding pocket, which is formed by L209, E213, and S216 in CXCR7. L-Pro5 is buried in the hydrophobic pocket consisting of F294 and L297 in CXCR7. To provide further insights into the SAR between 1e and 3c, which bound to CXCR7 in essentially the same binding pose, we compared the energy-minimized structures of peptides 1e and 3c in a vacuum environment (Figure 3). The backbone structure of 1e was slightly different from the one in the CXCR7-1e complex (see Supporting Information). The NH groups in Tyr1-Arg2 and Arg2-Arg3 peptide bonds formed polar interactions with a carbonyl group of the Nal4-Pro5 peptide bond (Figure ACS Paragon Plus 7 Environment


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

Page 8 of 30

3B). The backbone conformation in the energy-minimized structure of 3c was identical to that in the calculated CXCR7-3c complex, in which the carbonyl groups of D-Tyr1, L-Arg2, and L-MeArg3 were directed away from the receptor pocket (Figure 3D). The N-methyl group of MeArg3 in 3c may prevent intramolecular hydrogen bonds. These observations indicate that the more potent bioactivity of 3c for CXCR7 is attributed to the minimal energy loss that occurs upon transforming to the favorable bioactive conformation. Peptide 1a exhibits potent CXCR4 receptor binding, whereas peptides 1e and 3c show potent and selective activity towards CXCR7. These selective bioactivities were derived from a number of characteristic interactions at the ligand binding pocket(s) in each receptor, in which the interactive residues are not conserved between CXCR4 and CXCR7. For example, there are no key Asp residues in CXCR7 that contribute to the binding of Arg2 of 1a to CXCR4 in our CXCR4-1a model (D97 and D187 in CXCR4).34 In addition, two water molecules were found to mediate a key hydrogen bond network for the interaction between CXCR4:E288 and 1a, which was also observed in the CXCR4CVX15 crystal structure.37 The corresponding CXCR7:Q301 would presumably alter the hydrogen bonding network (Figure 2B). These would result in significantly weaker binding of 1a to CXCR7, although a hypothetical model of the CXCR7-1a complex could be generated without inappropriate violations. The interactions between Pro5 of peptides 1e and 3c and the counterpart hydrophobic pocket in CXCR7 would compensate these unfavorable interfaces, leading to the development of potent and selective CXCR7 ligands.

Conclusions Considering the possible similar molecular recognitions between CXCR4 and CXCR7, a series of CXCR4 antagonist peptide 1a and the derivatives with a cyclic pentapeptide scaffold were screened for inhibition of SDF-1 binding to CXCR7. A derivative 1e with L-Pro5 exhibited potent CXCR7-selective binding, suggesting that the receptor selectivity of cyclic pentapeptide ligands was altered by a local conformational restriction at this position. Peptide 3c with an Nα-methylation of L-Arg3 in 1e also ACS Paragon Plus 8 Environment

Page 9 of 30

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


showed potent CXCR7 binding and β-arrestin recruitment. It was demonstrated by bioevaluation for three CXCR7 mutants that D179 is a pivotal interactive residue for binding of peptides 1e and 3c. The molecular modeling study using a homology-modeled CXCR7 structure revealed that the binding mode and principal interactions of peptide 3c with the receptor were partially conserved when compared with the CXCR4-1a model. The slightly more potent binding of peptide 3c than peptide 1e is rationalized by the similar conformations of 3c in the free and receptor-bound states. Taken together, we developed novel CXCR7 agonist peptides with an identical combination of functional groups (phenol, two guanidine groups, and naphthalene) through an SAR study of a CXCR4 antagonist 1a. Peptide 3c (FC313) could be a promising lead peptide for further optimization to design more potent and effective CXCR7 ligands.



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

Page 10 of 30

Experimental Section General. 1H NMR spectra were recorded using a JEOL ECA-500 spectrometer. Chemical shifts are reported in δ (ppm) relative to Me4Si as an internal standard.

13

C NMR spectra were referenced to the

residual DMSO signal. For analytical HPLC, a Cosmosil 5C18-ARII column (4.6 × 250 mm, Nacalai Tesque, Inc., Kyoto, Japan) was employed with a linear gradient of CH3CN containing 0.1% (v/v) TFA at a flow rate of 1 mL/min. Preparative HPLC was performed using a Cosmosil 5C18-ARII preparative column (20 × 250 mm, Nacalai Tesque, Inc) with a linear gradient of CH3CN containing 0.1% (v/v) TFA at a flow rate of 8 mL/min. All peptides were characterized by MALDI-TOF-MS analysis and the purity of the peptides was determined by HPLC analysis (>95%).

Fmoc-based Solid-phase Peptide Synthesis. The protected linear peptides were constructed by Fmoc-based solid-phase synthesis on H-L-Pro-(2-Cl)Trt resin (0.72 mmol/g, 139 mg, 0.10 mmol), H-LNal-(2-Cl)Trt resin (0.38 mmol/g, 263 mg, 0.10 mmol), or H-L-Arg(Pbf)-(2-Cl)Trt resin (0.55 mmol/g, 182 mg, 0.10 mmol). Pbf and t-Bu groups were employed for side-chain protection of Arg and Tyr, respectively. Fmoc-protected amino acids (0.50 mmol) were coupled by using O-(7-aza-1Hbenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 190 mg, 0.50 mmol) and (i-Pr)2NEt (85.0 µL, 0.50 mmol) in DMF for the coupling to N-methylamino acid, or N,N'diisopropylcarbodiimide (77.4 µL, 0.50 mmol) and HOBt·H2O (76.6 mg, 0.50 mmol) in DMF for the coupling to other amino acids. The Fmoc-protecting group was removed by treating the resin with 20% piperidine in DMF.

On-resin N-Methyl Modification of N-Terminal α-Amino Group. The peptide resin (0.10 mmol) was treated with o-nitrobenzenesulfonyl chloride (111 mg, 0.50 mmol) and 2,4,6-collidine (132 µL, 1.0 mmol) in NMP for 30 min (× 2) at room temperature. The N-Ns-protected resin in anhydrous THF were added MeOH (40.5 µL, 1.0 mmol), PPh3 (262 mg, 1.0 mmol) and diethyl diazodicarboxylate (455 µL,


Page 11 of 30

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


1.0 mmol) at room temperature. The suspension was shaken for 30 min (× 2) at room temperature. The N-methylated resin was treated with DBU (74.8 µL, 0.50 mmol) and 2-mercaptoethanol (70.1 µL, 1.0 mmol) for 5 min (× 2) at room temperature to give the N-methylated peptide resin.

Cleavage of Protected Peptide from the Resin, Cyclization, and Final Deprotection: Synthesis of cyclo(-D-Tyr-L-Arg-L-Arg-L-Nal-L-Pro-) (1e). The protected peptide resin (0.10 mmol) was treated with HFIP/CH2Cl2 (2:8, 20 mL) at room temperature for 2 h. After filtration of the residual resin, the filtrate was concentrated under reduced pressure to give a crude linear peptide. To a mixture of the linear peptide and NaHCO3 (42.0 mg, 0.50 mmol) in DMF (150 mL) was added diphenylphosphoryl azide (53.9 µL, 0.25 mmol) at –40 °C. The mixture was stirred for 28 h at room temperature and then filtered. The filtrate was concentrated under reduced pressure, followed by flash chromatography over silica gel with CHCl3–MeOH (90:10) to give the protected cyclic peptide. The peptide was treated with TFA/H2O (95:5, 10 mL) at room temperature for 2 h and the mixture was poured into ice-cold dry Et2O. The resulting powder was collected by centrifugation. The crude product was purified by preparative HPLC to give the title cyclic peptide 1e (bistrifluoroacetate, 6.6 mg, 6.6%): 1H NMR (500 MHz, DMSO-d6) δ: 1.37-1.55 (10H, m, CH2), 1.66-1.78 (2H, m, CH2), 2.66-2.76 (2H, m, CH2), 3.04-3.09 (4H, m, CH2), 3.15-3.23 (3H, m, CH2), 3.37 (1H, m, CH2, overlapped with residual H2O), 3.79 (1H, m, CHα), 4.14 (1H, m, CHα), 4.27 (1H, m, CHα), 4.35-4.38 (2H, m, CHα), 6.64 (2H, d, J = 8.6 Hz, Ar), 6.99-7.00 (3H, m, Ar, CONH), 7.37 (1H, d, J = 8.6 Hz, Ar), 7.43-7.49 (3H, m, Ar, CONH), 7.66-7.71 (3H, m, Ar, NH), 7.80-7.87 (3H, m, Ar), 8.54 (1H, d, J = 7.4 Hz, CONH), 8.66 (1H, d, J = 5.7 Hz, CONH), 9.24 (1H, s, OH). 13C NMR (125 MHz, DMSO-d6) δ: 21.7, 24.9, 25.1, 27.3, 28.6, 31.6, 36.1, 36.2, 40.1 (overlapped with DMSO), 40.3, 46.2, 52.3, 53.9, 54.8, 54.9, 59.1, 114.9 (2C), 125.4, 125.9, 127.26 (2C), 127.34, 127.37, 127.41, 127.8, 129.9 (2C), 131.7, 133.0, 135.6, 155.9, 156.76, 156.84, 168.4, 170.2, 172.0, 172.1, 173.2.



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

Page 12 of 30

cyclo(-D-Tyr-L-MeArg-L-Arg-L-Nal-L-Pro-) (3b): 1H NMR (500 MHz, DMSO-d6; 6:4 mixture of conformers) δ: 1.06-1.24 (4.4H, m, CH2), 1.42-1.88 (7.6H, m, CH2), 2.64-2.68 (1.6H, m, CH2, CONCH3), 2.77-2.85 (4.4H, m, CH2, CONCH3), 3.03-3.43 (7H, m, CH2, overlapped with H2O), 3.95 (0.4H, m, CHα), 4.09 (0.6H, m, CHα), 4.28 (0.6H, m, CHα), 4.42 (0.6H, m, CHα), 4.50 (0.4H, m, CHα), 4.58 (0.6H, m, CHα), 4.70-4.76 (1H, m, CHα), 4.99 (0.4H, m, CHα), 5.09 (0.4H, m, CHα), 6.61-6.73 (2.6H, m, Ar, CONH), 7.00-7.02 (2H, m, Ar), 7.35-7.70 (6.4H, m, Ar, CONH, NH), 7.80-7.86 (4H, m, Ar, CONH), 7.99 (0.4H, d, J = 8.5 Hz, CONH), 8.78 (0.6H, d, J = 3.5 Hz, CONH), 9.20 (0.4H, s, OH), 9.29 (0.6H, s, OH).

13

C NMR (125 MHz, DMSO-d6; 6:4 mixture of conformers) δ: 21.2, 21.5, 24.1,

24.3, 24.4, 24.8, 24.9, 25.3, 28.1, 29.0, 29.9, 30.1, 31.3, 31.6, 35.9, 36.0, 36.1, 38.1, 39.9 (overlapped with DMSO), 40.0 (overlapped with DMSO), 40.4, 40.5, 45.6, 45.9, 50.4, 50.6, 51.3, 51.6, 52.7, 54.0, 55.5, 57.9, 59.0, 59.5, 114.8 (2C), 115.0 (2C), 116.0, 118.4, 125.37, 125.40, 125.9 (2C), 126.6, 127.25, 127.32 (2C), 127.4 (3C), 127.7, 127.87, 127.95, 130.1 (2C), 130.2 (2C), 131.7, 131.8, 132.8, 133.0, 135.2, 136.0, 155.8, 156.1, 156.69, 156.75, 156.8, 156.9, 168.1, 168.6, 169.9, 170.1 (2C), 170.4, 170.6, 171.0, 172.2, 174.2.

cyclo(-D-Tyr-L-Arg-L-MeArg-L-Nal-L-Pro-) (3c): 1H NMR (500 MHz, DMSO-d6) δ: 1.08 (1H, m, CH2), 1.24-1.53 (8H, m, CH2), 1.75-1.93 (3H, m, CH2), 2.35 (3H, s, CONCH3), 2.80 (1H, dd, J = 5.0, 13.7 Hz, CH2), 2.92-3.01 (2H, m, CH2), 3.07-3.23 (5H, m, CH2), 3.31-3.43 (2H, m, CH2, overlapped with residual H2O), 4.04 (1H, m, CHα), 4.09 (1H, m, CHα), 4.18 (1H, m, CHα), 4.39 (1H, m, CHα), 5.00 (1H, m, CHα), 6.64 (2H, d, J = 8.6 Hz, Ar), 6.97 (2H, d, J = 8.6 Hz, Ar), 7.42-7.48 (3H, m, Ar), 7.63 (2H, t, J = 5.4 Hz, NH), 7.69 (1H, d, J = 6.9 Hz, CONH), 7.72 (1H, s, Ar), 7.78-7.85 (3H, m, Ar), 8.21 (1H, d, J = 9.2 Hz, CONH), 8.30 (1H, d, J = 8.0 Hz, CONH), 9.18 (1H, s, OH). 13C NMR (125 MHz, DMSO-d6) δ: 20.3, 25.2, 25.5, 26.2, 29.1, 30.1, 31.1, 32.7, 38.3, 40.4, 40.8, 46.5, 49.4, 52.6, 54.3, 58.5, 60.0, 114.8 (2C), 125.4, 125.9, 127.2, 127.3, 127.4, 127.6, 128.1, 128.3, 130.1 (2C), 131.7, 132.8, 136.1, 155.7, 156.7, 156.8, 168.1, 169.0, 169.9, 170.2, 172.0.


Page 13 of 30

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


SDF-1 Binding and Displacement. Membrane extracts were prepared from HEK293 cell lines expressing CXCR4 or CHO cell lines expressing CXCR7. For ligand binding, 50 µL of the peptide, 25 µL of [125I]-SDF-1α (0.20 nM for CXCR4 or 0.25 nM for CXCR7, Perkin-Elmer Life Sciences) and 25 µL of the membrane/beads mixture [CXCR4: 10−20 µg/well of membrane, 0.25 mg/well of PVT WGA beads (Perkin-Elmer Life Sciences); CXCR7: 10−20 µg/well of membrane, 0.25 mg/well of PVT WGA beads] in assay buffer (25 mM HEPES pH 7.4, 1 mM CaCl2, 5 mM MgCl2, 140 mM NaCl, 250 mM sucrose, 0.5% BSA) were incubated in the wells of an Optiplate (Perkin-Elmer Life Sciences) at room temperature for 1 h. The bound radioactivity was counted for 1 min/well in a TopCount (Perkin-Elmer Life Sciences). Inhibitory activity of the test compounds was determined based on the inhibition of [125I]-SDF-1 binding to the receptors (IC50). The IC50 values of unlabeled SDF-1 for CXCR7 were in the range of 1.21-3.92 nM.

BRET Measurements. CXCR7-mediated β-arrestin recruitment was measured by BRET essentially as described previously.20,32 HEK293E cells were co-transfected with 1 µg of receptor-eYFP construct with 0.05 µg of β-arrestin 2-Rluc. For [acceptor]/[donor] titrations, 0.05 µg of β-arrestin 2-Rluc was cotransfected with increasing amounts of the receptor-eYFP construct. All transfections were completed to 2 µg/well with an empty vector. Following overnight culture, transiently transfected HEK293 cells were seeded in 96-well, white, clear-bottom microplates (ViewPlate; PerkinElmer Life Sciences) coated with poly(D-lysine). After additional incubation for 24 h, the culture medium was removed from the plate and 30 µL of BRET buffer (PBS, 0.5 mM MgCl2, 0.01% BSA) was added. The bottom of the plate was sealed with a white sticker and 10 µL of ligand solution in BRET buffer was added. After incubation for 5 min at 37 °C, the Rluc substrate coelenterazine h (NanoLight Technology) was added at a final concentration of 5 µM to the BRET buffer (PBS, 0.5 mM MgCl2, 0.1% glucose). BRET readings were collected using a Mithras LB940 plate reader (Berthold Technologies) and MicroWin2000 ACS Paragon Plus 13 Environment


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

Page 14 of 30

software. BRET measurements between Rluc and YFP were obtained by sequential integration of the signals in the 460–500 nm (Rluc) and 510–550 nm (YFP) ranges. The BRET signal was calculated as the ratio of light emitted by the acceptor (YFP) over the light emitted by the donor (Rluc). The values were corrected to net BRET by subtracting the background BRET signal obtained in cells transfected with the Rluc construct alone. β-Arrestin recruitment was measured 30 min after ligand addition.

Molecular Modeling. The CXCR7-ligand complex structures were developed by simulations and molecular manipulations using the Molecular Operating Environment (MOE).39 Initially, the CXCR7 structure was built by the homology modeling method using the CXCR4-1a complex structure34 as the template. CXCR7 ligand conformations were manually generated based on the structure of 1a in the CXCR4-1a complex model. After placing the ligand structures in the CXCR7 homology models, the side chain conformations of the receptor and ligands were manually optimized to avoid bumps and distortions, where needed. Subsequently, energy minimization calculations of the complex structures were carried out with MMFF94x forcefield. The backbone structures of CXCR7 were fixed during the minimization.

ASSOCIATED CONTENT Supporting Information The data of characterization, bioassay, binding mode analysis, and NMR spectra. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors *For S. Oishi: phone, +81-75-753-4561; fax, +81-75-753-4570; E-mail, [email protected]. *For N. Fujii: E-mail, [email protected].


Page 15 of 30

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


ACKNOWLEDGMENTS This work was supported by a Core-to-Core Program from JSPS, Japan; Grants-in-Aid for Scientific Research from JSPS, Japan; Platform for Drug Discovery, Informatics, and Structural Life Science from MEXT, Japan; and the Canadian Institutes for Health Research (grant MOP123421). R.M. and Y.K. are grateful for JSPS Research Fellowships for Young Scientists.

ABBREVIATIONS USED ACKR3, atypical chemokine receptor 3; BRET, bioluminescence resonance energy transfer; CXCL11, CXC chemokine ligand 11; CXCL12 CXC chemokine ligand 12; CXCR4, CXC chemokine receptor 4; CXCR7, CXC chemokine receptor 7; ECL, extracellular loop; I-TAC, interferon-inducible T-cell α chemoattractant; MeAla, Nα-methylalanine; MeArg, Nα-methylarginine; MeNal, 3-(2-naphthyl)-Nαmethylalanine; MeTyr, Nα-methyltyrosine; Nal, 3-(2-naphthyl)alanine; Pic, pipecolic acid; RLuc, Renilla luciferase; SDF-1, stromal cell-derived factor 1; TM, transmembrane domain; YFP, yellow fluorescent protein.



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

Page 16 of 30

References and footnotes (1) Balabanian, K.; Lagane, B.; Infantino, S.; Chow, K. Y. C.; Harriague, J.; Moepps, B.; ArenzanaSeisdedos, F.; Thelen, M.; Bachelerie, F. The chemokine SDF-1/CXCL12 binds to and signals through the orphan receptor RDC1 in T lymphocytes. J. Biol. Chem. 2005, 280, 35760-35766. (2) Burns, J. M.; Summers, B. C.; Wang, Y.; Melikian, A.; Berahovich, R.; Miao, Z.; Penfold, M. E. T.; Sunshine, M. J.; Littman, D. R.; Kuo, C. J.; Wei, K.; McMaster, B. E.; Wright, K.; Howard, M. C.; Schall, T. J. A novel chemokine receptor for SDF-1 and I-TAC involved in cell survival, cell adhesion, and tumor development. J. Exp. Med. 2006, 203, 2201-2213. (3) Kalatskaya, I.; Berchiche, Y. A.; Gravel, S.; Limberg, B. J.; Rosenbaum, J. S.; Heveker, N. AMD3100 is a CXCR7 ligand with allosteric agonist properties. Mol. Pharmacol. 2009, 75, 1240-1247. (4) Rajagopal, S.; Kim, J.; Ahn, S.; Craig, S.; Lam, C. M.; Gerard, N. P.; Gerard, C.; Lefkowitz, R. J. β-Arrestin- but not G protein-mediated signaling by the "decoy" receptor CXCR7. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 628-632. (5) Sierro, F.; Biben, C.; Martínez-Muñoz, L.; Mellado, M.; Ransohoff, R. M.; Li, M.; Woehl, B.; Leung, H.; Groom, J.; Batten, M.; Harvey, R. P.; Martínez-A, C.; Mackay, C. R.; Mackay, F. Disrupted cardiac development but normal hematopoiesis in mice deficient in the second CXCL12/SDF-1 receptor, CXCR7. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 14759-14764. (6) Levoye, A.; Balabanian, K.; Baleux, F.; Bachelerie, F.; Lagane, B. CXCR7 heterodimerizes with CXCR4 and regulates CXCL12-mediated G protein signaling. Blood 2009, 113, 6085-6093. (7) Miao, Z.; Luker, K. E.; Summers, B. C.; Berahovich, R.; Bhojani, M. S.; Rehemtulla, A.; Kleer, C. G.; Essner, J. J.; Nasevicius, A.; Luker, G. D.; Howard, M. C.; Schall, T. J. CXCR7 (RDC1) promotes breast and lung tumor growth in vivo and is expressed on tumor-associated vasculature. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 15735-15740. ACS Paragon Plus 16 Environment

Page 17 of 30

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


(8) Wang, J.; Shiozawa, Y.; Wang, J.; Wang, Y.; Jung, Y.; Pienta, K. J.; Mehra, R.; Loberg, R.; Taichman, R. S. The role of CXCR7/RDC1 as a chemokine receptor for CXCL12/SDF-1 in prostate cancer. J. Biol. Chem. 2008, 283, 4283-4294. (9) Hattermann, K.; Held-Feindt, J.; Lucius, R.; Müerköster, S. S.; Penfold, M. E. T.; Schall, T. J.; Mentlein, R. The chemokine receptor CXCR7 is highly expressed in human glioma cells and mediates antiapoptotic effects. Cancer Res. 2010, 70, 3299-3308. (10) Dambly-Chaudière C.; Cubedo N.; Ghysen, A. Control of cell migration in the development of the posterior lateral line: antagonistic interactions between the chemokine receptors CXCR4 and CXCR7/RDC1. BMC Dev. Biol. 2007, 7, 23. (11) Boldajipour, B.; Mahabaleshwar, H.; Kardash, E.; Reichman-Fried, M.; Blaser, H.; Minina, S.; Wilson, D.; Xu, Q.; Raz, E. Control of chemokine-guided cell migration by ligand sequestration. Cell 2008, 132, 463-473. (12) For a review regarding immune regulation by CXCR7, see: Nibbs, R. J.; Graham, G. J. Immune regulation by atypical chemokine receptors. Nat. Rev. Immunol. 2013, 13, 815-829. (13) Valentin, G.; Haas, P.; Gilmour, D. The chemokine SDF1a coordinates tissue migration through the spatially restricted activation of Cxcr7 and Cxcr4b. Curr. Biol. 2007, 17, 1026-1031. (14) Donà, E.; Barry, J. D.; Valentin, G.; Quirin, C.; Khmelinskii, A.; Kunze, A.; Durdu, S.; Newton, L. R.; Fernandez-Minan, A.; Huber, W.; Knop, M.; Gilmour, D. Directional tissue migration through a self-generated chemokine gradient. Nature 2013, 503, 285-289. (15) Wang, Y.; Li, G.; Stanco, A.; Long, J. E.; Crawford, D.; Potter, G. B.; Pleasure, S. J.; Behrens, T.; Rubenstein, J. L. CXCR4 and CXCR7 have distinct functions in regulating interneuron migration. Neuron 2011, 69, 61-76. (16) Sánchez-Alcañiz, J. A.; Haege, S.; Mueller, W.; Pla, R.; Mackay, F.; Schulz, S.; López-Bendito, ACS Paragon Plus 17 Environment


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

Page 18 of 30

G.; Stumm, R.; Marín, O. Cxcr7 controls neuronal migration by regulating chemokine responsiveness. Neuron 2011, 69, 77-90. (17) Mazzinghi, B.; Ronconi, E.; Lazzeri, E.; Sagrinati, C.; Ballerini, L.; Angelotti, M. L.; Parente, E.; Mancina, R.; Netti, G. S.; Becherucci, F.; Gacci, M.; Carini, M.; Gesualdo, L.; Rotondi, M.; Maggi E.; Lasagni, L.; Serio, M.; Romagnani, S.; Romagnani, P. Essential but differential role for CXCR4 and CXCR7 in the therapeutic homing of human renal progenitor cells. J. Exp. Med. 2008, 205, 479-490. (18) Ikeda, Y.; Kumagai, H.; Skach, A.; Sato, M.; Yanagisawa, M. Modulation of circadian glucocorticoid oscillation via adrenal opioid-CXCR7 signaling alters emotional behavior. Cell 2013, 155, 1323-1336. (19) Recently, a number of CXCR7 ligands have been reported, see ref 40 and references therein. (20) Gravel, S.; Malouf, C.; Boulais, P. E.; Berchiche, Y. A.; Oishi, S.; Fujii, N.; Leduc, R.; Sinnett, D.; Heveker, N. The peptidomimetic CXCR4 antagonist TC14012 recruits β-arrestin to CXCR7: roles of receptor domains. J. Biol. Chem. 2010, 285, 37939-37943. (21) Ehrlich, A.; Ray, P.; Luker, K. E.; Lolis, E. J.; Luker, G. D. Allosteric peptide regulators of chemokine receptors CXCR4 and CXCR7. Biochem. Pharmacol. 2013, 86, 1263-1271. (22) Fujii, N.; Oishi, S.; Hiramatsu, K.; Araki, T.; Ueda, S.; Tamamura, H.; Otaka, A.; Kusano, S.; Terakubo, S.; Nakashima, H.; Broach, J. A.; Trent, J. O.; Wang, Z.; Peiper, S. C. Molecular-size reduction of a potent CXCR4-chemokine antagonist using orthogonal combination of conformation- and sequence-based libraries. Angew. Chem., Int. Ed. 2003, 42, 3251-3253. (23) Ueda, S.; Oishi, S.; Wang, Z.; Araki, T.; Tamamura, H.; Cluzeau, J.; Ohno, H.; Kusano, S.; Nakashima, H.; Trent, J. O.; Peiper, S. C.; Fujii, N. Structure-activity relationships of cyclic peptidebased chemokine receptor CXCR4 antagonists: disclosing the importance of side-chain and backbone functionalities. J. Med. Chem. 2007, 50, 192-198. ACS Paragon Plus 18 Environment

Page 19 of 30

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


(24) Narumi, T.; Hayashi, R.; Tomita, K.; Kobayashi, K.; Tanahara, N.; Ohno, H.; Naito, T.; Kodama, E.; Matsuoka, M.; Oishi, S.; Fujii, N. Synthesis and biological evaluation of selective CXCR4 antagonists containing alkene dipeptide isosteres. Org. Biomol. Chem. 2010, 8, 616-621. (25) Inokuchi, E.; Oishi, S.; Kubo, T.; Ohno, H.; Shimura, K.; Matsuoka, M.; Fujii, N. Potent CXCR4 antagonists containing amidine-type peptide bond isosteres. ACS Med. Chem. Lett. 2011, 2, 477-480. (26) Demmer, O.; Dijkgraaf, I.; Schumacher, U.; Marinelli, L.; Cosconati, S.; Gourni, E.; Wester, H. J.; Kessler, H. Design, synthesis, and functionalization of dimeric peptides targeting chemokine receptor CXCR4. J. Med. Chem. 2011, 54, 7648-7662. (27) Kobayashi, K.; Oishi, S.; Hayashi, R.; Tomita, K.; Kubo, T.; Tanahara, N.; Ohno, H.; Yoshikawa, Y.; Furuya, T.; Hoshino, M.; Fujii, N. Structure–activity relationship study of a CXC chemokine receptor type 4 (CXCR4) antagonist FC131 using a series of alkene dipeptide isosteres. J. Med. Chem. 2012, 55, 2746-2757. (28) Mungalpara, J.; Thiele, S.; Eriksen, Ø.; Eksteen, J.; Rosenkilde, M. M.; Våbenø, J. Rational design of conformationally constrained cyclopentapeptide antagonists for C-X-C chemokine receptor 4 (CXCR4). J. Med. Chem. 2012, 55, 10287-10291. (29) Mungalpara, J.; Zachariassen, Z. G.; Thiele, S.; Rosenkilde, M. M.; Våbenø, J. Structure-activity relationship studies of the aromatic positions in cyclopentapeptide CXCR4 antagonists. Org. Biomol. Chem. 2013, 11, 8202-8208. (30) Gombert, F. O.; Lederer, A.; Obrecht, D.; Romagnoli, B.; Loewe, R.; Zimmermann, J. Templatefixed peptidomimetics with CXCR7 modulating activity. PCT Int. Appl. WO 2011095218 A1, 2011. (31) Gombert, F. O.; Lederer, A.; Loewe, R.; Obrecht, D.; Romagnoli, B.; Zimmermann, J.; Patel, K. Template-fixed peptidomimetics with CXCR7 modulating activity. PCT Int. Appl. WO 2011095607 A1, 2011. ACS Paragon Plus 19 Environment


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

Page 20 of 30

(32) Montpas, N.; Cabana, J.; St-Onge, G.; Gravel, S.; Morin, G.; Kuroyanagi, T.; Sylvain-Drolet, G.; Lavigne, P.; Fujii, N.; Oishi, S.; Heveker, N. The binding mode of the cyclic agonist peptide TC14012 to CXCR7 – identification of receptor and compound determinants. Biochemistry 2015, 54, 1505-1515. (33) The apparent discrepancy between binding inhibition activity (IC50 values) and CXCR7 agonistic activity (EC50 values) may be derived from the different ability of receptor occupancies of the compounds. In the functional assay of β-arrestin recruitment, maximal functional responses may be achieved when a small fraction of the receptor population is occupied in the presence of low concentrations of the compounds: see ref. 32, 41 and 42. (34) Yoshikawa, Y.; Kobayashi, K.; Oishi, S.; Fujii, N.; Furuya, T. Molecular modeling study of cyclic pentapeptide CXCR4 antagonists: new insight into CXCR4-FC131 interactions. Bioorg. Med. Chem. Lett. 2012, 22, 2146-2150. (35) Thiele, S.; Mungalpara, J.; Steen, A.; Rosenkilde, M. M.; Våbenø, J. Determination of the binding mode for the cyclopentapeptide CXCR4 antagonist FC131 using a dual approach of ligand modifications and receptor mutagenesis. Br. J. Pharmacol. 2014, 171, 5313-5329. (36) Våbenø, J.; Nikiforovich, G. V.; Marshall, G. R. Insight into the binding mode for cyclopentapeptide antagonists of the CXCR4 receptor. Chem. Biol. Drug Des. 2006, 67, 346-354. (37) Wu, B.; Chien, E. Y.; Mol, C. D.; Fenalti, G.; Liu, W.; Katritch, V.; Abagyan, R.; Brooun, A.; Wells, P.; Bi, F. C.; Hamel, D. J.; Kuhn, P.; Handel, T. M.; Cherezov, V.; Stevens, R. C. Structures of the CXCR4 chemokine GPCR with small-molecule and cyclic peptide antagonists. Science 2010, 330, 1066-1071. (38) Of note, the template CXCR4 structure for the homology model of CXCR7 is in the antagonist (1a)-bound inactive state. (39) MOE Molecular Operating Environment; Chemical Computing Group Inc.: Montreal, Quebec, ACS Paragon Plus 20 Environment

Page 21 of 30

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


Canada, 2007. (40) Yoshikawa, Y.; Oishi, S.; Kubo, T.; Tanahara, N.; Fujii, N.; Furuya, T. An optimized method of G-protein coupled receptor homology modeling: its application to the discovery of novel CXCR7 ligands. J. Med. Chem. 2013, 56, 4236-4251. (41) Wilson, S.; Chambers, J. K.; Park, J. E.; Ladurner, A.; Cronk, D. W.; Chapman, C. G.; Kallender, H.; Browne, M. J.; Murphy, G. J.; Young, P. W. Agonist potency at the cloned human β3 adrenoceptor depends on receptor expression level and nature of assay. J. Pharmacol. Exp. Ther. 1996, 279, 214-221. (42) Galandrin, S.; Bouvier, M. Distinct signaling profiles of β1 and β2 adrenergic receptor ligands toward adenylyl cyclase and mitogen-activated protein kinase reveals the pluridimensionality of efficacy. Mol. Pharmacol. 2006, 70, 1575-1584.



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

Page 22 of 30

Table 1. Initial Screening of Cyclic Pentapeptides for Inhibitory Activities against SDF-1 Binding to CXCR7 and CXCR4 cyclo(-D-Tyr-L-Arg-L-Arg-L-Nal-Xaa-) peptide

Xaa

IC50 (µM)a CXCR7

CXCR4

1a

Gly

>30

1.2 ± 0.27

1b

L-Ala

18 ± 2.9

23 ± 2.1

1c

D-Ala

>30

2.2 ± 0.09

1d

β-Ala

>30

6.0 ± 0.72

1e

L-Pro

1.5 ± 0.50

>30

1f

D-Pro

>30

>30

1g

L-Pic

13 ± 5.3

>30

1h

D-Pic

>30

>30

1i

L-MeAla

15 ± 7.0

>30

1j

D-MeAla

>30

4.6 ± 0.23

a

IC50 values are the concentrations for 50% inhibition of the [125I]SDF-1α binding to CXCR7 and CXCR4.


Page 23 of 30

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


Table 2. Inhibitory Activities of Cyclic Pentapeptides against SDF-1 Binding to CXCR7 peptide

Sequence

IC50 (µM)a

1e

cyclo(-D-Tyr-L-Arg-L-Arg-L-Nal-L-Pro-)

1.5 ± 0.50

2a

cyclo(-L-Tyr-L-Arg-L-Arg-L-Nal-L-Pro-)

>30

2b

cyclo(-D-Tyr-D-Arg-L-Arg-L-Nal-L-Pro-)

4.6 ± 1.1

2c

cyclo(-D-Tyr-L-Arg-D-Arg-L-Nal-L-Pro-)

>30

2d

cyclo(-D-Tyr-L-Arg-L-Arg-D-Nal-L-Pro-)

>30

3a

cyclo(-D-MeTyr-L-Arg-L-Arg-L-Nal-L-Pro-) 18 ± 4.9

3b

cyclo(-D-Tyr-L-MeArg-L-Arg-L-Nal-L-Pro-) 2.4 ± 0.89

3c

cyclo(-D-Tyr-L-Arg-L-MeArg-L-Nal-L-Pro-) 0.80 ± 0.30

3d

cyclo(-D-Tyr-L-Arg-L-Arg-L-MeNal-L-Pro-) 3.4 ± 1.5

4a

cyclo(-L-MeTyr-L-Arg-L-Arg-L-Nal-L-Pro-)

4b

cyclo(-D-Tyr-D-MeArg-L-Arg-L-Nal-L-Pro-) 13 ± 2.9

4c

cyclo(-D-Tyr-L-Arg-D-MeArg-L-Nal-L-Pro-) 4.6 ± 0.40

4d

cyclo(-D-Tyr-L-Arg-L-Arg-D-MeNal-L-Pro-) >30

23 ± 3.5

a

IC50 values are the concentrations for 50% inhibition of the [125I]SDF-1α binding to CXCR7. IC50 values of all the peptides for [125I]SDF-1α binding to CXCR4 were >30 µM.



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

Page 24 of 30

Table 3. Alanine-Scanning Experiment of a Potent CXCR7 Ligand peptide

Sequence

IC50 (µM)a

3c

cyclo(-D-Tyr-L-Arg-L-MeArg-L-Nal-L-Pro-)

0.80 ± 0.30

5a

cyclo(-D-Ala-L-Arg-L-MeArg-L-Nal-L-Pro-) >30

5b

cyclo(-D-Tyr-L-Ala-L-MeArg-L-Nal-L-Pro-)

16 ± 5.3

5c

cyclo(-D-Tyr-L-Arg-L-MeAla-L-Nal-L-Pro-)

11 ± 5.5

5d

cyclo(-D-Tyr-L-Arg-L-MeArg-L-Ala-L-Pro-)

>30

a

IC50 values are the concentrations for 50% inhibition of the [125I]SDF-1α binding to CXCR7. IC50 values of all the peptides for [125I]SDF-1α binding to CXCR4 were >30 µM.


Page 25 of 30

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


Table 4. Biological Activities of Potent CXCR7 Ligands peptide

IC50 (µM)a

EC50 (µM) (pEC50 ± SD) b

1a

>30

2.4 (5.7 ± 0.36)

6c

1.7 ± 0.24

0.036 (7.5 ± 0.28)

1e

1.5 ± 0.50

0.074 (7.1 ± 0.18)

3c

0.80 ± 0.30

0.095 (7.0 ± 0.23)

a

IC50 values are the concentrations for 50% inhibition of the [125I]SDF-1α binding to CXCR7. The data are from the previous tables. bEC50 values are the concentrations needed for 50% induction of β-arrestin recruitment in human CXCR7 expressing HEK293 cells. cPeptide sequence: cyclo(-L-Ile-D-Arg-L-ArgL-Nal-D-Pro-L-Thr-).



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

Page 26 of 30

Table 5. Biological Activities of Potent CXCR7 Ligands for Mutant CXCR7 Receptors EC50 (µM) (pEC50 ± SD)a peptide WT

D179N

S198R

D275N

SDF-1

0.020 (7.7 ± 0.10)

0.057 (7.2 ± 0.14)

0.046 (7.3 ± 0.16)

0.16 (6.8 ± 0.07)

1e

0.19 (6.9 ± 0.46)

3.5 (5.4 ± 0.16)

0.36 (6.4 ± 0.10)

1.5 (5.8 ± 0.07)

3c

0.24 (6.8 ± 0.44)

2.0 (5.7 ± 0.08)

0.36 (6.4 ± 0.11)

0.65 (6.2 ± 0.11)

a

EC50 values are the concentrations needed for 50% induction of β-arrestin recruitment in human CXCR7 expressing HEK293 cells.


Page 27 of 30

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


Figure 1. Structures of a cyclic pentapeptide CXCR4 antagonist



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

Page 28 of 30

Figure 2. The binding pose of peptide 3c within CXCR7. (A) Overall binding mode. (B) Interactions of L-Arg2. (C) Interactions of L-MeArg3 and L-Nal.

ACS Paragon Plus Environment

28

Page 29 of 30

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


Figure 3. The energy-minimized structures of peptides 1e and 3c in a vacuum environment. The lateral views of peptides 1e (A) and 3c (C), and the axial views of peptides 1e (B) and 3c (D).

ACS Paragon Plus Environment

29


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

Table of Contents Graphic


Page 30 of 30

Development of Novel CXC Chemokine Receptor ... - ACS Publications

Recommend Documents