Structure–activity Relationship Study of Cyclic Pentapeptide Ligands

4 hours ago - The atypical chemokine receptor 3 (ACKR3)/CXC chemokine receptor 7 (CXCR7) recognizes stromal cell-derived factor 1 (SDF-1)/CXCL12 and i...
0 downloads 11 Views 529KB Size
Download PDF
Subscriber access provided by - Access paid by the | UCSB Libraries

Structure–activity Relationship Study of Cyclic Pentapeptide Ligands for Atypical Chemokine Receptor 3 (ACKR3) Haruka Sekiguchi, TOMOKO KUROYANAGI, David Rhainds, Kazuya Kobayashi, Yuka Kobayashi, Hiroaki Ohno, Nikolaus Heveker, kenichi akaji, Nobutaka Fujii, and Shinya Oishi J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 02 Apr 2018 Downloaded from http://pubs.acs.org on April 2, 2018

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 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 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.

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 7 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

Structure–activity Relationship Study of Cyclic Pentapeptide Ligands for Atypical Chemokine Receptor 3 (ACKR3) Haruka Sekiguchi,† Tomoko Kuroyanagi,† David Rhainds,‡,§ Kazuya Kobayashi,|| Yuka Kobayashi,† Hiroaki Ohno,† Nikolaus Heveker,‡,§ Kenichi Akaji,|| Nobutaka Fujii,*,† and Shinya Oishi*,† †

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 || Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607-8412, Japan §

ABSTRACT: The atypical chemokine receptor 3 (ACKR3)/CXC chemokine receptor 7 (CXCR7) recognizes stromal cell-derived factor 1 (SDF-1)/CXCL12 and is involved in a number of physiological and pathological processes. Here, we investigated the SAR of the component amino acids in an ACKR3-selective ligand, FC313 [cyclo(-D-Tyr-L-Arg-L-MeArg-L-Nal(2)-L-Pro-)], for the development of highly active ACKR3 ligands. Notably, modification at the L-Pro position with a bulky hydrophobic side chain led to improved bioactivity toward ACKR3.

INTRODUCTION Stromal cell-derived factor-1 (SDF-1)/CXCL12 is an endogenous ligand that interacts with two chemokine receptors, CXC chemokine receptor 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3)/CXC chemokine receptor 7 (CXCR7).1,2 CXCR4 is a Gi-coupled receptor that decreases cAMP levels via adenylate cyclase inhibition, whereas ACKR3 is an atypical receptor that does not activate G protein-mediated signaling but induces β-arrestin recruitment.3 ACKR3 forms a heterodimer with CXCR4 to indirectly regulate CXCL12-mediated G protein signaling.4 The structural basis for CXCL12 binding and β-arrestin recruitment by ACKR3 has been revealed through mutational analyses of ACKR3,5,6 although the crystal structure of ACKR3 has not been determined. ACKR3 may act as a scavenger receptor for CXCL12 through receptor internalization to eliminate CXCL12 from its surrounding environment.7 Under physiological conditions, ACKR3 functions independently or cooperatively with CXCR4 to regulate cell migration during developmental processes,8,9,10 brain development11,12 and regenerative processes.13 ACKR3 expression is also observed in activated endothelial cells and a variety of cancer cells,14 in which ACKR3 signaling regulates cell growth, survival, adhesion and invasion of many types of cancers,2,15,16,17 as well as in angiogenesis via cytokine production.18 Accordingly, selective ACKR3 ligands represent potential therapeutic agents for the treatment of cancers.19,20 Recently, we identified a novel ACKR3-selective ligand FC313 (1) with a cyclic pentapeptide scaffold (Figure 1) via a selectivity switch from a CXCR4-selective cyclic pentapeptide FC131 [2, cyclo(-D-Tyr-L-Arg-L-Arg-L-Nal(2)-Gly-), (LNal(2): L-β-(2-naphthyl)alanine)].21 Taking advantage of a common Arg-Arg-Nal(2) substructure in peptide 222 and ACKR3-selective cyclic hexapeptides,23,24 a number of cyclic pentapeptides were designed, which contained one or more structural motif(s) for global conformational restriction of the

peptide macrocycle. Substitution of Gly5 with L-Pro and Nmethylation at L-Arg3 of 2 switched the receptor preference of the peptides from CXCR4 to ACKR3. The resulting peptide 1 induced the recruitment of β-arrestin to ACKR3. Alaninescanning of peptide 1 also suggested that the aromatic groups of D-Tyr1 and L-Nal(2)4 were favorable for bioactivity, whereas the two guanidino groups of L-Arg2 and L-MeArg3 could be optimized to improve the activity of peptide 1. Here, in an effort to extend the SARs of ACKR3 ligands, we have designed a series of cyclic pentapeptides with modification of residues in peptide 1. The selectivity profiles of 1 for opioid receptors25 and the possible binding modes of several ACKR3 ligands by molecular modeling studies are also discussed. Figure 1. Structure of an ACKR3-selective ligand, FC313 (1).

RESULTS AND DISCUSSION Modification of Two Arginine Residues in FC313. We investigated the SARs of two Arg residues in peptide 1. Because the previous alanine-scanning experiment of peptide 1 indicated that the two Arg positions could be substituted with other amino acids,21 we expected that more active ACKR3 ligands might be identified through a SAR study of these positions. All peptides were synthesized according to our previous procedure, which employs standard Fmoc-based solid-phase

ACS Paragon Plus Environment

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

synthesis on (2-Cl)Trt resin followed by diphenylphosphoryl azide-mediated macrocyclization.21 The binding of the peptides to the receptors was assessed based on the inhibitory activity against radiolabeled CXCL12 binding to ACKR3 or CXCR4.21 Peptides 3 and 4 were designed by substituting LArg2 or L-MeArg3 (L-MeArg: L-Nα-methylarginine) in peptide 1 with charged and hydrophilic amino acids (Table 1). Substitution with acidic amino acids such as Asp and Glu led to a loss of activity (IC50: >30 µM). Although Asn2-containing peptide was also inactive, the other neutral carboxamide and urea (Asn, Gln or Cit) derivatives (peptides 3b,c and 4a,b,c) showed micromolar affinity to ACKR3 [IC50(3b): 9.5 µM; IC50(3c): 3.6 µM; IC50(4a): 4.1 µM; IC50(4b): 5.8 µM; IC50(4c): 13 µM]. The ornithine (Orn)-containing derivatives 3d and 4d, in which the Arg guanidine group in peptide 1 was replaced with a δ-amino group, had 3.2-fold and 1.6-fold less activity, respectively [IC50(3d): 2.6 µM; IC50(4d): 1.3 µM]. Similarly, peptides 3e and 4e containing norarginine (Nar) with a shorter side chain tether also exhibited slightly decreased activities [IC50(3e): 2.6 µM; IC50(4e): 1.5 µM]. In contrast, peptides 3f and 4f having homoarginine (Har) with a longer side chain tether showed slightly higher activity when compared with those of the parent peptide 1 [IC50(3f): 0.84 µM; IC50(4f): 0.56 µM]. These results imply that a longer side chain tether should be favorable for the interaction of the basic guanidino groups at the L-Arg2 and L-MeArg3 positions with ACKR3. TABLE 1. Biological Activity of FC313 Derivatives with Modification at Two Arginine Residues

peptide FC313

cyclo(-D-Tyr-L-Xaa-L-MeYaa-L-Nal(2)-L-Pro-) L-Xaaa L-MeYaaa IC50 (µM)b L-Arg L-MeArg 0.86 ± 0.38

Page 2 of 7

appropriate functional groups at the L-Arg2 position for the favorable interaction with ACKR3. Among the series of amino acids examined, modification at the L-Arg2 position of peptide 1 to cysteine (Cys) to yield peptide 3g gave the best result [IC50(3g): 0.25 µM], whereas the other peptides 3j-u showed micromolar affinity to ACKR3 (IC50: 2.0–24 µM) (Table 1 and Table S2 in the Supporting Information). These results suggested that both L-Har and L-Cys are appropriate for the LArg2 position of peptide 1. Modification of Two Aromatic Amino Acids in FC313. Next, we aimed to obtain insights into the role of the two aromatic amino acids in defining the affinity of peptide 1 to ACKR3. The previous alanine-scanning experiment of peptide 1 indicated that D-Tyr1 and L-Nal(2)4 positions were important for bioactivity.21 Substitution of L-Nal(2) with L-Tyr, L-Phe and the derivatives led to a significant decrease or loss of bioactivity, whereas bulky bicyclic aromatic amino acids such as L-Trp and the derivatives maintained micromolar affinity to ACKR3 (see Table S3 in the Supporting Information). For the D-Tyr1 position in peptide 1, any substituted phenylalanine and bicyclic aromatic amino acids were tolerable (Table 2). Notably, D-2-fluorophenylalanine [D-Phe(2-F)]containing peptide 5c showed 2-fold stronger binding compared with that of peptide 1 [IC50(5c): 0.41 µM]. Peptide 5i with the photoreactive D-Bpa residue also possessed comparable activity and could therefore be a useful photoaffinity probe for ACKR3 [IC50(5i): 0.72 µM]. Substitution with other amino acids including D-4-chlorophenylalanine [D-Phe(4-Cl)], D-4bromophenylalanine [D-Phe(4-Br)], D-Nal(2), and D-Ala(3Bzt) resulted in an increase in ACKR3 binding [IC50(5f): 0.79 µM; IC50(5g): 0.72 µM; IC50(5j): 0.69 µM; IC50(5l): 0.54 µM]. These results demonstrate that bulky bicyclic amino acids were preferable at the L-Nal(2)4 position in peptide 1, whereas a variety of aromatic amino acids are appropriate for the DTyr1 position. TABLE 2. Optimization of the D-Tyr1 Residue

3b

L-Gln

L-MeArg

9.5 ± 2.1

3c

L-Cit

L-MeArg

3.6 ± 0.8

3d

L-Orn

L-MeArg

2.6 ± 1.0

3e

L-Nar

L-MeArg

2.6 ± 0.3

3f

L-Har

L-MeArg

0.84 ± 0.27

3g

L-Cys

L-MeArg

0.25 ± 0.04

4a

L-Arg

L-MeAsn

4.1 ± 1.3

5a

D-Phe

1.1 ± 0.3

D-Hph

5.0 ± 1.2

cyclo(-D-Xaa-L-Arg-L-MeArg-L-Nal(2)-L-Pro-) peptide D-Xaaa IC50 (µM)b FC313 D-Tyr 0.86 ± 0.38

4b

L-Arg

L-MeGln

5.8 ± 1.5

5b

4c

L-Arg

L-MeCit

13 ± 3.0

5c

D-Phe(2-F)

0.41 ± 0.11

1.3 ± 0.3

5d

D-Phe(3-F)

1.3 ± 0.2

D-Phe(4-F)

1.2 ± 0.2

4d

L-Arg

L-MeOrn

4e

L-Arg

L-MeNar

1.5 ± 0.2

5e

4f

L-Arg

L-MeHar

0.56 ± 0.18

5f

D-Phe(4-Cl)

0.79 ± 0.21

5g

D-Phe(4-Br)

0.72 ± 0.07

5h

D-Phe(4-NO2)

2.0 ± 0.3

5i

D-Bpa

0.72 ± 0.10

5j

D-Nal(2)

0.69 ± 0.16

5k

D-Trp

3.2 ± 0.5

5l

D-Ala(3-Bzt)

0.54 ± 0.12

a

L-Cit: L-citrulline; L-Orn: L-ornithine; L-Nar: (S)-2-amino-4guanidinobutyric acid; L-Har: (S)-2-amino-6-guanidinohexanoic acid; L-MeArg: L-Nα-methylarginine; L-MeAsn: L-Nαmethylasparagine; L-MeGln: L-Nα-methylglutamine; L-MeCit: LNα-methylcitrulline; L-MeOrn: L-Nα-methylornithine; L-MeNar: (S)-2-(N-methylamino)-4-guanidinobutyric acid; L-MeHar: (S)-2-

(N-methylamino)-6-guanidinohexanoic acid. bIC50 values are the concentrations for 50% inhibition of [125I]SDF-1α binding to ACKR3. IC50 values of all peptides for [125I]SDF-1α binding to CXCR4 were >30 µM. Substitution with L-Asn2 (3a), L-Asp2 (3h), L-Glu2 (3i), L-MeAsp3 (4h), L-MeGlu3 (4i) showed no inhibitory activity at 30 µM.

In our previous modeling study, no charge-charge interaction between L-Arg2 of peptide 1 and ACKR3 was revealed,21 which contributed to the strong binding between peptide 2 and CXCR4.26 Based on this insight, we further explored more

a

D-Hph: (R)-2-amino-4-phenylbutanoic acid; D-Phe(2-F): 2fluoro-D-phenylalanine; D-Phe(3-F): 3-fluoro-D-phenylalanine; D-Phe(4-F): 4-fluoro-D-phenylalanine; D-Phe(4-Cl): 4-chloro-Dphenylalanine; D-Phe(4-Br): 4-bromo-D-phenylalanine; D-Phe(4NO2): 4-nitro-D-phenylalanine; D-Bpa: D-4benzoylphenylalanine; D-Nal(2): D-β-(2-naphthyl)alanine; DAla(3-Bzt): D-β-(3-benzo[b]thienyl)alanine. bIC50 values are the concentrations for 50% inhibition of [125I]SDF-1α binding to

ACS Paragon Plus Environment

Page 3 of 7 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 ACKR3. IC50 values of all peptides for [125I]SDF-1α binding to CXCR4 were >30 µM.

Modification of the L-Pro5 Position in FC313. L-Pro5 in peptide 1 is favorable for high receptor selectivity toward ACKR3.21 The Pro5 pyrrolidine structure contributes both to the global conformations of peptide 1 and to the interaction with the hydrophobic pocket consisting of F294 and L297 in ACKR3. On the basis of these observations, we speculated that the φ and ψ angles of the backbone macrocycle would be optimized by modification at the L-Pro5 position, leading to the development of more active ACKR3 ligands. Peptides 6a– – d have ring-fused proline and pipecolic acid derivatives at the L-Pro5 position of peptide 1 (Table 3). Octahydro-1H-indole2-carboxylic acid 6a and indoline-2-carboxylic acid 6b showed improved binding to ACKR3 [IC50(6a): 0.35 µM; IC50(6b): 0.19 µM]. Fused piperidine derivatives 6c,d showed less activity than peptides 6a,b [IC50(6c): 4.2 µM; IC50(6d): 2.1 µM], which corresponds with the observed lower activity of pipecolic acid-containing peptides in our previous study.21 Peptides containing a variety of α,α-disubstituted amino acids at the L-Pro5 position were also designed and synthesized. However, modification with α,α-disubstituted amino acids, regardless of whether they are cyclic or acyclic, were not appropriate for the L-Pro5 position of peptide 1 (see Table S4 in the Supporting Information). TABLE 3. Modification at the L-Pro5 Position with RingFused Proline or Pipecolic Acid Derivatives cyclo(-D-Tyr-L-Arg-L-MeArg-L-Nal(2)-L-Xaa-) peptide L-Xaa IC50 (µM)a FC313 L-Pro 0.86 ± 0.38 6a

6b

0.35 ± 0.09

0.19 ± 0.06

show that L-Pro5 in peptide 1 is buried in the large hydrophobic binding pocket of ACKR3,21 and suggest that the hydrophobic side chains of L-MePhe, L-MeTrp and L-MeNal(1) provide favorable interactions with the same binding site. To investigate the contribution of the N-methyl group to the activity of peptide 7g, peptide 8g with an unmethylated L-Trp at the L-Pro5 position was designed. Although peptide 8g showed slightly weaker binding toward ACKR3 than 7g [IC50(8g): 0.85 µM], no binding to CXCR4 was observed at 30 µM. Noticeably, several peptides recovered the micromolar activity toward CXCR4 [CXCR4 IC50(7a): 21 µM; IC50(7g): 10 µM; IC50(7i): 17 µM], which is consistent with our previous reports,21,27 but the activity was significantly less than peptide 2. TABLE 4. Substitution at the L-Pro5 Position with NMethylamino Acids cyclo(-D-Tyr-L-Arg-L-MeArg-L-Nal(2)-L-Xaa-) IC50 (µM)b peptide L-Xaaa ACKR3 CXCR4 FC313 L-Pro 0.86 ± 0.38 >30 7a

L-MeCys

4.6 ± 0.5

21 ± 3

7b

L-MeIle

2.2 ± 0.3

>30

7c

L-MeLeu

1.9 ± 0.3

>30

7d

L-MeMet

1.8 ± 0.3

>30

7e

L-MePhe

0.60 ± 0.06 >30

7f

L-MeVal

4.9 ± 0.5

7g

L-MeTrp

0.52 ± 0.06 10 ± 3.0

7h

L-MeTyr

1.8 ± 0.4

7i

L-MeNal(1)

0.17 ± 0.05 17 ± 4.0

7j

L-MeNal(2)

2.6 ± 0.5

>30

7k

L-MeAla(2-Bztz) 1.0 ± 0.4

>30

7l

L-MeBph

4.0 ± 1.5

>30

8g

L-Trp

0.85 ± 0.05 >30

>30 >30

a

6c

4.2 ± 1.0

6d

2.1 ± 0.6

a

IC50 values are the concentrations for 50% inhibition of [ I]SDF-1α binding to ACKR3. IC50 values of all peptides for [125I]SDF-1α binding to CXCR4 were >30 µM. 125

We also explored appropriate N-methylamino acids for the L-Pro5 position of peptide 1, which could append functional

groups on the side chain accompanied with the N-methyl group-mediated global conformation restriction. A number of hydrophilic amino acids were not acceptable for this position (see Table S5 in the Supporting Information). Peptides 7a-h with a hydrophobic amino acid at the L-Pro5 position maintained ACKR3 binding in the micromolar range (Table 4, IC50: 0.52–4.9 µM). In particular, L-MePhe (7e) and L-MeTrp (7g) substitutions slightly improved the binding of peptide 1 [IC50(7e): 0.60 µM; IC50(7g): 0.52 µM]. Among the peptides 7i–l with unnatural aromatic amino acid side chains, L-Nmethyl-β-(1-naphthyl)alanine [L-MeNal(1)] derivative 7i showed six-fold stronger binding toward ACKR3 when compared with that of peptide 1 [IC50(7i): 0.17 µM]. These results

L-MeCys: L-N-methylcysteine; L-MeIle: L-Nmethylisoleucine; L-MeLeu: L-N-methylleucine; L-MeMet: L-Nmethylmethionine; L-MePhe: L-N-methylphenylalanine; LMeVal: L-N-methylvaline; L-MeTrp: L-Nα-methyltryptophane; LMeTyr: L-N-methyltyrosine; L-MeNal(1): L-N-methyl-β-(1naphthyl)alanine; L-MeNal(2): L-N-methyl-β-(2-naphthyl)alanine; L-MeAla(2-Bztz): L-N-methyl-β-(3-benzo[b]thiazolyl)alanine; LMeBph: N-methyl-β-(4-biphenylyl)-L-alanine. bIC50 values are the concentrations for 50% inhibition of [125I]SDF-1α binding to ACKR3 and CXCR4.

Finally, we investigated the multiplied effect of all favorable residues from the above investigations on the bioactivity of highly active cyclic pentapeptides toward ACKR3. Peptide 9 includes D-Phe(2-F)1, L-Har2, L-MeHar3 and L-MeNal(1)5, which refers to the amino acid residues of 5c, 3f, 4f and 7i, respectively. The IC50 value of peptide 9 for CXCL12 binding inhibition of ACKR3 was 0.45 µM (see Table S6 in the Supporting Information), which is comparable to those of the parent peptides with a single amino acid substitutions. This result indicates that a multiplied effect was not observed, implying that the structure of the binding pocket(s) in ACKR3 may be altered by binding of highly active ligands with a single substitution and therefore additional modifications might not work well to improve the bioactivity as expected.28 ACKR3-Mediated β-Arrestin Recruitment Activity of Highly Active Ligands. The ACKR3-mediated β-arrestin recruitment by highly active peptide 7i was assessed (Table 5).

ACS Paragon Plus Environment

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

The association between ACKR3-yellow fluorescent protein (YFP) and β-arrestin2–Renilla luciferase (RLuc) constructs were detected by a bioluminescence resonance energy transfer (BRET)-based assay.3,29,30 Peptides 7i induced ACKR3mediated β-arrestin recruitment with an EC50 of 0.49 µM, which was 5.3-fold less active when compared with that of the parent peptide 1 [EC50(1): 0.091 µM]. This discrepancy between CXCL12 displacement and β-arrestin recruitment abilities may be due to the different receptor occupancies of the ligands. That is, the maximal functional responses of β-arrestin recruitment could be induced by lower receptor occupancies of peptide 1. These observations suggest that different factor(s) among cyclic peptide ligands contribute to receptor binding (CXCL12 displacement) and β-arrestin recruitment. TABLE 5. Biological Activities of ACKR3 Ligands for Mutant ACKR3 Receptors ACKR3 WT D179N S198R D275N

EC50 (µM)a (pEC50 ± SD) CXCL12 peptide 1 0.014 0.091 (7.8 ± 0.10) (7.0 ± 0.12) 0.043 2.2 (7.3 ± 0.10) (5.6 ± 0.14) 0.034 0.27 (7.4 ± 0.06) (6.5 ± 0.07) 0.021 0.36 (7.6 ± 0.06) (6.4 ± 0.06)

peptide 7i 0.49 (6.3 ± 0.09) 0.11 (6.9 ± 0.63) 1.9 (5.7 ± 0.09) 5.5 (5.2 ± 0.09)

tencies between peptides 1 and 7i for the ACKR3/D275N mutant compared with that of WT ACKR3 indicated that LMeNal(1) in 7i may have some interaction(s) with the pocket around ACKR3:D275. Binding Mode Analysis of Cyclic Pentapeptide ACKR3 Ligands. In our previous study,21 the possible binding modes of ACKR3 ligands including peptide 1 was estimated by molecular modeling studies using ACKR3 homology models, which were obtained from the structure of the CXCR4-2 complex.26 Taking advantage of this protocol, we investigated the plausible binding modes of peptides 3g, 4f and 7i with ACKR3 to rationalize the SAR (Figure 2, and Figures S1 and S4 in the Supporting Information). Initially, we manually generated the structures of each peptide ligand based on the structure of 2 in the CXCR4–2 complex model.26 The peptide ligands were placed in the ACKR3 model in the ACKR3–1 complex,21 and the peptide–ACKR3 complex structures were optimized by energy minimization.31 Figure 2. Representative binding poses of cyclic peptides with ACKR3. The ACKR3 homology model for simulation was built from the CXCR4–2 complex model,21,32 which was constructed from the CXCR4–CVX15 complex (PDB ID: 3OE0).33 (A) Peptide 3g

a

EC50 values are the concentrations needed for 50% induction of β-arrestin recruitment in WT ACKR3 expressing HEK293 cells.

To estimate the possible interface residue(s) on ACKR3 for peptide 7i, the β-arrestin recruitment in three cell lines of ACKR3 mutants (D179N, S198R and D275N) was evaluated. D179 in the transmembrane domain 4 (TM4) and D275 in TM6 are involved in CXCL12 binding to ACKR3, which was demonstrated by the radio-ligand binding assay.6 D179 also contributes to the interaction with peptide 1 via the formation of a salt bridge between the carboxylate and a guanidine group in L-MeArg3.21 S198 in extracellular loop 2 (ECL2) is a key residue to the β-arrestin recruitment induced by a 14-residue peptide ligand 1o [TC14012: H-Arg-Arg-Nal-Cys-Tyr-CitLys-D-Cit-Pro-Tyr-Arg-Cit-Cys-Arg-NH2 (S-S bridged)], which binds CXCR4 and ACKR3.29,30 CXCL12 induced the βarrestin recruitment even via mutant ACKR3 receptors, whereas peptide 1 had a different bioactivity profile, as previously reported.21 Interestingly, peptide 7i showed 20-fold higher activity toward ACKR3/D179N-mediated β-arrestin recruitment [ACKR3/D179N EC50(7i): 0.11 µM] when compared with that of peptide 1. The activity of 7i for the ACKR3/D179N mutant was slightly higher than for WT ACKR3, despite the loss of the salt bridge to L-MeArg3 in peptide 7i. L-MeNal(1) in 7i appears to provide indirectly favorable effect(s) on the interaction with ACKR3/D179N by an unknown mechanism(s). The S198R mutation in ACKR3 resulted in a similar decrease of the bioactivity of CXCL12, peptides 1 and 7i [ACKR3/S198R EC50(CXCL12): 0.034 µM; EC50(1): 0.27 µM; EC50(7i): 1.9 µM]. The parallel decrease in the β-arrestin recruitment was assumed to be due to the S198R mutation and that there was no characteristic interaction between ACKR3:S198 and the cyclic peptide ligands such as 7i. In contrast, a 15-fold lower activity of peptide 7i for the ACKR3/D275N-β-arrestin pathway compared with that of peptide 1 was observed [ACKR3/D275N EC50(1): 0.36 µM; EC50(7i): 5.5 µM]. The more significant difference of the po-

(B) Peptide 4f

(C) Peptide 7i

ACS Paragon Plus Environment

Page 4 of 7

Page 5 of 7 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

Peptide 3g with the L-Cys2-substitution bound to ACKR3 displayed a similar binding mode to that of the parent peptide 1 (Figure 2A). The key interactions of L-MeArg3 in 2 with H121, S125, and D179 in ACKR3 were retained in peptide 3g. The naphthyl group of L-Nal(2)4 in 3g is buried in the ligand binding pocket consisting of L209, E213 (side chain alkyl group) and S216 in ACKR3 to form a nonpolar interaction, as observed in the interaction of peptide 1. L-Pro5 also contributed to binding by interacting with the hydrophobic pocket of F294 and L297 in ACKR3. In the plausible binding model of peptide 1, there were no characteristic charge-charge interaction of L-Arg2 with ACKR3.21 This was supported by the finding that a variety of side chain functional groups were acceptable for this position (Table 1). The interaction between the L-Cys2 mercapto group of 3g and the aromatic ring of ACKR3:H121 would reimburse the loss of a number of hydrogen bonds between the L-Arg2 guanidino group in peptide 1 and ACKR3. The overall binding mode of another peptide 4f with LMeHar3 was also conserved in peptide 1. For example, the guanidino group of L-Arg2 in 4f interacted with the side chain of S103 and the backbone carbonyl group of C196 in ACKR3. In contrast, substitution with L-MeHar3 having an elongated hydrocarbon side chain tether slightly altered the binding mode. New interactions of the guanidine group of L-MeHar3 with the side chains of Q301 in ACKR3 were observed and accompanied with the conserved interaction with H121. The interactions of the L-MeHar3 side chain with S125 and D179, which were observed in peptide 1, were missing in 4f. Peptide 7i with L-MeNal(1) adopted a similar binding mode to that of peptide 1, in which the spatial orientations of the backbone peptide bonds were maintained. The naphthalene ring of L-MeNal(1) was buried in the hydrophobic pocket, in which the F294 and L297 of ACKR3 contributed to favorable hydrophobic interactions. Additional interactions by the peptide backbone of 7i was observed between the methyl group of L-MeNal(1) and D275 of ACKR3, as well as between the carbonyl oxygen of L-MeNal(1) and the side chain of ACKR3:Q301. Since Q301 is involved in ACKR3 β-arrestin signaling, this new interaction probably rationalizes the increased activity of 7i. The lower conformational restriction by the acyclic amino acid at the L-Pro5 position of 1 was compensated by these characteristic interactions, leading to the improved binding of 7i toward ACKR3. CONCLUSIONS In this study, we investigated the SAR of an ACKR3 ligand, FC313 (1), with a cyclic pentapeptide scaffold. Among a variety of peptides with modification at the L-Arg2 and LMeArg3 positions of 1, L-Har2 (3f), L-Cys2 (3g) and L-

MeHar3 (4f) derivatives exhibited higher affinity toward ACKR3. The wide variety of functional groups at the L-Arg2 position, including nonpolar groups, was acceptable, which was rationalized by the binding modes of peptides 1 and 3g. For the L-Nal(2)4 residue, bicyclic aromatic groups with an appropriate arrangement for occupation of the hydrophobic pocket of ACKR3 were favorable. In contrast, the D-Tyr1 position was more tolerable to modification with any aromatic amino acid, and such modifications yielded similar binding as observed for peptide 1. The most effective modifications to regulate bioactivity were at the L-Pro5 position of 1. Peptides bearing proline analogues (6a,b) and L-MeNal(1) (7i) showed improved ACKR3 binding. The profile of peptide 7i for ACKR3-mediated β-arrestin recruitment was different from that of peptide 1, indicating that peptide 7i could interact with ACKR3 by an alternative mode of receptor activation. Binding mode analysis using a homology model of ACKR3 demonstrated that the interaction between the 1-naphthyl group of LMeNal(1)5 in peptide 7i and the hydrophobic pocket in ACKR3 could facilitate the higher affinity toward ACKR3. Taken together, we successfully improved the bioactivity of cyclic pentapeptide ACKR3 ligands by optimization of residues of peptide 1. EXPERIMENTAL SECTION General. For analytical HPLC, a Cosmosil 5C18-ARII column (4.6 mm × 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 mm × 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 MALDITOF-MS analysis, and the purity of the peptides was determined by HPLC analysis (>95%). cyclo[-D-Tyr-L-Arg-L-MeArg-L-Nal(2)-L-MeNal(1)-] (7i). The protected peptide resin (0.10 mmol) was treated with 1,1,1,3,3,3-hexafluoro-2-propanol/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 15 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/TIS (95:2.5:2.5, 4 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 7i (bistrifluoroacetate, 10.4 mg, 9.2%). SDF-1 Binding and Displacement.21 Membrane extracts were prepared from HEK293 cell lines expressing CXCR4 or CHO cell lines expressing ACKR3. For ligand binding, 50 µL of the peptide solution, 25 µL of [125I]SDF-1α solution (0.20 nM for CXCR4 or 0.25 nM for CXCR7, PerkinElmer 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 (PerkinElmer Life Sciences); ACKR3, 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

ACS Paragon Plus Environment

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

incubated in the wells of an Optiplate (PerkinElmer Life Sciences) at room temperature for 1 h. The bound radioactivity was counted for 1 min/well in a TopCount (PerkinElmer 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 ACKR3 were in the range of 1.21−3.92 nM. BRET Measurements. ACKR3-mediated β-arrestin recruitment was measured by BRET essentially as described previously.29,30 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 receptoreYFP 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 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.21 The ACKR3-ligand complex structures were developed by simulations and molecular manipulations using the Molecular Operating Environment (MOE)34 according to the procedure in our previous reports.21 ACKR3 ligand structures were manually generated based on the structure of 2 in the CXCR4–2 complex model.32 Subsequently, conformational searches of the ligands were performed by varying the torsion angles newly incorporated side chains to generate one or several initial structures. After placing the ligand structures into the ACKR3 homology models,21 energy minimization calculations of the complex structures were carried out with MMFF94x forcefield. The backbone structures of ACKR3 were fixed during the minimization.

ASSOCIATED CONTENT Supporting Information. The supporting information is available free of charge on the ACS Publications website at DOI: #############. Experimental procedures, the data of characterization, bioassay, binding mode analysis, NMR spectra (PDF); molecular formula strings (CSV); and atomic coordinates of ACKR3–peptide complexes by binding mode analysis (PDB).

AUTHOR INFORMATION Corresponding Author *For N. Fujii: E-mail, [email protected].

Page 6 of 7

*For S. Oishi: phone, +81-75-753-4561; fax, +81-75-753-4570; E-mail, [email protected].

ACKNOWLEDGMENT This work was supported by a Core-to-Core Program from JSPS, Japan; a Grant-in-Aid for Scientific Research from JSPS, Japan (15H04654); the Platform Project for Supporting Drug Discovery and Life Science Research from AMED, Japan; the Suzuki Memorial Foundation. N.H. was supported by grant MOP123421 from the Canadian Institutes of Health Research (CIHR). Y.K. is grateful for JSPS Research Fellowships for Young Scientists.

ABBREVIATIONS ACKR3, atypical chemokine receptor 3; Ala(3-Bzt), β-(3benzo[b]thienyl)alanine; Ala(2-Bztz), β-(3benzo[b]thiazolyl)alanine; Bpa, 4-benzoylphenylalanine; Cit, citrulline; BRET, bioluminescence resonance energy transfer; CXCL12, CXC chemokine ligand 12; CXCR4, CXC chemokine receptor 4; CXCR7, CXC chemokine receptor 7; ECL, extracellular loop; Har, 2-amino-6-guanidinohexanoic acid; Hph, 2-amino4-phenylbutanoic acid; Nar, 2-amino-4-guanidinobutyric acid; HATU, O-(7-aza-1H-benzotriazol-1-yl)-N,N,N’,N’tetramethyluronium hexafluorophosphate; Nal(1), β-(1naphthyl)alanine; Nal(2), β-(2-naphthyl)alanine; Orn, ornithine; RLuc, Renilla luciferase; SDF-1, stromal cell-derived factor 1; TM, transmembrane domain; YFP, yellow fluorescent protein.

REFERENCES (1) Balabanian, K.; Lagane, B.; Infantino, S.; Chow, K. Y. C.; Harriague, J.; Moepps, B.; Arenzana-Seisdedos, 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) Levoye, A.; Balabanian, K.; Baleux, F.; Bachelerie, F.; Lagane, B. CXCR7 heterodimerizes with CXCR4 and regulates CXCL12mediated G protein signaling. Blood 2009, 113, 6085-6093. (5) Gustavsson, M.; Wang, L.; van Gils, N.; Stephens, B. S.; Zhang, P.; Schall, T. J.; Yang, S.; Abagyan, R.; Chance, M. R.; Kufareva, I.; Handel, T. M. Structural basis of ligand interaction with atypical chemokine receptor 3. Nat. Commun. 2017, 8, 14135. (6) Benredjem, B.; Girard, M.; Rhainds, D.; St-Onge, G.; Heveker, N. Mutational analysis of atypical chemokine receptor 3 (ACKR3/CXCR7) interaction with its chemokine ligands CXCL11 and CXCL12. J. Biol. Chem. 2017, 292, 31-42. (7) Naumann, U.; Cameroni, E.; Pruenster, M.; Mahabaleshwar, H.; Raz, E.; Zerwes, H. G.; Rot, A.; Thelen, M. CXCR7 functions as a scavenger for CXCL12 and CXCL11. PLoS ONE 2010, 5, e9175. (8) 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. (9) 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. (10) Boldajipour, B.; Mahabaleshwar, H.; Kardash, E.; ReichmanFried, M.; Blaser, H.; Minina, S.; Wilson, D.; Xu, Q.; Raz, E. Control of chemokine-guided cell migration by ligand sequestration. Cell

ACS Paragon Plus Environment

Page 7 of 7 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 2008, 132, 463-473. (11) 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. (12) Sánchez-Alcañiz, J. A.; Haege, S.; Mueller, W.; Pla, R.; Mackay, F.; Schulz, S.; López-Bendito, G.; Stumm, R.; Marín, O. Cxcr7 controls neuronal migration by regulating chemokine responsiveness. Neuron 2011, 69, 77-90. (13) 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. (14) For a review, see: Hattermann, K.; Mentlein, R. An infernal trio: the chemokine CXCL12 and its receptors CXCR4 and CXCR7 in tumor biology. Ann Anat. 2013, 195, 103-110. (15) 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 tumorassociated vasculature. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 15735-15740. (16) 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. (17) Singh R. K.; Lokeshwar, B. L. The IL-8-regulated chemokine receptor CXCR7 stimulates EGFR signaling to promote prostate cancer growth. Cancer Res. 2011, 71, 3268-3277. (18) 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. (19) Xu, D.; Li, R.; Wu, J.; Jiang, L.; Zhong, H. A. Drug design targeting the CXCR4/CXCR7/CXCL12 pathway. Curr. Top. Med. Chem. 2016, 16, 1441-1451. (20) Barbieri, F.; Bajetto, A.; Thellung, S.; Würth, R.; Florio, T. Drug design strategies focusing on the CXCR4/CXCR7/CXCL12 pathway in leukemia and lymphoma. Expert Opin. Drug Discov. 2016, 11, 1093-1109. (21) Oishi, S.; Kuroyanagi, T.; Kubo, T.; Montpas, N.; Yoshikawa, Y.; Misu, R.; Kobayashi, Y.; Ohno, H.; Heveker, N.; Furuya, T.; Fujii, N. Development of novel CXC chemokine receptor 7 (CXCR7) ligands: selectivity switch from CXCR4 antagonists with a cyclic pentapeptide scaffold. J. Med. Chem. 2015, 58, 5218-5225. (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) Gombert, F. O.; Lederer, A.; Obrecht, D.; Romagnoli, B.; Loewe, R.; Zimmermann, J. Template-fixed Peptidomimetics with CXCR7 Modulating Activity. PCT Int. Appl. WO 2011095218 A1, 2011. (24) 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. (25) A recent report revealed that opioid peptides bind to CXCR7 to modulate the circadian glucocorticoid oscillation, resulting in anxiolytic-like behavior.35 However, peptide 1 had no binding for any opioid receptor (see Table S1 in the Supporting Information). (26) 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. (27) 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 peptide-based chemokine receptor CXCR4 antagonists: disclosing the importance of side-chain and backbone functionalities. J. Med. Chem. 2007, 50, 192-198. (28) Although the improved bioactivity of peptide 9 with four favorable residues was not observed, a combination of two or three substitutions might result in the expected additive effects. (29) 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. (30) 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. (31) Alternative binding sites and binding mode may be possible, if significant conformational change of ACKR3 occurs upon ligand binding. (32) 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. (33) 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. (34) MOE Molecular Operating Environment; Chemical Computing Group Inc.: Montreal, Quebec, Canada, 2007. (35) Ikeda, Y.; Kumagai, H.; Skach, A.; Sato, M.; Yanagisawa, M. Modulation of circadian glucocorticoid oscillation via adrenal opioidCXCR7 signaling alters emotional behavior. Cell 2013, 155, 13231336.

Table of Contents (TOC) Graphic

H2N

NH2

NH

HN

HN

HN

N

L-Nal(2)4

O

L-MeArg3 O N

H2N

NH

HS

F

HN NH

O O

L-Arg2 HN

HN Me

NH O

Me N

N H N L-Pro5

O

O

H2N HO

D-Tyr1

FC313

ACS Paragon Plus Environment