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Elucidating the structure-activity relationship of the pentaglutamic acid sequence of minigastrin with the cholecystokinin receptor subtype 2 Andreas Ritler, Michal Shoshan, Xavier Deupi, Patrick Wilhelm, Roger Schibli, Helma Wennemers, and Martin Behe Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00849 • Publication Date (Web): 04 Jan 2019 Downloaded from http://pubs.acs.org on January 7, 2019
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Bioconjugate Chemistry
Elucidating the structure-activity relationship of the pentaglutamic acid sequence of minigastrin with the cholecystokinin receptor subtype 2 Andreas Ritler,†,‡,* Michal S. Shoshan,† Xavier Deupi,# Patrick Wilhelm,† Roger Schibli,‡,* Helma Wennemers,† Martin Béhé* †Department of Chemistry and Applied Biosciences (D-CHAB), Laboratory of Organic Chemistry (LOC), ETH, CH-8093 Zurich, Switzerland ‡Department
of Chemistry and Applied Biosciences (D-CHAB), Institute of Pharmaceutical Sciences (IPW), ETH, CH-8093 Zurich, Switzerland *Research Department Biology and Chemistry, Center for Radiopharmaceutical Sciences (CRS), Paul Scherrer Institute, CH-5232 Villigen, Switzerland #Laboratory
of Biomolecular Research, and Condensed Matter Theory Group, Paul Scherrer Institute, CH-5232 Villigen, Switzerland KEYWORDS minigastrin, cholecystokinin, cholecystokinin receptor subtype 2 (CCK2R), neuroendocrine tumors, radiolabeled peptides, Lutetium-177, non-natural amino acids
ABSTRACT: Derivatized minigastrin analogues are a promising class of candidates to target the cholecystokinin receptor subtype 2 (CCK2R), which is overexpressed on cancer cells of various neuroendocrine tumors (NETs). The pentaglutamic acid sequence of minigastrin influences its biological properties. In particular, it plays a crucial role in the kidney reuptake mechanism. However, the importance for the binding affinity and interaction of this region with the receptor on a molecular level remains unclear. In order to elucidate its structure-activity relationship with the CCK2R, we replaced this sequence with various linkers differing in their amount of anionic charges, structural characteristics, and flexibility. Specifically, a flexible aliphatic linker, a linker with only three D-Glu residues and a structured linker with four adjacent β3-glutamic acid residues were evaluated and compared to the lead compound PP-F11N (DOTA-[D-Glu1-6,Nle11]gastrin-13). 1,4,7,10Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was conjugated to the minigastrin derivatives, which allowed radiolabeling with Lutetium-177. In vitro internalization into MZ-CRC1 cells and in vivo tumor uptake, as well as human blood plasma stability increased in the following order: aliphatic linker < three D-Glu < (β3-Glu)4 < (D-Glu)6. The in vitro and in vivo behavior was therefore significantly increased with anionic charges. Computational modeling of a CCK2 receptor-ligand complex revealed ionic interactions between cationic residues (Arg and His) of the receptor with the anionic residues of the ligand in the linker.
INTRODUCTION The cholecystokinin receptor subtype 2 (CCK2R) is a peptide-binding G protein-coupled receptor (GPCR) often overexpressed on the cell surface of neuroendocrine tumors (NETs), especially of medullary thyroid and small cell lung cancer. Thus, the CCK2R has been used for radionuclide imaging and is a suitable target for peptide receptor radionuclide therapy (PRRT).(1, 2) Cholecystokinin (CCK) and gastrin peptides are endogenous ligands of the CCK2R and have the amidated C-terminal binding sequence Trp-Met-Asp-Phe-NH2 in common.(3) Both peptide variants exist in various lengths in the human body (gastrin-34, gastrin-17, gastrin-14, CCK4, CCK-8, CCK-12, CCK-22, CCK-33, and CCK-58).(4) CCK and gastrin structurally differ by a common Tyr residue
closer to the C-terminal binding sequence in gastrin peptides compared to its position in CCK and the lack of a (L-Glu)5 sequence in CCK derivatives. Native sulfation of this Tyr residue in cholecystokinin enhances its affinity towards CCK1R, but not CCK2R. In fact, CCK1R binds sulfated CCK-8 with a 500- to 1000-fold higher affinity than gastrin (sulfated or non-sulfated) and non-sulfated CCK. On the other hand CCK2R discriminates less between cholecystokinin and gastrin forms with different length and sulfation state.(4, 5) Sulfation of cholecystokinine derivatives led to increased tumor targeting properties, whereas minigastrin derivatives are not affected to the same extent by this modification.(6, 7)
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DOTA-D-Glu-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2
MG11N* (no linker) O
DOTA
O
OH
O
H N
O
O
H N
N H
O
O
H N
N H
O
OH
OH
OH
D-Glu-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2
N H
O OH
O
PP-F11N* (lead compound) O
O
OH
OH O
O
O
N D-Glu-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2 H
N H
N H
N H
N H
N H
O
OH
O
O
DOTA
O
OH
AR060 (β3-Glu linker) O O
DOTA
N H
N H
OH
O
H N
N H
O
O
H N
N H
O O
H N O
O N D-Glu-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2 H
OH
AR059 (reduced amount of charges within the linker)
DOTA
H N
H N O
H N O
O N H
H N O
O N D-Glu-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2 H
AR058 (uncharged linker) Chart 1 Synthesized minigastrin derivatives with different linkers between DOTA and the C-terminal binding sequence. *The “N” in these compounds stands for the replacement of the terminal methionine of gastrin by a norleucine in order to avoid possible oxidation of the side chain. Several N-terminally truncated and/or modified cholecystokinin and minigastrin analogs have been investigated regarding their in vitro and in vivo properties for imaging and therapy of NETs over the last decades.(79) MG11, a short N-terminally truncated minigastrin derivative that lacks the pentaglutamic acid sequence and bears only one D-Glu residue instead (Chart 1), displayed good tumor-to-kidney ratio in vivo but a low stability in human blood and a low overall uptake in the tumor.(10-13) Ligands containing the pentaglutamic acid sequence showed higher tumor uptake(8, 14), but also an undesired accumulation in the kidneys. Kidney uptake can be reduced by co-injection of polyglutamic acids(15) or by strategies to replace the L-glutamates by other linkers like D-glutamate residues (PP-F11)(16). PP-F11 (alternative name: CP04) retains high tumor uptake properties but has a significantly reduced kidney uptake which allows an application for peptide receptor radionuclide therapy.(11, 16) These previous studies showed that the charged, anionic pentaglutamate sequence plays an important role in the
pharmacokinetic and pharmacodynamic properties of the minigastrin peptides, since it is not only related to renal uptake but also to stability. Successive truncation of the Nterminal glutamates correlates with decreased stability towards proteases in human blood serum.(12) One reason for the increased tumor uptake of ligands with glutamic acid residues in vivo is therefore their increased stability towards proteases. However, the role of additional factors, e.g. intermolecular interactions between the Glu residues and the receptor in correlation with the in vitro and in vivo properties, is unclear. Several essential amino acids within the binding site of the CCK2 receptor have been identified by site-directed mutagenesis studies, photoaffinity-labeling and molecular modeling, but did not shed light on the molecular interplay between the pentaglutamic acid sequence of minigastrin and CCK2 receptor in direct correlation to the in vitro and in vivo performance.(17) Herein we explored the role of the pentaglutamic acid sequence on the activity of minigastrin by replacing this
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Bioconjugate Chemistry
region with different linkers. We show that anionic charges influence the binding affinity, cell internalization, and stability of minigastrin derivatives. Furthermore, we correlated these findings to structural features obtained by molecular docking studies. RESULTS AND DISCUSSION Synthesized Peptides. We started our investigations by replacing the pentaglutamic acid sequence of native minigastrin with linkers that have approximately the same length but different degrees of flexibility, amounts of negative charges, and varying positions of those charges (Chart 1). This included the use of proteinogenic -Lamino acids as well as -D- and -amino acids. More specifically the region after the N-terminal D-Glu of MG11 was extended by a flexible, uncharged Gly/GABA linker (AR058), by a flexible linker still containing two D-Glu residues that may form ionic interactions with the receptor according to a previous computational model(18) (AR059), and by a structured β3-glutamic acid sequence with the potential to form a stable 314 helix(19) (AR060). PP-F11N, containing six consecutive D-Glu residues, was used as the reference compound together with MG11N, without the D-Glu residues, for comparison. DOTA was coupled to the different ligands and radiolabeling with [177Lu]Lu3+ allowed us to evaluate their in vitro and in vivo properties. Lu-177 is used because it is applied routinely and successfully in therapeutic applications and studies for PRRT. The emitting gamma rays allow the use for preclinical studies as well as for imaging and diagnostic purposes. The peptides were synthesized by solid phase peptide synthesis following the standard Fmoc/tBu-protocol. Structural investigations of the peptides included NMR and CD spectroscopy (for details, see supporting information). Human blood plasma stability. The stability of the minigastrin derivatives was evaluated by incubation in human blood plasma and HPLC analysis. The minigastrin derivative MG11N (DOTA-[dGlu1,Nle6]gastrin-8) is known to be rapidly degraded in human blood plasma.(10, 12) Incubation of the newly prepared minigastrin analogs with human blood plasma showed that all of them display a significantly better stability than MG11N with the derivatives bearing negatively charged residues as the most stable ones (Figure 1). Among them, PP-F11N and AR060 exhibit the highest stability as expected for peptides containing D-glutamic and β3-glutamic acid residues, respectively.(20) Interestingly, the aliphatic linker without any additional negatively charged amino acid residues (AR058) displayed an increased stability compared to MG11N as well. Several cleavage sites of minigastrin derivatives by human blood serum proteases have been identified and are located between Asp12-Phe13, Tyr8-Gly9 and Gly9-Trp10 within minigastrin-13.(21) These cleavage sites are distant from the N-terminus of minigastrin where we implemented the different linkers. Thus, our studies show that the proteolytic stability of minigastrin can be significantly enhanced even by remote modifications.
Figure 1 Stability experiments of minigastrin derivatives performed in human blood plasma. Determination of IC50 values. The binding affinity of the minigastrin derivatives towards the CCK2R was evaluated by performing displacement experiments with 177Lu-labeled PP-F11N on MZ-CRC-1 cells. For all tested peptides IC50 values in the range of 10-110 nM were obtained (Table 1 and Figure 2). The lowest IC50 of 103 ± 36 nM was observed for AR058, which shows that an elongated aliphatic linker that lacks anionic charge significantly reduced the binding affinity. AR060 with the (β3-Glu)4 linker has an IC50 of 25 ± 11 nM, which is comparable to that of the lead compound PP-F11N (IC50 = 16 ± 3 nM).
Figure 2 Determination of IC50 values on MZ-CRC-1 cells with 175Lu[Lu]-labeled minigastrin ligands. Comparison of the sequence of AR058 with AR059, AR060 and PP-F11N emphasizes the importance of anionic charges in the pentaglutamic acid region, revealing a trend towards lower IC50 values with an increased amount of anionic charges. Table 1 Determination of IC50 values on MZ-CRC-1 cells with the 175Lu[Lu]-labeled minigastrin ligands MG11N, AR058, AR059, AR060 and the lead compound PP-F11N.a Ligand
IC50 [nM] b
MG11N
28 ± 14
AR058
103 ± 36
AR059
35 ± 11
AR060
25 ± 11
PP-F11N
16 ± 3
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Bioconjugate Chemistry Values represent mean ± standard deviation (SD). Displacement experiments of [177Lu]Lu-PP-F11N. a
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b
In vitro cell internalization studies. In vitro studies with MZ-CRC-1 cancer cells were performed to determine the internalization of the minigastrin derivatives (Figure 3 and Table 2). The in vitro internalization studies showed that the uptake of AR060 bearing four β3-Glu residues (52.4 ± 7.4 % after 4 h) is in the same range of the reference ligand PP-F11N with six D-Glu residues (62.1 ± 8.3 % after 4 h, Table 2). The in vitro internalization studies also revealed a clear trend of increasing uptake in CCK2Rpositive cells with the amount of negative charges (Table 2). Ligands with similar amounts of anionic charges to the pentaglutamic acid motif of native minigastrin have a higher uptake than N-terminally truncated analogs (MG11N, 35.4 ± 2.6 % after 4 h), non-charged analogs (AR058, 38.9 ± 6.2 % after 4 h), and analogs with fewer charges (AR059, 45.3 ± 6.9 % after 4 h). MG11N displays the lowest cellular uptake values, despite its good binding affinity. Since internalization relies not only on the binding affinity of the ligand to the receptor but activation of the receptor by the ligands, this result suggests that the linker region has an influence on receptor activation.
Figure 3 Time dependent internalization into MZ-CRC-1 cells of 177Lu[Lu]-labeled minigastrin ligands. The internalization is presented as the percentage of the total added radiolabeled peptide. The surface bound fraction for all five minigastrin derivatives was very low, since binding lead preferentially to internalization (
