PESIN Conjugates for Multimodal Imaging: Can Multimerization Compensate Charge Influences on Cell Binding Properties? A Case Study

Abstract

Recently, anionic charges were found to negatively influence the in vitro gastrin-releasing peptide receptor (GRPR) binding parameters of dually radioisotope and fluorescent dye labeled GRPR-specific peptide dimers. From this, the question arose if this adverse impact on in vitro GRP receptor affinities could be mitigated by a higher valency of peptide multimerization. For this purpose, we designed two different hybrid multimodal imaging units (MIUs), comprising either one or two click chemistry-compatible functional groups and reacted them with PESIN (PEG3-BBN7–14, PEG = polyethylene glycol) dimers to obtain a dually labeled peptide homodimer or homotetramer. Using this approach, other dually labeled peptide monomers, dimers, and tetramers can also be obtained, and the chelator and fluorescent dye can be adapted to specific requirements. The MIUs, as well as their peptidic conjugates, were evaluated in terms of their photophysical properties, radiolabeling efficiency with 68Ga and 64Cu, hydrophilicity, and achievable GRP receptor affinities. Here, the hydrophilicity and the GRP receptor binding affinities were found to be especially strongly influenced by the number of negative charges and peptide copies, showing logD (1-octanol-water-distribution coefficient) and IC50 (half maximal inhibitory concentration) values of −2.2 ± 0.1 and 59.1 ± 1.5 nM for the homodimer, and −1.9 ± 0.1 and 99.8 ± 3.2 nM for the homotetramer, respectively. From the obtained data, it can be concluded that the adverse influence of negatively charged building blocks on the in vitro GRP receptor binding properties of dually labeled PESIN multimers can, at least partly, be compensated for by the number of introduced peptide binding motives and the used molecular design.

Synthesis, Characterization and In Vitro Evaluation of Hybrid Monomeric Peptides Suited for Multimodal Imaging by PET/OI: Extending the Concept of Charge—Cell Binding Correlation

“…Taken together, these data demonstrate that the conjugation of the MIUs to the monomeric PESIN peptide results in principle in only a minor reduction of binding affinity of the conjugates, which were still able to specifically interact with the GRPR with high affinity and the negative influence of anionically charged fluorescent dyes on GRPR binding affinities was confirmed. As expected, the adverse influence of the negatively charged dye on GRPR affinities was more pronounced in case of the peptide monomer compared to the peptide homodimer, confirming the theory that a higher number of peptide copies within the bioconjugates is at least in part able to compensate the adverse effect of the negative charge of the dye [22].…”

“…To be more specific, it was found that the higher the number of negative charges in the selected dye was, the worse the resulting binding affinity to the targeted receptor. This concept was confirmed using diverse PESIN-homodimer conjugates presenting different dye units [20,21], as well as with a PESIN-homotetramer conjugate, being characterized by a higher peptide valency [22]. In this latter case, it was interestingly observed that the adverse influence of the negatively charged synthons on the target receptor affinity was in part compensated by the higher number of peptide units in the molecule, arguing a possible intramolecular spatial shielding effect of the peptides on the adverse charges.…”

The Exception That Proves the Rule: How Sodium Chelation Can Alter the Charge‐Cell Binding Correlation of Fluorescein‐Based Multimodal Imaging Agents

“…Therefore, it was assumed that the higher the number of anionic charges carried by the conjugate (being determined by the fluorescent dye used), the lower the resulting GRPR binding affinity of the resulting multimodal imaging agent. This theory was confirmed upon various PESINmonomer conjugates [1] presenting different dye units, as well as with the corresponding PESIN-homodimer [3,4] and -homotetramer [2] conjugates. By comparing the binding profiles within these series, it was also observed that the GRPR affinities of the conjugates carrying a higher number of peptide copies were less affected by the anionic charges of the introduced fluorescent dyes than their corresponding conjugates exhibiting a lower peptide valency.…”

“…This was demonstrated by analyzing the absorption spectra of the two compounds at the fixed pH value of the medium (measured pH of 8.23): both spectra presented main absorption peaks at 496/498 nm, with a shoulder around 475 nm, corresponding to the di‐anionic proteolytic form of fluorescein, being in line with data reported in literature. [18] As confirmation, in the context of the charge‐cell binding correlation, the PESIN dimer derivative 2 a showed a GRPR binding affinity comparable to other dimeric PESIN conjugates carrying two anionic charges within the dye moiety,[ 2 , 3 ] including the tartrazine‐based conjugate 2 b being evaluated under the same conditions for direct comparison (IC 50 values of 62.07±3.87 nM and 58.44±1.47 nM for 2 a and 2 b , respectively; Table 1 ). These results demonstrate a clear correlation between the number of negative charges carried by the dye molecule within multimodal imaging agent and its GRPR binding properties.…”

“…Recent studies[ 1 , 2 , 3 , 4 ] revealed that, when aiming to synthesize peptide‐based dually labelled imaging probes suited for both positron emission tomography[ 5 , 6 ] and optical imaging[ 7 , 8 ] (PET/OI), the choice of the fluorescent dye for the optical detection can have a significant influence on the in vitro binding profile of the resulting multimodal imaging agent. At first, this was observed for dually labelled PESIN derivatives for gastrin‐releasing peptide receptor (GRPR)‐specific imaging (PESIN: PEG 3 ‐BBN 7–14 , BBN 7–14 : truncated peptide sequence of the endogenous GRPR ligand bombesin, Figure 1 ), whose receptor affinities showed to be negatively affected by the introduction of negative charges being introduced by the respective fluorescent dye.…”