PhpC modulates G-quadruplex-RNA landscapes in human cells.

Stabilizing DNA/RNA G-quadruplexes (G4s) using small molecules (ligands) has proven an efficient strategy to decipher G4 biology. Quite paradoxically, this search has also highlighted the need for finding molecules able to disrupt G4s to tackle G4-associated cellular dysfunctions. We report here on both qualitative and quantitative investigations that validate the G4-RNA-destabilizing properties of the leading compound PhpC in human cells.

The multivalent G-quadruplex (G4)-ligands MultiTASQs allow for versatile click chemistry-based investigations.

Chemical biology hinges on multivalent molecular tools that can specifically interrogate and/or manipulate cellular circuitries from the inside. The success of many of these approaches relies on molecular tools that make it possible to visualize biological targets in cells and then isolate them for identification purposes. To this end, click chemistry has become in just a few years a vital tool in offering practically convenient solutions to address highly complicated biological questions. We report here on two clickable molecular tools, the biomimetic G-quadruplex (G4) ligands MultiTASQ and azMultiTASQ, which benefit from the versatility of two types of bioorthogonal chemistry, CuAAC and SPAAC (the discovery of which was very recently awarded the Nobel Prize of chemistry). These two MultiTASQs are used here to both visualize G4s in and identify G4s from human cells. To this end, we developed click chemo-precipitation of G-quadruplexes (G4-click-CP) and in situ G4 click imaging protocols, which provide unique insights into G4 biology in a straightforward and reliable manner.

Side-by-side comparison of G-quadruplex (G4) capture efficiency of the antibody BG4 versus the small-molecule ligands TASQs.

The search for G-quadruplex (G4)-forming sequences across the genome is motivated by their involvement in key cellular processes and their putative roles in dysregulations underlying human genetic diseases. Sequencing-based methods have been developed to assess the prevalence of DNA G4s genome wide, including G4-seq to detect G4s in purified DNA (in vitro) using the G4 stabilizer PDS, and G4 chromatin immunoprecipitation sequencing (G4 ChIP-seq) to detect G4s in in situ fixed chromatin (in vivo) using the G4-specific antibody BG4. We recently reported on G4-RNA precipitation and sequencing (G4RP-seq) to assess the in vivo prevalence of RNA G4 landscapes transcriptome wide using the small molecule BioTASQ. Here, we apply this technique for mapping DNA G4s in plants (rice) and compare the efficiency of this new technique, G4-DNA precipitation and sequencing, G4DP-seq, to that of BG4-DNA-IP-seq that we developed for mapping of DNA G4s in rice using BG4. By doing so, we compare the G4 capture ability of small-sized ligands (BioTASQ and BioCyTASQ) versus the antibody BG4.

Modular One-Pot Strategy for the Synthesis of Heterobivalent Tracers.

Bivalent ligands, i.e., molecules having two ligands covalently connected by a linker, have been gathering attention since the first description of their pharmacological potential in the early 80s. However, their synthesis, particularly of labeled heterobivalent ligands, can still be cumbersome and time-consuming. We herein report a straightforward procedure for the modular synthesis of labeled heterobivalent ligands (HBLs) using dual reactive 3,6-dichloro-1,2,4,5-tetrazine as a starting material and suitable partners for sequential SNAr and inverse electron-demand Diels-Alder (IEDDA) reactions. This assembly method conducted in a stepwise or in a sequential one-pot manner provides quick access to multiple HBLs. A conjugate combining ligands toward the prostate-specific membrane antigen (PSMA) and the gastrin-releasing peptide receptor (GRPR) was radiolabeled, and its biological activity was assessed in vitro and in vivo (receptor binding affinity, biodistribution, imaging) as an illustration that the assembly methodology preserves the tumor targeting properties of the ligands.

Theranostics of Primary Prostate Cancer: Beyond PSMA and GRP-R.

The imaging of Prostate-Specific Membrane Antigen (PSMA) is now widely used at the initial staging of prostate cancers in patients with a high metastatic risk. However, its ability to detect low-grade tumor lesions is not optimal. METHODS: First, we prospectively performed neurotensin receptor-1 (NTS1) IHC in a series of patients receiving both [68Ga]Ga-PSMA-617 and [68Ga]Ga-RM2 before prostatectomy. In this series, PSMA and GRP-R IHC were also available (n = 16). Next, we aimed at confirming the PSMA/GRP-R/NTS1 expression profile by retrospective autoradiography (n = 46) using a specific radiopharmaceuticals study and also aimed to decipher the expression of less-investigated targets such as NTS2, SST2 and CXCR4. RESULTS: In the IHC study, all samples with negative PSMA staining (two patients with ISUP 2 and one with ISUP 3) were strongly positive for NTS1 staining. No samples were negative for all three stains-for PSMA, GRP-R or NTS1. In the autoradiography study, binding of [111In]In-PSMA-617 was high in all ISUP groups. However, some samples did not bind or bound weakly to [111In]In-PSMA-617 (9%). In these cases, binding of [111n]In-JMV 6659 and [111In]In-JMV 7488 towards NTS1 and NTS2 was high. CONCLUSIONS: Targeting PSMA and NTS1/NTS2 could allow for the detection of all intraprostatic lesions.

Click-Chemistry-Based Biomimetic Ligands Efficiently Capture G-Quadruplexes In Vitro and Help Localize Them at DNA Damage Sites in Human Cells.

Interrogating G-quadruplex (G4) biology at its deepest roots in human cells relies on the design, synthesis, and use of ever smarter molecular tools. Here, we demonstrate the versatility of biomimetic G4 ligands referred to as TASQ (template assembled synthetic G-quartet) in which a biotin handle was incorporated for G4-focused chemical biology investigations. We have rethought the biotinylated TASQ design to make it readily chemically accessible via an efficient click-chemistry-based strategy. The resulting biotinylated, triazole-assembled TASQ, or BioTriazoTASQ, was thus shown to efficiently isolate both DNA and RNA G4s from solution by affinity purification protocols, for identification purposes. Its versatility was then further demonstrated by optical imaging that provided unique mechanistic insights into the actual strategic relevance of G4-targeting strategies, showing that ligand-stabilized G4 sites colocalize with and, thus, are responsible for DNA damage foci in human cells.

1,5-Disubstituted 1,2,3-Triazoles as Amide Bond Isosteres Yield Novel Tumor-Targeting Minigastrin Analogs.

1,5-Disubstituted 1,2,3-triazoles (1,5-Tz) are considered bioisosteres of cis-amide bonds. However, their use for enhancing the pharmacological properties of peptides or proteins is not yet well established. Aiming to illustrate their utility, we chose the peptide conjugate [Nle15]MG11 (DOTA-dGlu-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2) as a model compound since it is known that the cholecystokinin-2 receptor (CCK2R) is able to accommodate turn conformations. Analogs of [Nle15]MG11 incorporating 1,5-Tz in the backbone were synthesized and radiolabeled with lutetium-177, and their pharmacological properties (cell internalization, receptor binding affinity and specificity, plasma stability, and biodistribution) were evaluated and compared with [Nle15]MG11 as well as their previously reported analogs bearing 1,4-disubstituted 1,2,3-triazoles. Our investigations led to the discovery of novel triazole-modified analogs of [Nle15]MG11 with nanomolar CCK2R-binding affinity and 2-fold increased tumor uptake. This study illustrates that substitution of amides by 1,5-disubstituted 1,2,3-triazoles is an effective strategy to enhance the pharmacological properties of biologically active peptides.

Biomimetic, Smart, and Multivalent Ligands for G-Quadruplex Isolation and Bioorthogonal Imaging.

G-quadruplexes (G4s) continue to gather wide attention in the field of chemical biology as their prevalence in the human genome and transcriptome strongly suggests that they play key regulatory roles in cell biology. G4-specific, cell-permeable small molecules (G4-ligands) innovatively permit the interrogation of cellular circuitries in order to assess to what extent G4s influence cell fate and functions. Here, we report on multivalent, biomimetic G4-ligands referred to as TASQs that enable both the isolation and visualization of G4s in human cells. Two biotinylated TASQs, BioTASQ and BioCyTASQ, are indeed efficient molecular tools to isolate G4s from mixtures of nucleic acids through simple affinity capture protocols and to image G4s in cells via a biotin/avidin pretargeted imaging system first applied here to G4s, found to be a reliable alternative to in situ click chemistry.

Fluorogenic Enzyme-Triggered Domino Reactions Producing Quinoxalin-2(1H)-one-based Heterocycles.

A simple and effective biocompatible domino reaction triggered by a model protease and leading to the formation of strongly fluorescent quinoxalin-2(1H)-one N-heterocycles is described. Some positive attributes including versatility and the ability to provide outstanding fluorescence "OFF-ON" responses were revealed by this work. They open the way for practical applications of this novel type of "covalent-assembly"-based fluorescent probe in the fields of sensing and bioimaging.

Single Peptide Backbone Surrogate Mutations to Regulate Angiotensin GPCR Subtype Selectivity.

Mutating the side-chains of amino acids in a peptide ligand, with unnatural amino acids, aiming to mitigate its short half-life is an established approach. However, it is hypothesized that mutating specific backbone peptide bonds with bioisosters can be exploited not only to enhance the proteolytic stability of parent peptides, but also to tune its receptor subtype selectivity. Towards this end, four [Y]6 -Angiotensin II analogues are synthesized where amide bonds have been replaced by 1,4-disubstituted 1,2,3-triazole isosteres in four different backbone locations. All the analogues possessed enhanced stability in human plasma in comparison with the parent peptide, whereas only two of them achieved enhanced AT2 R/AT1 R subtype selectivity. This diversification has been studied through 2D NMR spectroscopy and unveiled a putative more structured microenvironment for the two selective ligands accompanied with increased number of NOE cross-peaks. The most potent analogue, compound 2, has been explored regarding its neurotrophic potential and resulted in an enhanced neurite growth with respect to the established agent C21.

Synthesis and evaluation of zirconium-89 labelled and long-lived GLP-1 receptor agonists for PET imaging.

INTRODUCTION: Lately, zirconium-89 has shown great promise as a radionuclide for PET applications of long circulating biomolecules. Here, the design and synthesis of protracted and long-lived GLP-1 receptor agonists conjugated to desferrioxamine and labelled with zirconium-89 is presented with the purpose of studying their in vivo distribution by PET imaging. The labelled conjugates were evaluated and compared to a non-labelled GLP-1 receptor agonist in both in vitro and in vivo assays to certify that the modification did not significantly alter the peptides' structure or function. Finally, the zirconium-89 labelled peptides were employed in PET imaging, providing visual verification of their in vivo biodistribution. METHODS: The evaluation of the radiolabelled peptides and comparison to their non-labelled parent peptide was performed by in vitro assays measuring binding and agonistic potency to the GLP-1 receptor, physicochemical studies aiming at elucidating change in peptide structure upon bioconjugation and labelling as well as an in vivo food in-take study illustrating the compounds' pharmacodynamic properties. The biodistribution of the labelled GLP-1 analogues was determined by ex vivo biodistribution and in vivo PET imaging. RESULTS: The results indicate that it is surprisingly feasible to design and synthesize a protracted, zirconium-89 labelled GLP-1 receptor agonist without losing in vitro potency or affinity as compared to a non-labelled parent peptide. Physicochemical properties as well as pharmacodynamic properties are also maintained. The biodistribution in rats shows high accumulation of radiolabelled peptide in well-perfused organs such as the liver, kidney, heart and lungs. The PET imaging study confirmed the findings from the biodistribution study with a significant high uptake in kidneys and presence of activity in liver, heart and larger blood vessels. CONCLUSIONS AND ADVANCES IN KNOWLEDGE: This initial study indicates the potential to monitor the in vivo distribution of long-circulating incretin hormones using zirconium-89 based PET.

Amide-to-triazole switch vs. in vivo NEP-inhibition approaches to promote radiopeptide targeting of GRPR-positive tumors.

INTRODUCTION: Radiolabeled bombesin (BBN)-analogs have been proposed for diagnosis and therapy of gastrin-releasing peptide receptor (GRPR)-expressing tumors, such as prostate, breast and lung cancer. Metabolic stability represents a crucial factor for the success of this approach by ensuring sufficient delivery of circulating radioligand to tumor sites. The amide-to-triazole switch on the backbone of DOTA-PEG4-[Nle14]BBN(7-14) (1) was reported to improve the in vitro stability of resulting 177Lu-radioligands. On the other hand, in-situ inhibition of neutral endopeptidase (NEP) by coinjection of phosphoramidon (PA) was shown to significantly improve the in vivo stability and tumor uptake of biodegradable radiopeptides. We herein compare the impact of the two methods on the bioavailability and localization of 177Lu-DOTA-PEG4-[Nle14]BBN(7-14) analogs in GRPR-positive tumors in mice. METHODS: The 1,4-disubstituted [1-3]-triazole was used to replace one (2: Gly11-His12; 3: Ala9-Val10) or two (4: Ala9-Val10 and Gly11-His12) peptide bonds in 1 (reference) and all compounds were labeled with 177Lu. Each of [177Lu]1-[177Lu]4 was injected without (control) or with PA in healthy mice. Blood samples collected 5min post-injection (pi) were analyzed by HPLC. Biodistribution of [177Lu]1-[177Lu]4 was conducted in SCID mice bearing human prostate adenocarcinoma PC-3 xenografts at 4h pi. Groups of 4 animals were injected with radioligand, alone (controls), or with coinjection of PA, or of a mixture of PA and excess and [Tyr4]BBN to determine GRPR-specificity of uptake (Block). RESULTS: The in vivo stability of the radioligands was: [177Lu]1 (25% intact), [177Lu]2 (45% intact), [177Lu]3 (30% intact) and [177Lu]4 (40% intact). By PA-coinjection these values notably increased to 90%-93%. Moreover, treatment with PA induced an impressive and GRPR-specific uptake of all radioligands in the PC-3 xenografts at 4h pi: [177Lu]1: 4.7±0.4 to 24.8±4.9%ID/g; [177Lu]2: 8.3±1.2 to 26.0±1.1%ID/g; [177Lu]3: 6.6±0.4 to 21.3±4.4%ID/g; and [177Lu]4: 4.8±1.6 to 13.7±3.8%ID/g. CONCLUSIONS: This study has shown that amide-to-triazole substitutions in 177Lu-DOTA-PEG4-[Nle14]BBN(7-14) induced minor effects on bioavailability and tumor uptake in mice models, whereas in-situ NEP-inhibition(s) by PA impressively improved in vivo profiles.

Comparison of the octadentate bifunctional chelator DFO*-pPhe-NCS and the clinically used hexadentate bifunctional chelator DFO-pPhe-NCS for 89Zr-immuno-PET.

PURPOSE: All clinical 89Zr-immuno-PET studies are currently performed with the chelator desferrioxamine (DFO). This chelator provides hexadentate coordination to zirconium, leaving two coordination sites available for coordination with, e.g., water molecules, which are relatively labile ligands. The unsaturated coordination of DFO to zirconium has been suggested to result in impaired stability of the complex in vivo and consequently in unwanted bone uptake of 89Zr. Aiming at clinical improvements, we report here on a bifunctional isothiocyanate variant of the octadentate chelator DFO* and the in vitro and in vivo comparison of its 89Zr-DFO*-mAb complex with 89Zr-DFO-mAb. METHODS: The bifunctional chelator DFO*-pPhe-NCS was prepared from previously reported DFO* and p-phenylenediisothiocyanate. Subsequently, trastuzumab was conjugated with either DFO*-pPhe-NCS or commercial DFO-pPhe-NCS and radiolabeled with Zr-89 according to published procedures. In vitro stability experiments were carried out in saline, a histidine/sucrose buffer, and blood serum. The in vivo performance of the chelators was compared in N87 tumor-bearing mice by biodistribution studies and PET imaging. RESULTS: In 0.9 % NaCl 89Zr-DFO*-trastuzumab was more stable than 89Zr-DFO-trastuzumab; after 72 h incubation at 2-8 °C 95 % and 58 % intact tracer were left, respectively, while in a histidine-sucrose buffer no difference was observed, both products were ≥ 92 % intact. In vivo uptake at 144 h post injection (p.i.) in tumors, blood, and most normal organs was similar for both conjugates, except for skin, liver, spleen, ileum, and bone. Tumor uptake was 32.59 ± 11.95 and 29.06 ± 8.66 % ID/g for 89Zr-DFO*-trastuzumab and 89Zr-DFO-trastuzumab, respectively. The bone uptake was significantly lower for 89Zr-DFO*-trastuzumab compared to 89Zr-DFO-trastuzumab. At 144 h p.i. for 89Zr-DFO*-trastuzumab and 89Zr-DFO-trastuzumab, the uptake in sternum was 0.92 ± 0.16 and 3.33 ± 0.32 % ID/g, in femur 0.78 ± 0.11 and 3.85, ± 0.80 and in knee 1.38 ± 0.23 and 8.20 ± 2.94 % ID/g, respectively. The uptake in bone decreased from 24 h to 144 h p.i. about two fold for the DFO* conjugate, while it increased about two fold for the DFO conjugate. CONCLUSIONS: Zr-DFO*-trastuzumab showed superior in vitro stability and in vivo performance when compared to 89Zr-DFO-trastuzumab. This makes the new octadentate DFO* chelator a candidate successor of DFO for future clinical 89Zr-immuno-PET.

Novel chemoselective (18)F-radiolabeling of thiol-containing biomolecules under mild aqueous conditions.

We report a novel prosthetic group based on a heterocyclic methylsulfone derivative for the rapid, stable, and chemoselective (18)F-labeling of thiol-containing (bio)molecules under mild aqueous reaction conditions. Compared to established maleimide approaches, the new methodology displays some clear advantages for imaging probe development.

Toward the Optimization of Bombesin-Based Radiotracers for Tumor Targeting.

The peptide bombesin (BBN) is a peptide with high affinity for the gastrin-releasing peptide receptor (GRPr), a receptor that is overexpressed by, for example, breast and prostate cancers. Thus, GRPr agonists can be used as cancer-targeting vectors to shuttle diagnostic and therapeutic agents into tumor cells. With the aim of optimizing the tumor targeting properties of a radiolabeled [Nle(14)]BBN(7-14) moiety, novel BBN(7-14)- and BBN(6-14)-based radioconjugates were synthesized, labeled with Lu-177, and fully evaluated in vitro and in vivo. The effect of residue and backbone modification on several parameters such as the internalization of the radiolabeled peptides into PC3 and AR42J tumor cells, their affinity toward the human GRPr, metabolic stability in blood plasma, and biodistribution in mice bearing GRPr-expressing PC3 xenografts was studied. As a result of our investigations, a novel radiolabeled GRPr agonist with a high tumor uptake and a high tumor-to-kidney ratio was identified.

Structure-Activity Relationship Studies of Amino Acid Substitutions in Radiolabeled Neurotensin Conjugates.

Radiolabeled derivatives of the peptide neurotensin (NT) and its binding sequence NT(8-13) have been studied as potential imaging probes and therapeutics for NT-1-receptor-positive cancer. However, a direct comparison of reported NT analogues, even if radiolabeled with the same radionuclide, is difficult because different techniques and models have been used for preclinical evaluations. In an effort to identify a suitable derivative of NT(8-13) for radiotracer development, we herein report a side-by-side in vitro comparison of radiometallated NT derivatives bearing some of the most commonly reported amino acid substitutions in their sequence. Performed investigations include cell internalization experiments, determinations of receptor affinity, measurements of the distribution coefficient, and blood serum stability studies. Of the [(177)Lu]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-labeled examples studied, analogues of NT(8-13) containing a short hydrophilic tetraethylene glycol (PEG4 ) spacer between the peptide and the radiometal complex, and a minimum number of substitutions of amino acid residues, exhibited the most promising properties in vitro.

1,2,3-Triazole Stabilized Neurotensin-Based Radiopeptidomimetics for Improved Tumor Targeting.

Neurotensin (NT) is a regulatory peptide with nanomolar affinity toward NT receptors, which are overexpressed by different clinically relevant tumors. Its binding sequence, NT(8-13), represents a promising vector for the development of peptidic radiotracers for tumor imaging and therapy. The main drawback of the peptide is its short biological half-life due to rapid proteolysis in vivo. Herein, we present an innovative strategy for the stabilization of peptides using nonhydrolizable 1,4-disubstituted, 1,2,3-triazoles as amide bond surrogates. A "triazole scan" of the peptide sequence yielded novel NT(8-13) analogues with enhanced stability, retained receptor affinity, and improved tumor targeting properties in vivo. The synthesis of libraries of triazole-based peptidomimetics was achieved efficiently on solid support by a combination of Fmoc-peptide chemistry, diazo transfer reactions, and the Cu(I)-catalyzed alkyne azide cycloaddition (CuAAC) employing methods that are fully compatible with standard solid phase peptide synthesis (SPPS) chemistry. Thus, the amide-to-triazole substitution strategy may represent a general methodology for the metabolic stabilization of biologically active peptides.

Development of 68Ga- and 89Zr-Labeled Exendin-4 as Potential Radiotracers for the Imaging of Insulinomas by PET.

UNLABELLED: Clinical studies have demonstrated the potential of radiometallated exendin-4 derivatives for the imaging of glucagonlike peptide-1 receptor-overexpressing insulinomas. Recently investigated exendin-4 derivatives were radiolabeled with the SPECT isotopes 99mTc or 111In. Despite promising results, the low spatial resolution associated with SPECT and the occasional need to perform imaging several days after injection for the demarcation of insulinomas from the kidneys represent current limitations. The aim of this work was the development of exendin-4 derivatives for the imaging of insulinomas by high-resolution PET at early or late time points after injection of the radiotracer. METHODS: An exendin-4 derivative conjugated to desferrioxamine (DFO) was used for radiolabeling with the PET isotopes 68Ga and 89Zr. Both radiotracers were evaluated in vitro with RIN-m5F cells for their cell internalization properties as well as affinities and specificities toward the glucagonlike peptide-1 receptor. Serum stabilities of the radiopeptides were assessed in blood serum, and their distribution coefficient was determined by the shake-flask method. Biodistribution experiments were performed with nude mice bearing RIN-m5F xenografts. For all experiments, clinically evaluated [Lys40-(AHX-DTPA-111In)NH2]exendin-4 was used as a reference compound. RESULTS: [Lys40-(AHX-DFO)NH2]exendin-4 was labeled with 89Zr and 68Ga in high radiochemical yield and purity. In vitro experiments showed favorable cell uptake and receptor affinity for [Lys40-(AHX-DFO-68Ga)NH2]exendin-4, and [Lys40-(AHX-DFO-89Zr)NH2]exendin-4 and [Lys40-(AHX-DTPA-111In)NH2]exendin-4 performed similarly well. In biodistribution experiments, [Lys40-(AHX-DFO-68Ga)NH2]exendin-4 exhibited a significantly enhanced tumor uptake 1 h after injection in comparison to the other 2 radiotracers. Tumor uptake of [Lys40-(AHX-DFO-89Zr)NH2]exendin-4 was comparable to that of [Lys40-(AHX-DTPA-111In)NH2]exendin-4 at 1-48 h after injection. All compounds showed a fast blood clearance and low accumulation in receptor-negative organs and tissue with the exception of the kidneys, a known characteristic for exendin-4-based radiotracers. CONCLUSION: 68Ga- and 89Zr-radiolabeled [Lys40-(AHX-DFO)NH2]exendin-4 exhibit characteristics comparable or superior to the clinically tested reference compound [Lys40-(AHX-DTPA-111In)NH2]exendin-4 and, thus, represent potential new tracers for the imaging of insulinomas by PET.

Probing the Backbone Function of Tumor Targeting Peptides by an Amide-to-Triazole Substitution Strategy.

Novel backbone-modified radiolabeled analogs based on the tumor targeting peptide bombesin were synthesized and fully evaluated in vitro and in vivo. We have recently introduced the use of 1,4-disubstituted 1,2,3-triazoles as metabolically stable trans-amide bond surrogates in radiolabeled peptides in order to improve their tumor targeting. As an extension of our approach, we now report several backbone-modified analogs of the studied bombesin peptide bearing multiple triazole substitutions. We investigated the effect of the modifications on several biological parameters including the internalization of the radiopeptidomimetics into tumor cells, their affinity toward the gastrin releasing peptide receptor (GRPr), metabolic stability in blood plasma, and biodistribution in mice bearing GRPr-expressing xenografts. The backbone-modified radiotracers exhibited a significantly increased resistance to proteolytic degradation. In addition, some of the radiopeptidomimetics retained a nanomolar affinity toward GRPr, resulting in an up to 2-fold increased tumor uptake in vivo in comparison to a (all amide bond) reference compound.

Radiolabeled antagonistic bombesin peptidomimetics for tumor targeting.

The replacement of amide bonds in the backbone of peptides by proteolytically stable 1,2,3-triazole isosteres can provide novel peptidomimetics with promising properties for the development of tumor-targeting radiopeptides. On the basis of our previous work with radiolabeled agonistic bombesin (BBN) derivatives of the sequence [Nle(14) ]BBN(7-14), we substituted selected amide bonds of the structurally closely related antagonistic peptide analog JMV594. With the exception of the C-terminal modification, amide-to-triazole substitutions tolerated by [Nle(14) ]BBN(7-14) without loss of biological function led to abolished receptor affinity in the case of JMV594. These findings provide an additional piece of evidence for the currently disputed differences in the modes of action of agonistic and antagonistic gastrin-releasing peptide receptor (GRPR)-targeting radiopeptides.