Jürgen Einsiedel

Labeling and Glycosylation of Peptides Using Click Chemistry: A General Approach to 18F-Glycopeptides as Effective Imaging Probes for Positron Emission Tomography

In the field of molecular imaging, positron emission tomography (PET) has emerged as an imaging modality with excellent sensitivity for in vivo studies. PET labeling is challenging since short-lived positron-emitting isotopes such as F and C are used as labeling agents. The optimization and efficient application of rapid and reliable labeling strategies are prerequisites for obtaining access to new radiopharmaceuticals for both research and clinical trials. Bioactive peptides that specifically address molecular targets in vivo represent an important class of PET tracers to facilitate predictive imaging and PET-guided therapy. Diverse strategies for the synthesis of peptide-based radiopharmaceuticals using F-labeled prosthetic groups have been elaborated, including chemoselective oxime conjugation and the use of F-labeled maleimide derivatives as cysteinereactive reagents. Following the concept of click chemistry introduced by Sharpless et al., the Huisgen [3+2] azide– alkyne cycloaddition has been adapted to F-radiosynthetic methods in order to take advantage of its selectivity, reliability, and speed under aqueous mild Cupromoted reaction conditions. The versatility of peptide imaging agents is frequently hampered by their instability in vivo because of rapid degradation by endogenous peptidases. As an example, the synthesis of radiolabeled peptide-based imaging agents for the neurotensin receptor-1 (NTR-1), which is overexpressed in a number of human cancers, requires modifications to improve the metabolic stability. Synthetic approaches to RGD tracers targeting avb3 integrin, which plays a key role in angiogenesis, capitalize on the pioneering studies by Kessler et al. , who successfully developed cyclic pentameric RGD peptides that selectively recognize integrin avb3. [9] Various radiolabeled cyclic RGD peptides have been described. Among these, [F]galactoRGD has been extensively evaluated in clinical studies. Since glycosylation of peptides is known to frequently improve the biokinetic and in vivo clearance properties, [F]galacto-RGD and further radiopeptides have been approached. 13] However, the multistep radiosynthesis of [F]galacto-RGD is time-consuming and laborious. In proposals to overcome this drawback, F-labeling by 2-deoxy-2[F]fluoroglucose (FDG) has been discussed. 15] The major disadvantages of the F-peptide-labeling strategies currently used are 1) harsh reaction conditions, 2) laborious multiple-step syntheses with a limited decayuncorrected radiochemical yield (RCY), which would complicate the automation for large-scale production, and 3) lipophilic derivatization, which impair the biokinetic properties of the tracer. Based on our previous work on click chemistry in drug discovery and the synthesis of b-mannosyl azides, we herein present an efficient strategy toward F-labeling with concomitant glycosylation for the synthesis of F-glycopeptides as imaging agents for PET. We combined this strategy with the development of a metabolically stable glycopeptoid analogue of NT(8–13), which is the highly potent C-terminal hexapeptide of the natural agonist neurotensin (NT). As a proof of concept, two F-glycopeptides derived from NT(8– 13) and c(RGDfPra), respectively, were applied to biodistribution studies and mPET for imaging NTR and avb3-integrin expression in vivo using xenograft nude mice models. In detail, 2-deoxy-2-fluoroglucosyl azide (3) could be obtained starting from tetraacetylated 2-deoxy-2-fluoroglucose. The glucosyl azide 3 was applied for the Cu-catalyzed azide–alkyne coupling with a series of alkyne-functionalized peptides to evaluate the influence of the appended glycosyl residue on receptor recognition. Commercially available propargylglycine (Pra) was introduced by solid-phase-supported synthesis at position X into the sequence of the bioactive peptide c(RGDfX) and at the N terminus of NT(8– 13) and metabolically stabilized derivatives thereof. Considering our studies on the influence of peptide backbone modifications and ligand conformation on affinity changes for a series of NT(8–13) analogues, metabolic stabilization was envisioned by alteration of three amino acids in the sequence [*] Dr. S. Maschauer, Dr. C. Hocke, Prof. T. Kuwert, Dr. O. Prante Nuklearmedizinische Klinik, Labor f r Molekulare Bildgebung Friedrich-Alexander-Universit t Erlangen-N rnberg Schwabachanlage 6, 91054 Erlangen (Germany) Fax: (+ 49)9131-853-4325 E-mail: olaf.prante@uk-erlangen.de

Discovery of highly potent and neurotensin receptor 2 selective neurotensin mimetics.

The neurotensin receptor subtype 2 (NTS2) is involved in the modulation of tonic pain sensitivity and psychiatric diseases and is, therefore, regarded as a highly attractive pharmacological target protein. Aiming to discover NTS2 selective ligands, we herein describe the identification of screening hits and the chemical synthesis of structural variants leading to the highly potent and NTS2 selective peptide-peptoid hybrids of type 3. The neurotensin mimetics 3a and 3e-g incorporating an N-(4-hydroxyphenethyl)glycine substructure exhibit single digit nanomolar affinity (K(i) = 4.3-8.8 nM) and 1900-12000 fold selectivity over the neurotensin receptor subtype 1 (NTS1). According to functional experiments, the test compounds 3a and 3e-g displayed an inhibition of constitutive mitogen-activated protein kinase (MAPK) activity exceeding 2.6-4.6 times the inverse agonist activity of the endogenous ligand neurotensin.

Phenyloxazoles and phenylthiazoles as benzamide bioisosteres: synthesis and dopamine receptor binding profiles ☆

Conformationally restricted benzamide bioisosteres were investigated when the aminomethylpyrrolidine derivative 4o proved D3 as well as D4 binding properties which were comparable to those of the atypical neuroleptics sulpiride and clozapine, respectively.

Novel insights into GPCR—Peptide interactions: Mutations in extracellular loop 1, ligand backbone methylations and molecular modeling of neurotensin receptor 1

Investigating prototypical interactions between NT(8-13) and the human neurotensin receptor 1 (hNTR1), we created a receptor-ligand model that was validated by site-directed mutagenesis and structure-activity relationship studies. Stabilization of the extracellular loop 1 (EL1) by pi-stacking clusters proved to be important for agonist binding when substitution of six conserved amino acids by alanine resulted in an agonist specific loss of maximal binding capacity. In agreement with our modeling studies, EL1 seems to adopt a clamp-type border area controlling the shape of the binding site crevice. Employing chemically manipulated peptide analogs as molecular probes, the impact of backbone modifications on receptor-ligand interaction, especially the influence on ligand conformation, was examined in binding studies and explained by in silico analysis.

Benzamide bioisosteres incorporating dihydroheteroazole substructures: EPC synthesis and SAR leading to a selective dopamine D4 receptor partial agonist (FAUC 179)

Conformationally restricted benzamide bioisosteres were investigated when the chiral phenyldihydroimidazole derivative 4e (FAUC 179) showed strong and highly selective dopamine D4 receptor binding (K(i)high=0.95nM). Mitogenesis experiments indicated partial agonist properties (42%). EPC syntheses of the target compounds of type 4 were performed starting from alpha-amino acids.

Peptide backbone modifications on the C-terminal hexapeptide of neurotensin.

To compare backbone-induced susceptibilities with affinity changes that are caused by side-chain modifications in the respective positions, structure activity relationship studies on a series of NT(8-13) analogues were performed providing valuable insights into the major requirement for neurotensin receptor recognition and activation. The data led us to highly potent NTR1 ligands and the generation of a pharmacophore model that will be helpful for the discovery of therapeutically relevant non-peptidic NTR1 agonists.

Functional characterization of a partial loss-of-function mutation of the epithelial sodium channel (ENaC) associated with atypical cystic fibrosis.

Loss-of-function mutations of the epithelial sodium channel (ENaC) may contribute to pulmonary symptoms resembling those of patients with atypical cystic fibrosis (CF). Recently, we identified a loss-of-function mutation in the α-subunit of ENaC (αF61L) in an atypical CF patient without mutations in CFTR. To investigate the functional effect of this mutation, we expressed human wild-type αβγ-ENaC or mutant α F61Lβγ-ENaC in Xenopus laevis oocytes. The αF61L mutation reduced the ENaC mediated whole-cell currents by ñ90%. In contrast, the mutation decreased channel surface expression only by ñ40% and did not alter the single-channel conductance. These findings indicate that the major effect of the mutation is a reduction of the average channel open probability (Po). This was confirmed by experiments using the βS520C mutant ENaC which can be converted to a channel with a Po of nearly one, and by experiments using chymotrypsin to proteolytically activate the channel. These experiments revealed that the mutation reduced the average Po of ENaC by ñ75%. Na+ self inhibition of the mutant channel was significantly enhanced, but the observed effect was too small to account for the large reduction in average channel Po. The ENaC-activator S3969 partially rescued the loss-of-function phenotype of the αF61L mutation. We conclude that the αF61L mutation may contribute to respiratory symptoms in atypical CF patients.

Cyclic Amidines as Benzamide Bioisosteres: EPC Synthesis and SAR Studies Leading to the Selective Dopamine D4 Receptor Agonist FAUC 312

Investigation of conformationally restricted benzamide bioisosteres led to the chiral phenyltetrahydropyrimidine derivative ent2a (FAUC 312) displaying strong and highly selective dopamine D4 receptor binding (K(i(high))=1.5 nM). Mitogenesis experiments indicated 83% ligand efficacy when compared to the unselective agonist quinpirole. The target compounds of type 2 and 3 were synthesized in enantiopure form starting from asparagine.

18F- and 68Ga-Labeled Neurotensin Peptides for PET Imaging of Neurotensin Receptor 1

The neurotensin (NT) receptor-1 (NTS1) is overexpressed in a variety of carcinomas and is therefore an interesting target for imaging with positron emission tomography (PET). The aim of this study was the development of new NT derivatives based on the metabolically stable peptide sequence NLys-Lys-Pro-Tyr-Tle-Leu suitable for PET imaging. The NT peptides were synthesized by solid-phase supported peptide synthesis and elongated with respective chelators (NODA-GA, DOTA) for (68)Ga-labeling or propargylglycine for (18)F-labeling via copper-catalyzed azide-alkyne cycloaddition. Receptor affinities of the peptides for NTS1 were in the range of 19-110 nM. Biodistribution studies using HT29 tumor-bearing mice showed highest tumor uptake for [(68)Ga]6 and [(68)Ga]8 and specific binding in small-animal PET studies. The tumor uptake of (68)Ga-labeled peptides in vivo significantly correlated with the in vitro Ki values for NTS1. [(68)Ga]8 displayed an excellent tumor-to-background ratio and could therefore be considered as an appropriate molecular probe for NTS1 imaging by PET.

Stereocontrolled dopamine receptor binding and subtype selectivity of clebopride analogues synthesized from aspartic acid.

Employing the achiral 4-aminopiperidine derivative clebopride as a lead compound, chiral analogues were developed displaying dopamine receptor binding profiles that proved to be strongly dependent on the stereochemistry. Compared to the D1 receptor, the test compounds showed high selectivity for the D2-like subtypes including D2(long), D2(short), D3 and D4. The highest D4 and D3 affinities were observed for the cis-3-amino-4-methylpyrrolidines 3e and the enantiomer ent3e resulting in K(i) values of 0.23 and 1.8 nM, respectively. The benzamides of type 3 and 5 were synthesized in enantiopure form starting from (S)-aspartic acid and its unnatural optical antipode.