Rationalizing the Binding Modes of PET Radiotracers Targeting the Norepinephrine Transporter

Abstract

Purpose: A new PET radiotracer 18F-AF78 showing great potential for clinical application has been reported recently. It belongs to a new generation of phenethylguanidine-based norepinephrine transporter (NET)-targeting radiotracers. Although many efforts have been made to develop NET inhibitors as antidepressants, systemic investigations of the structure–activity relationships (SARs) of NET-targeting radiotracers have rarely been performed. Methods: Without changing the phenethylguanidine pharmacophore and 3-fluoropropyl moiety that is crucial for easy labeling, six new analogs of 18F-AF78 with different meta-substituents on the benzene-ring were synthesized and evaluated in a competitive cellular uptake assay and in in vivo animal experiments in rats. Computational modeling of these tracers was established to quantitatively rationalize the interaction between the radiotracers and NET. Results: Using non-radiolabeled reference compounds, a competitive cellular uptake assay showed a decrease in NET-transporting affinity from meta-fluorine to iodine (0.42 and 6.51 µM, respectively), with meta-OH being the least active (22.67 µM). Furthermore, in vivo animal studies with radioisotopes showed that heart-to-blood ratios agreed with the cellular experiments, with AF78(F) exhibiting the highest cardiac uptake. This result correlates positively with the electronegativity rather than the atomic radius of the meta-substituent. Computational modeling studies revealed a crucial influence of halogen substituents on the radiotracer–NET interaction, whereby a T-shaped π–π stacking interaction between the benzene-ring of the tracer and the amino acid residues surrounding the NET binding site made major contributions to the different affinities, in accordance with the pharmacological data. Conclusion: The SARs were characterized by in vitro and in vivo evaluation, and computational modeling quantitatively rationalized the interaction between radiotracers and the NET binding site. These findings pave the way for further evaluation in different species and underline the potential of AF78(F) for clinical application, e.g., cardiac innervation imaging or molecular imaging of neuroendocrine tumors.

Navigating new horizons: Prospects of NET-targeted radiopharmaceuticals in precision medicine

“…By forging partnerships between academia, industry, and healthcare systems, we can enhance the availability and application of these tracers, ensuring equitable access to this crucial diagnostic technology. Moreover, yet in the early development stage, with tracers such as 18 F-AF78 72 , 73 and 18 F-PHPG/MHPG 75 demonstrating high NET affinity, refining imaging protocols becomes possible, which may significantly improve disease detection and characterization. By integrating these imaging results with molecular and genetic data, we could potentially further tailor the diagnosis and treatment of NET-related disorders, moving closer to the personalized medicine paradigm.…”

“…(D) Comparative PET scans of non-human primates (NHPs), with and without the NET-selective inhibitor desipramine, demonstrate the specificity of 18 F-AF78 in cardiac imaging. Adapted with permission from 73 , Copyright 2023, MDPI.…”

“…The placement of radionuclides within the molecular structures is highlighted in red. Adapted with permission from 73 , Copyright 2023, MDPI.…”

“…This was a significant step forward in the cardiac applications of PET imaging, building on the foundations laid by its predecessors. The introduction of 18 F-AF78 [71][72][73] and 18 F-PHPG/MHPG [74,75] is a testament to the rapid progress in this domain, delivering tracers with pronounced NET affinity [72], potentially reshaping the diagnostic landscape for a variety of NET-related disorders. The high binding affinity of 18 F-AF78 (Figure 2) [73] could lead to superior sensitivity and specificity in disease detection, especially in conditions where NET dysfunction is implicated, including certain neurodegenerative pathologies and neuroendocrine tumors.…”

“…The introduction of 18 F-AF78 [71][72][73] and 18 F-PHPG/MHPG [74,75] is a testament to the rapid progress in this domain, delivering tracers with pronounced NET affinity [72], potentially reshaping the diagnostic landscape for a variety of NET-related disorders. The high binding affinity of 18 F-AF78 (Figure 2) [73] could lead to superior sensitivity and specificity in disease detection, especially in conditions where NET dysfunction is implicated, including certain neurodegenerative pathologies and neuroendocrine tumors. The evolution of these PET tracers showcases a decisive march toward imaging modalities that are not only more efficient but also molecularly astute, aligning with the overarching objectives of precision medicine.…”