Search Results (18)

Background/Objectives: α-carbonic anhydrase (α-TcCA) has emerged as a promising drug target in T. cruzi, the causative agent of Chagas disease in the Americas. Sulfonamides, known inhibitors of CAs, bind to the zinc ion on the enzyme’s active site. This study proposes the repositioning of sulfonamide-based drugs to identify new trypanocidal agents. Method: Ligand-based virtual screening and molecular docking analysis were performed on FDA-approved drugs targeting α-TcCA. These compounds were evaluated in vitro and ex vivo against the A1 and NINOA strains, followed by enzymatic assays. Results: Four sulfonylureas were selected: glimepiride (Glim), acetohexamide (Ace), gliclazide (Glic), and tolbutamide (Tol). Ace and Tol had half-maximal inhibitory concentration (IC₅₀) values similar or better than reference drugs against the NINOA strain in the epimastigote and trypomastigote stages, while Glic and Glim had the highest activity against the A1 strain (epimastigotes and amastigotes). Notably, Ace had the highest trypanocidal activity against all stages in NINOA, with IC₅₀ values of 6.5, 46.5, and 46 μM for epimastigotes, trypomastigotes, and amastigotes, respectively. Additionally, Ace inhibited α-TcCA with K_I = 5.6 μM, suggesting that its trypanocidal effect is associated to the enzyme inhibition. Conclusions: This study supports the repositioning of FDA-approved sulfonamide-based hypoglycaemic agents as trypanocidal compounds. Future studies should focus on structural modifications to improve selectivity. Integrating docking, parasitological, and enzymatic data is crucial for optimizing drug candidates for Chagas disease. Full article

Background/Objectives: Gastrin-releasing peptide receptor is a promising target for cancer diagnosis and therapy. However, the high pancreas uptake of reported GRPR-targeted radioligands limits their clinical applications. Our group previously reported one ⁶⁸Ga-labeled GRPR antagonist, [⁶⁸Ga]Ga-TacsBOMB5 (⁶⁸Ga-DOTA-Pip-[D-Phe⁶,NMe-Gly¹¹,Leu¹³ψThz¹⁴]Bombesin(6–14)), and two agonists, [⁶⁸Ga]Ga-LW01110 (⁶⁸Ga-DOTA-Pip-[D-Phe⁶,Tle¹⁰,NMe-His¹²,Thz¹⁴]Bombesin(6–14)) and [⁶⁸Ga]Ga-LW01142 (⁶⁸Ga-DOTA-Pip-[D-Phe⁶,His⁷,Tle¹⁰,NMe-His¹²,Thz¹⁴]Bombesin(6–14)) showing minimal pancreas uptake. Thus, in this study, we prepared their ¹⁷⁷Lu-labeled analogs, evaluated their therapeutic potentials, and compared them with the clinically evaluated [¹⁷⁷Lu]Lu-AMBA. Methods: GRPR binding affinities were determined by in vitro competition binding assay using PC-3 prostate cancer cells. Longitudinal SPECT/CT imaging and ex vivo biodistribution studies were conducted in PC-3 tumor-bearing mice. Dosimetry data were calculated from the biodistribution results. Results: The K_i(GRPR) values of Lu-TacsBOMB5, Lu-LW01110, Lu-LW01142, and Lu-AMBA were 12.6 ± 1.02, 3.07 ± 0.15, 2.37 ± 0.28, and 0.33 ± 0.16 nM, respectively. SPECT/CT images and biodistribution results demonstrated good tumor accumulation of [¹⁷⁷Lu]Lu-TacsBOMB5, [¹⁷⁷Lu]Lu-LW01110, and [¹⁷⁷Lu]Lu-LW01142 at early time points with rapid clearance over time. The pancreas uptake of all three [Thz¹⁴]Bombesin(6–14)-derived ligands was significantly lower than that of [¹⁷⁷Lu]Lu-AMBA at all time points. The calculated absorbed doses of [¹⁷⁷Lu]Lu-TacsBOMB5, [¹⁷⁷Lu]Lu-LW01110, and [¹⁷⁷Lu]Lu-LW01142 in PC-3 tumor xenografts were 87.1, 312, and 312 mGy/MBq, respectively, higher than that of [¹⁷⁷Lu]Lu-AMBA (79.1 mGy/MBq), but lower than that of the previously reported [¹⁷⁷Lu]Lu-RM2 (429 mGy/MBq). Conclusions: Our data suggest that [¹⁷⁷Lu]Lu-TacsBOMB5 and [¹⁷⁷Lu]Lu-LW01142 reduce radiation exposure to the pancreas. However, further optimizations are needed for both radioligands to prolong their tumor retention and enhance treatment efficacy. Full article

Background/Objectives: Overexpressed in various solid tumors, the gastrin-releasing peptide receptor (GRPR) is a promising target for cancer diagnosis and therapy. However, the high pancreas uptake of the current clinically evaluated GRPR-targeted radiopharmaceuticals limits their applications. In this study, we replaced the Pro¹⁴ residue in our previously reported GRPR-targeted LW02056 and ProBOMB5 with 4,4-difluoroproline (diF-Pro) to obtain an agonist LW02060 (DOTA-Pip-[D-Phe⁶,Tle¹⁰,NMe-His¹²,diF-Pro¹⁴]Bombesin(6–14)) and an antagonist LW02080 (DOTA-Pip-[D-Phe⁶,NMe-Gly¹¹,Leu¹³(ψ)diF-Pro¹⁴]Bombesin(6–14)), respectively. Methods/Results: The binding affinities (K_i) of Ga-LW02060, Ga-LW02080, Lu-LW02060, and Lu-LW02080 were measured by in vitro competition binding assays using PC-3 cells and were found to be 5.57 ± 2.47, 21.7 ± 6.69, 8.00 ± 2.61, and 32.1 ± 8.14 nM, respectively. The ⁶⁸Ga- and ¹⁷⁷Lu-labeled ligands were obtained in 36–75% decay-corrected radiochemical yields with >95% radiochemical purity. PET imaging, SPECT imaging, and ex vivo biodistribution studies were conducted in PC-3 tumor-bearing mice. Both [⁶⁸Ga]Ga-LW02060 and [⁶⁸Ga]Ga-LW02080 enabled clear tumor visualization in PET images at 1 h post-injection (pi). Tumor uptake values of [⁶⁸Ga]Ga-LW02060 and [⁶⁸Ga]Ga-LW02080 at 1 h pi were 16.8 ± 2.70 and 7.36 ± 1.33 %ID/g, respectively, while their pancreas uptake values were 3.12 ± 0.89 and 0.38 ± 0.04 %ID/g, respectively. Compared to [¹⁷⁷Lu]Lu-LW02080, [¹⁷⁷Lu]Lu-LW02060 showed higher tumor uptake at all time points (1, 4, 24, 72, and 120 h pi). However, fast tumor clearance was observed for both [¹⁷⁷Lu]Lu-LW02060 and [¹⁷⁷Lu]Lu-LW02080. Conclusions: Our data demonstrate that [⁶⁸Ga]Ga-LW02060 is promising for clinical translation for the detection of GRPR-expressing tumor lesions. However, further optimizations are needed for [¹⁷⁷Lu]Lu-LW02060 and [¹⁷⁷Lu]Lu-LW02080 to prolong tumor retention for therapeutic applications. Full article

We developed ligandomics for the in vivo profiling of vascular ligands in mice, discovering secretogranin III (Scg3) as a novel angiogenic factor that selectively binds to retinal vessels of diabetic but not healthy mice. This discovery led to the development of anti-Scg3 therapy for ocular vasculopathies. However, in vivo ligandomics requires intracardial perfusion to remove unbound phage clones, limiting its use to vascular endothelial cells (ECs). To extend ligandomics to non-vascular cells, we investigated ex vivo ligandomics. We isolated ECs and retinal ganglion cells (RGCs) from diabetic and healthy mouse retinas by immunopanning. We quantified the binding of clonal phages displaying Scg3 and vascular endothelial growth factor (VEGF), confirming that their binding patterns to isolated diabetic versus healthy ECs matched in vivo patterns. Additionally, Scg3 and VEGF binding to isolated RGCs reflected their in vivo activity. These results support the feasibility of ex vivo ligandomics. We further mapped ligands binding to immunopanned diabetic and healthy ECs and RGCs by ligandomics, confirming that Scg3 was enriched with selective binding to diabetic ECs but not healthy ECs or diabetic/healthy RGCs. These findings demonstrate the feasibility of ex vivo ligandomics, which can be broadly applied to various cell types, tissues, diseases, and species. Full article

Omnipresent suppressive myeloid populations in the tumor microenvironment limit the efficacy of T-cell-directed immunotherapies, become more inhibitory after administration of T-cell checkpoint inhibitors, and are overall associated with worse survival of cancer patients. In early clinical trials, positive outcomes have been demonstrated for therapies aimed at repolarizing suppressive myeloid populations in the tumor microenvironment. We have previously described the key role of P-selectin glycoprotein ligand-1 (PSGL-1) in maintaining an inhibitory state of tumor-associated macrophages (TAMs), most of which express high levels of PSGL-1. Here we describe a novel, first-in-class humanized high-affinity monoclonal antibody VTX-0811 that repolarizes human macrophages from an M2-suppressive phenotype towards an M1 inflammatory phenotype, similar to siRNA-mediated knockdown of PSGL-1. VTX-0811 binds to PSGL-1 of human and cynomolgus macaque origins without inhibiting PSGL-1 interaction with P- and L-Selectins or VISTA. In multi-cellular assays and in patient-derived human tumor cultures, VTX-0811 leads to the induction of pro-inflammatory mediators. RNAseq data from VTX-0811 treated ex vivo tumor cultures and M2c macrophages show similar pathways being modulated, indicating that the mechanism of action translates from isolated macrophages to tumors. A chimeric version of VTX-0811, consisting of the parental murine antibody in a human IgG4 backbone, inhibits tumor growth in a humanized mouse model of cancer. VTX-0811 is exceptionally well tolerated in NHP toxicology assessment and is heading into clinical evaluation after successful IND clearance. Full article

The α-synucleinopathies are a group of neurodegenerative diseases characterized by the deposition of α-synuclein aggregates (α-syn) in the brain. Currently, there is no suitable tracer to enable a definitive early diagnosis of these diseases. We reported candidates based on 4,4′-disarylbisthiazole (DABTA) scaffold with a high affinity towards α-syn and excellent selectivity over Aβ and tau fibrils. Based on prior in silico studies, a focused library of 23 halogen-containing and O-methylated DABTAs was prepared. The DABTAs were synthesized via a modified two-step Hantzsch thiazole synthesis, characterized, and used in competitive binding assays against [³H]PiB and [³H]DCVJ. The DABTAs were obtained with an overall chemical yield of 15–71%, and showed a calculated lipophilicity of 2.5–5.7. The ligands demonstrated an excellent affinity to α-syn with both [³H]PiB and [³H]DCVJ: K_i 0.1–4.9 nM and up to 20–3900-fold selectivity over Aβ and tau fibrils. It could be concluded that in silico simulation is useful for the rational design of a new generation of DABTAs. Further investigation of the leads in the next step is encouraged: radiolabeling of the ligands with radioisotopes such as fluorine-18 or carbon-11 for in vivo, ex vivo, and translational research and for further in vitro experiments on human-derived protein aggregates. Full article

Claudins regulate paracellular permeability, contribute to epithelial polarization and are dysregulated during inflammation and carcinogenesis. Variants of the claudin-binding domain of Clostridium perfringens enterotoxin (cCPE) are highly sensitive protein ligands for generic detection of a broad spectrum of claudins. Here, we investigated the preferential binding of YFP- or GST-cCPE fusion proteins to non-junctional claudin molecules. Plate reader assays, flow cytometry and microscopy were used to assess the binding of YFP- or GST-cCPE to non-junctional claudins in multiple in vitro and ex vivo models of human and rat gastrointestinal epithelia and to monitor formation of a tight junction barrier. Furthermore, YFP-cCPE was used to probe expression, polar localization and dysregulation of claudins in patient-derived organoids generated from gastric dysplasia and gastric cancer. Live-cell imaging and immunocytochemistry revealed cell polarity and presence of tight junctions in glandular organoids (originating from intestinal-type gastric cancer and gastric dysplasia) and, in contrast, a disrupted diffusion barrier for granular organoids (originating from discohesive tumor areas). In sum, we report the use of cCPE fusion proteins as molecular probes to specifically and efficiently detect claudin expression, localization and tight junction dysregulation in cell lines, tissue explants and patient-derived organoids of the gastrointestinal tract. Full article

Prostate-specific membrane antigen (PSMA) is a well-validated prostate cancer marker but reported PSMA-targeted tracers derived from the Lys-urea-Glu pharmacophore including the clinically validated [^99mTc]Tc-EDDA/HYNIC-iPSMA have high off-target uptake in kidneys, spleen, and salivary glands. In this study, we synthesized and evaluated three novel ^99mTc-labeled PSMA-targeted tracers and investigated if the tracers derived from the Lys-urea-Aad pharmacophore could have minimized uptake in off-target organs/tissues. In vitro competition binding assays showed that compared with HYNIC-iPSMA, the three novel ligands had slightly weaker PSMA binding affinity (average K_i = 3.11 vs. 8.96–11.6 nM). Imaging and ex vivo biodistribution studies in LNCaP tumor-bearing mice showed that [^99mTc]Tc-EDDA/HYNIC-iPSMA and the three novel tracers successfully visualized LNCaP tumor xenografts in SPECT images and were excreted mainly via the renal pathway. The average tumor uptake at 1 h post-injection varied from 5.40 to 18.8%ID/g, and the tracers derived from the Lys-urea-Aad pharmacophore had much lower uptake in the spleen and salivary glands. Compared with the clinical tracer [^99mTc]Tc-EDDA/HYNIC-iPSMA, the Lys-urea-Aad-derived [^99mTc]Tc-EDDA-KL01127 had lower background uptake and superior tumor-to-background contrast ratios and is therefore promising for clinical translation to detect prostate cancer lesions with SPECT. Full article

The main aim of this study is to synthesize contrast microbubbles (MB) functionalized with engineered protein ligands using a microfluidic device to target breast cancer specific vascular B7-H3 receptor in vivo for diagnostic ultrasound imaging. We used a high-affinity affibody (ABY) selected against human/mouse B7-H3 receptor for engineering targeted MBs (TMBs). We introduced a C-terminal cysteine residue to this ABY ligand for facilitating site-specific conjugation to DSPE-PEG-2K-maleimide (M. Wt = 2.9416 kDa) phospholipid for MB formulation. We optimized the reaction conditions of bioconjugations and applied it for microfluidic based synthesis of TMBs using DSPE-PEG-ABY and DPPC liposomes (5:95 mole %). The binding affinity of TMBs to B7-H3 (MB_B7-H3) was tested in vitro in MS1 endothelial cells expressing human B7-H3 (MS1_B7-H3) by flow chamber assay, and by ex vivo in the mammary tumors of a transgenic mouse model (FVB/N-Tg (MMTV-PyMT)634Mul/J), expressing murine B7-H3 in the vascular endothelial cells by immunostaining analyses. We successfully optimized the conditions needed for generating TMBs using a microfluidic system. The synthesized MBs showed higher affinity to MS1 cells engineered to express higher level of hB7-H3, and in the endothelial cells of mouse tumor tissue upon injecting TMBs in a live animal. The average number (mean ± SD) of MB_B7-H3 binding to MS1_B7-H3 cells was estimated to be 354.4 ± 52.3 per field of view (FOV) compared to wild-type control cells (MS1_WT; 36.2 ± 7.5/FOV). The non-targeted MBs did not show any selective binding affinity to both the cells (37.7 ± 7.8/FOV for MS1_B7-H3 and 28.3 ± 6.7/FOV for MS1_WT cells). The fluorescently labeled MB_B7-H3 upon systemic injection in vivo co-localized to tumor vessels, expressing B7-H3 receptor, as validated by ex vivo immunofluorescence analyses. We have successfully synthesized a novel MB_B7-H3 via microfluidic device, which allows us to produce on demand TMBs for clinical applications. This clinically translatable MB_B7-H3 showed significant binding affinity to vascular endothelial cells expressing B7-H3 both in vitro and in vivo, which shows its potential for clinical translation as a molecular ultrasound contrast agent for human applications. Full article

Studies of the neurobiological causes of anxiety disorders have suggested that the γ-aminobutyric acid (GABA) system increases synaptic concentrations and enhances the affinity of GABA_A (type A) receptors for benzodiazepine ligands. Flumazenil antagonizes the benzodiazepine-binding site of the GABA/benzodiazepine receptor (BZR) complex in the central nervous system (CNS). The investigation of flumazenil metabolites using liquid chromatography (LC)-tandem mass spectrometry will provide a complete understanding of the in vivo metabolism of flumazenil and accelerate radiopharmaceutical inspection and registration. The main goal of this study was to investigate the use of reversed-phase high performance liquid chromatography (PR-HPLC), coupled with electrospray ionization triple-quadrupole tandem mass spectrometry (ESI-QqQ MS), to identify flumazenil and its metabolites in the hepatic matrix. Carrier-free nucleophilic fluorination with an automatic synthesizer for [¹⁸F]flumazenil, combined with nano-positron emission tomography (NanoPET)/computed tomography (CT) imaging, was used to predict the biodistribution in normal rats. The study showed that 50% of the flumazenil was biotransformed by the rat liver homogenate in 60 min, whereas one metabolite (M1) was a methyl transesterification product of flumazenil. In the rat liver microsomal system, two metabolites were identified (M2 and M3), as their carboxylic acid and hydroxylated ethyl ester forms between 10 and 120 min, respectively. A total of 10–30 min post-injection of [¹⁸F]flumazenil showed an immediate decreased in the distribution ratio observed in the plasma. Nevertheless, a higher ratio of the complete [¹⁸F]flumazenil compound could be used for subsequent animal studies. [¹⁸F] According to in vivo nanoPET/CT imaging and ex vivo biodistribution assays, flumazenil also showed significant effects on GABA_A receptor availability in the amygdala, prefrontal cortex, cortex, and hippocampus in the rat brain, indicating the formation of metabolites. We reported the completion of the biotransformation of flumazenil by the hepatic system, as well as [¹⁸F]flumazenil’s potential as an ideal ligand and PET agent for the determination of the GABA_A/BZR complex for multiplex neurological syndromes at the clinical stage. Full article

New biphenyl-based chimeric compounds containing pomalidomide were developed and evaluated for their activity to inhibit and degrade the programmed cell death-1/programmed cell death- ligand 1 (PD-1/PD-L1) complex. Most of the compounds displayed excellent inhibitory activity against PD-1/PD-L1, as assessed by the homogenous time-resolved fluorescence (HTRF) binding assay. Among them, compound 3 is one of the best with an IC₅₀ value of 60 nM. Using an ex vivo PD-1/PD-L1 blockade cell line bioassay that expresses human PD-1 and PD-L1, we show that compounds 4 and 5 significantly restore the repressed immunity in this co-culture model. Western blot data, however, demonstrated that these anti-PD-L1/pomalidomide chimeras could not reduce the protein levels of PD-L1. Full article

Nuclear and optical dual-modality probes can be of great assistance in prostate cancer localization, providing the means for both preoperative nuclear imaging and intraoperative surgical guidance. We developed a series of probes based on the backbone of the established GRPR-targeting radiotracer NeoB. The inverse electron demand of the Diels–Alder reaction was used to integrate the sulfo-cyanine 5 dye. Indium-111 radiolabeling, stability studies and a competition binding assay were carried out. Pilot biodistribution and imaging studies were performed in PC-3 tumor-bearing mice, using the best two dual-labeled probes. The dual-modality probes were radiolabeled with a high yield (>92%), were proven to be hydrophilic and demonstrated high stability in mouse serum (>94% intact labeled ligand at 4 h). The binding affinity for the GRPR was in the nanomolar range (21.9–118.7 nM). SPECT/CT images at 2 h p.i. clearly visualized the tumor xenograft and biodistribution studies, after scanning confirmed the high tumor uptake (8.47 ± 0.46%ID/g and 6.90 ± 0.81%ID/g for probe [¹¹¹In]In-12 and [¹¹¹In]In-15, respectively). Receptor specificity was illustrated with blocking studies, and co-localization of the radioactive and fluorescent signal was verified by ex vivo fluorescent imaging. Although optimal tumor-to-blood and tumor-to-kidney ratios might not yet have been reached due to the prolonged blood circulation, our probes are promising candidates for the preoperative and intraoperative visualization of GRPR-positive prostate cancer. Full article

Thyroid stimulating antibodies (TSAB) cause Graves’ disease and contribute to Graves’ Orbitopathy (GO) pathogenesis. We hypothesise that the presence of TSH binding proteins (truncated TSHR variants (TSHRv)) and/or nonclassical ligands such as thyrostimulin (α2β5) might provide a mechanism to protect against or exacerbate GO. We analysed primary human orbital preadipocyte-fibroblasts (OF) from GO patients and people free of GO (non-GO). Transcript (QPCR) and protein (western blot) expression levels of TSHRv were measured through an adipogenesis differentiation process. Cyclic-AMP production by TSHR activation was studied using luciferase-reporter and RIA assays. After differentiation, TSHRv levels in OF from GO were significantly higher than non-GO (p = 0.039), and confirmed in ex vivo analysis of orbital adipose samples. TSHRv western blot revealed a positive signal at 46 kDa in cell lysates and culture media (CM) from non-GO and GO-OF. Cyclic-AMP decreased from basal levels when OF were stimulated with TSH or Monoclonal TSAB (M22) before differentiation protocol, but increased in differentiated cells, and was inversely correlated with the TSHRv:TSHR ratio (Spearman correlation: TSH r = −0.55, p = 0.23, M22 r = 0.87, p = 0.03). In the bioassay, TSH/M22 induced luciferase-light was lower in CM from differentiated GO-OF than non-GO, suggesting that secreted TSHRv had neutralised their effects. α2 transcripts were present but reduced during adipogenesis (p < 0.005) with no difference observed between non-GO and GO. β5 transcripts were at the limit of detection. Our work demonstrated that TSHRv transcripts are expressed as protein, are more abundant in GO than non-GO OF and have the capacity to regulate signalling via the TSHR. Full article

(1) Background: Prostate-specific membrane antigen (PSMA) has been extensively studied in the last decade. It became a promising biological target in the diagnosis and therapy of PSMA-expressing cancer diseases. Although there are several radiolabeled PSMA inhibitors available, the search for new compounds with improved pharmacokinetic properties and simplified synthesis is still ongoing. In this study, we developed PSMA ligands with two different hybrid chelators and a modified linker. Both compounds have displayed a promising pharmacokinetic profile. (2) Methods: DATA^5m.SA.KuE and AAZTA⁵.SA.KuE were synthesized. DATA^5m.SA.KuE was labeled with gallium-68 and radiochemical yields of various amounts of precursor at different temperatures were determined. Complex stability in phosphate-buffered saline (PBS) and human serum (HS) was examined at 37 °C. Binding affinity and internalization ratio were determined in in vitro assays using PSMA-positive LNCaP cells. Tumor accumulation and biodistribution were evaluated in vivo and ex vivo using an LNCaP Balb/c nude mouse model. All experiments were conducted with PSMA-11 as reference. (3) Results: DATA^5m.SA.KuE was synthesized successfully. AAZTA⁵.SA.KuE was synthesized and labeled according to the literature. Radiolabeling of DATA^5m.SA.KuE with gallium-68 was performed in ammonium acetate buffer (1 M, pH 5.5). High radiochemical yields (>98%) were obtained with 5 nmol at 70 °C, 15 nmol at 50 °C, and 60 nmol (50 µg) at room temperature. [⁶⁸Ga]Ga-DATA^5m.SA.KuE was stable in human serum as well as in PBS after 120 min. PSMA binding affinities of AAZTA⁵.SA.KuE and DATA^5m.SA.KuE were in the nanomolar range. PSMA-specific internalization ratio was comparable to PSMA-11. In vivo and ex vivo studies of [¹⁷⁷Lu]Lu-AAZTA⁵.SA.KuE, [⁴⁴Sc]Sc-AAZTA⁵.SA.KuE and [⁶⁸Ga]Ga-DATA^5m.SA.KuE displayed specific accumulation in the tumor along with fast clearance and reduced off-target uptake. (4) Conclusions: Both KuE-conjugates showed promising properties especially in vivo allowing for translational theranostic use. Full article

There is a need for versatile in vivo nuclear imaging reporter systems to foster preclinical and clinical research. We explore the applicability of the SNAPTag and novel radiolabeled small-molecule ligands as a versatile reporter gene system for in vivo nuclear imaging. SNAPTag is a high-affinity protein tag used in a variety of biochemical research areas and based on the suicide DNA repair enzyme O⁶-methylguanine methyl transferase (MGMT). Its ligands are well suited for reporter gene imaging as the benzyl guanine core scaffold can be derivatized with fluorescent or radiolabeled moieties for various applications. Three guanine-based SNAPTag ligands ([¹⁸F]FBBG, [¹⁸F]pFBG and [¹⁸F]mFBG) were synthesized in high yields and were (radio)chemically characterized. HEK293 cells were engineered to express the SNAPTag on the cell surface and served as cell model to assess target affinity by radiotracer uptake assays, Western blotting and SDS-PAGE autoradiography. A subcutaneous HEK293-SNAPTag xenograft model in immunodeficient mice was used for in vivo evaluation of [¹⁸F]FBBG and [¹⁸F]pFBG while the biodistribution of [¹⁸F]mFBG was characterized in naïve animals. The results were validated by ex vivo biodistribution studies and immunofluorescence staining of the xenografts. All three radiotracers were produced in high radiochemical purity, molar activity and good yields. Western blot analysis revealed successful SNAPTag expression by the transfected HEK293 cells. In vitro testing revealed high target affinity of all three tracers with an up to 191-fold higher signal in the HEK293-SNAPTag cells compared to untransfected cells. This was further supported by a prominent radioactive protein band at the expected size in the SDS-PAGE autoradiograph of cells incubated with [¹⁸F]FBBG or [¹⁸F]pFBG. The in vivo studies demonstrated high uptake in HEK293-SNAP xenografts compared to HEK293 xenografts with excellent tumor-to-muscle ratios (7.5 ± 4.2 for [¹⁸F]FBBG and 10.6 ± 6.2 for [¹⁸F]pFBG). In contrast to [¹⁸F]pFBG and its chemical analogue [¹⁸F]mFBG, [¹⁸F]FBBG showed no signs of unspecific bone uptake and defluorination in vivo. Radiolabeled SNAPTag ligands bear great potential for clinical applications such as in vivo tracking of cell populations, antibody fragments and targeted radiotherapy. With excellent target affinity, good stability, and low non-specific binding, [¹⁸F]FBBG is a highly promising candidate for further preclinical evaluation. Full article