The development of highly selective and fast biocompatible reactions for ligation and cleavage has paved the way for new diagnostic and therapeutic applications of pretargeted in vivo chemistry. The concept of bioorthogonal pretargeting has attracted considerable interest, in particular for the targeted delivery of radionuclides and drugs. In nuclear medicine, pretargeting can provide increased target-to-background ratios at early time-points compared to traditional approaches. This reduces the radiation burden to healthy tissue and, depending on the selected radionuclide, enables better imaging contrast or higher therapeutic efficiency. Moreover, bioorthogonally triggered cleavage of pretargeted antibody–drug conjugates represents an emerging strategy to achieve controlled release and locally increased drug concentrations. The toolbox of bioorthogonal reactions has significantly expanded in the past decade, with the tetrazine ligation being the fastest and one of the most versatile in vivo chemistries. Progress in the field, however, relies heavily on the development and evaluation of (radio)labeled compounds, preventing the use of compound libraries for systematic studies. The rational design of tetrazine probes and triggers has thus been impeded by the limited understanding of the impact of structural parameters on the in vivo ligation performance. In this work, we describe the development of a pretargeted blocking assay that allows for the investigation of the in vivo fate of a structurally diverse library of 45 unlabeled tetrazines and their capability to reach and react with pretargeted trans-cyclooctene (TCO)-modified antibodies in tumor-bearing mice. This study enabled us to assess the correlation of click reactivity and lipophilicity of tetrazines with their in vivo performance. In particular, high rate constants (>50 000 M^–1 s^–1) for the reaction with TCO and low calculated logD_7.4 values (below −3) of the tetrazine were identified as strong indicators for successful pretargeting. Radiolabeling gave access to a set of selected ¹⁸F-labeled tetrazines, including highly reactive scaffolds, which were used in pretargeted PET imaging studies to confirm the results from the blocking study. These insights thus enable the rational design of tetrazine probes for in vivo application and will thereby assist the clinical translation of bioorthogonal pretargeting.

The serotonin 2A receptor (5-HT_2AR) is the mediator of the psychedelic effects of serotonergic psychedelics, which have shown promising results in clinical studies for several neuropsychiatric indications. The 5-HT_2AR is able to signal through the Gα_q and β-arrestin effector proteins, but it is currently not known how the different signaling pathways contribute to the therapeutic effects mediated by serotonergic psychedelics. In the present work, we have evaluated the subtype-selective 5-HT_2AR agonist 25CN-NBOH and a series of close analogues for biased signaling at this receptor. These ligands were designed to evaluate the role of interactions with Ser159^3×36. The lack of interaction between this hydroxyl moiety and Ser159^3×36 resulted in detrimental effects on potency and efficacy in both βarr2 and miniGα_q recruitment assays. Remarkably, Gα_q-mediated signaling was considerably more affected. This led to the development of the first efficacious βarr2-biased 5-HT_2AR agonists 4a-b and 6e-f, βarr2 preferring, relative to lysergic acid diethylamide (LSD).

Serotonergic psychedelics are described to have activation of the serotonin 2A receptor (5-HT_2A) as their main pharmacological action. Despite their relevance, the molecular mechanisms underlying the psychedelic effects induced by certain 5-HT_2A agonists remain elusive. One of the proposed hypotheses is the occurrence of biased agonism, defined as the preferential activation of certain signaling pathways over others. This study comparatively monitored the efficiency of a diverse panel of 4-position-substituted (and N-benzyl-derived) phenylalkylamines to induce recruitment of β-arrestin2 (βarr2) or miniGα_q to the 5-HT_2A, allowing us to assess structure-activity relationships and biased agonism. All test compounds exhibited agonist properties with a relatively large range of both EC₅₀ and E_max values. Interestingly, the lipophilicity of the 2C-X phenethylamines was correlated with their efficacy in both assays but yielded a stronger correlation in the miniGα_q- than in the βarr2-assay. Molecular docking suggested that accommodation of the 4-substituent of the 2C-X analogues in a hydrophobic pocket between transmembrane helices 4 and 5 of 5-HT_2A may contribute to this differential effect. Aside from previously used standard conditions (lysergic acid diethylamide (LSD) as a reference agonist and a 2 h activation profile to assess a compound's activity), serotonin was included as a second reference agonist, and the compounds' activities were also assessed using the first 30 min of the activation profile. Under all assessed circumstances, the qualitative structure-activity relationships remained unchanged. Furthermore, the use of two reference agonists allowed for the estimation of both "benchmark bias" (relative to LSD) and "physiology bias" (relative to serotonin).

Pretargeted nuclear imaging for the diagnosis of various cancers is an emerging and fast developing field. The tetrazine ligation is currently considered the most promising reaction in this respect. Monoclonal antibodies are often the preferred choice as pretargeting vector due to their outstanding targeting properties. In this work, we evaluated the performance of [⁶⁴Cu]Cu-NOTA-PEG₇-H-Tz using a setup we previously used for [¹¹¹In]In-DOTA-PEG₁₁-BisPy-Tz, thereby allowing for comparison of the performance of these two promising pretargeting imaging agents. The evaluation included a comparison of the physicochemical properties of the compounds and their performance in an ex vivo blocking assay. Finally, [⁶⁴Cu]Cu-NOTA-PEG₇-H-Tz was evaluated in a pretargeted imaging study and compared to [¹¹¹In]In-DOTA-PEG₁₁-BisPy-Tz. Despite minor differences, this study indicated that both evaluated tetrazines are equally suited for pretargeted imaging.

Targeted α-therapy (TAT) can eradicate tumor metastases while limiting overall toxicity. One of the most promising α-particle emitters is astatine-211 (²¹¹At). However, ²¹¹At-carbon bonds are notoriously unstable in vivo and no chelators are available. This hampers its adoption in TAT. In this study, the stability of ²¹¹At on the surface of gold nanoparticles (AuNPs) was investigated. The employed AuNPs had sizes in the 25–50 nm range. Radiolabeling by non-specific surface-adsorption in >99% radiochemical yield was achieved by mixing ²¹¹At and AuNPs both before and after polyethylene glycol (PEG) coating. The resulting ²¹¹At-AuNPs were first challenged by harsh oxidation with sodium hypochlorite, removing roughly 50% of the attached ²¹¹At. Second, incubation in mouse serum followed by a customized stability test, showed a stability of >95% after 4 h in serum. This high stability was further confirmed in an in vivo study, with comparison to a control group of free ²¹¹At. The AuNP-associated ²¹¹At showed low uptake in stomach and thyroid, which are hallmark organs of uptake of free ²¹¹At, combined with long circulation and high liver and spleen uptake, consistent with nanoparticle biodistribution. These results support that gold surface-adsorbed ²¹¹At has high biological stability and is a potentially useful delivery system in TAT.