Geranylgeranyltransferase type-I (GGTase-I) represents an important drug target since it contributes to the function of many proteins that are involved in tumor development and metastasis. This led to the development of GGTase-I inhibitors as anti-cancer drugs blocking the protein function and membrane association of e.g., Rap subfamilies that are involved in cell differentiation and cell growth. In the present study, we developed a new NanoBiT assay to monitor the interaction of human GGTase-I and its substrate Rap1B. Different Rap1B prenylation-deficient mutants (C181G, C181S, and ΔCQLL) were designed and investigated for their interaction with GGTase-I. While the Rap1B mutants C181G and C181S still exhibited interaction with human GGTase-I, mutant ΔCQLL, lacking the entire CAAX motif (defined by a cysteine residue, two aliphatic residues, and the C-terminal residue), showed reduced interaction. Moreover, a specific, peptidomimetic and competitive CAAX inhibitor was able to block the interaction of Rap1B with GGTase-I. Furthermore, activation of both Gα_s-coupled human adenosine receptors, A_2A (A_2AAR) and A_2B (A_2BAR), increased the interaction between GGTase-I and Rap1B, probably representing a way to modulate prenylation and function of Rap1B. Thus, A_2AAR and A_2BAR antagonists might be promising candidates for therapeutic intervention for different types of cancer that overexpress Rap1B. Finally, the NanoBiT assay provides a tool to investigate the pharmacology of GGTase-I inhibitors.

Connexin gap junctions (Cx GJs) enable the passage of small molecules and ions between cells and are therefore important for cell-to-cell communication. Their dysfunction is associated with diseases, and small molecules acting as modulators of GJs may therefore be useful as therapeutic drugs. To identify GJ modulators, suitable assays are needed that allow compound screening. In the present study, we established a novel assay utilizing HeLa cells recombinantly expressing Cx43. Donor cells additionally expressing the Gs protein-coupled adenosine A_2A receptor, and biosensor cells expressing a cAMP-sensitive GloSensor luciferase were established. Adenosine A_2A receptor activation in the donor cells using a selective agonist results in intracellular cAMP production. The negatively charged cAMP migrates via the Cx43 gap junctions to the biosensor cells and can there be measured by the cAMP-dependent luminescence signal. Cx43 GJ modulators can be expected to impact the transfer of cAMP from the donor to the biosensor cells, since cAMP transit is only possible via GJs. The new assay was validated by testing the standard GJ inhibitor carbenoxolon, which showed a concentration-dependent inhibition of the signal and an IC₅₀ value that was consistent with previously reported values. The assay was demonstrated to be suitable for high-throughput screening.

The adenosine A_2B receptor has been proposed as a novel therapeutic target in cancer, as its expression is drastically elevated in several tumors and cancer cells. Noninvasive molecular imaging via positron emission tomography (PET) would allow the in vivo quantification of this receptor in pathological processes and most likely enable the identification and clinical monitoring of respective cancer therapies. On the basis of a bicyclic pyridopyrimidine-2,4-dione core structure, the new adenosine A_2B receptor ligand 9 was synthesized, containing a 2-fluoropyridine moiety suitable for labeling with the short-lived PET radionuclide fluorine-18. Compound 9 showed a high binding affinity for the human A_2B receptor (K_i(A_2B) = 2.51 nM), along with high selectivities versus the A₁, A_2A, and A₃ receptor subtypes. Therefore, it was radiofluorinated via nucleophilic aromatic substitution of the corresponding nitro precursor using [¹⁸F]F^-/K_2.2.2./K₂CO₃ in DMSO at 120 °C. Metabolic studies of [¹⁸F]9 in mice revealed about 60% of radiotracer intact in plasma at 30 minutes p.i. A preliminary PET study in healthy mice showed an overall biodistribution of [¹⁸F]9, corresponding to the known ubiquitous but low expression of the A_2B receptor. Consequently, [¹⁸F]9 represents a novel PET radiotracer with high affinity and selectivity toward the adenosine A_2B receptor and a suitable in vivo profile. Subsequent studies are envisaged to investigate the applicability of [¹⁸F]9 to detect alterations in the receptor density in certain cancer-related disease models.

The G protein-coupled adenosine A_2B receptor is suggested to be involved in various pathological processes accompanied by increased levels of adenosine as found in inflammation, hypoxia, and cancer. Therefore, the adenosine A_2B receptor is currently in focus as a novel target for cancer therapy as well as for noninvasive molecular imaging via positron emission tomography (PET). Aiming at the development of a radiotracer labeled with the PET radionuclide fluorine-18 for imaging the adenosine A_2B receptor in brain tumors, one of the most potent and selective antagonists, the xanthine derivative PSB-603, was selected as a lead compound. As initial biodistribution studies in mice revealed a negligible brain uptake of [³H]PSB-603 (SUV_3min: 0.2), structural modifications were performed to optimize the physicochemical properties regarding blood–brain barrier penetration. Two novel fluorinated derivatives bearing a 2-fluoropyridine (5) moiety and a 4-fluoro-piperidine (6) moiety were synthesized, and their affinity towards the four adenosine receptor subtypes was determined in competition binding assays. Both compounds showed high affinity towards the adenosine A_2B receptor (K_i (5) = 9.97 ± 0.86 nM; K_i (6) = 12.3 ± 3.6 nM) with moderate selectivity versus the other adenosine receptor subtypes.