Exploring Alternative Pathways to Target Bacterial Type II Topoisomerases Using NBTI Antibacterials: Beyond Halogen-Bonding Interactions.

Novel bacterial topoisomerase inhibitors (NBTIs) are a new class of antibacterial agents that target bacterial type II topoisomerases (DNA gyrase and topoisomerase IV). Our recently disclosed crystal structure of an NBTI ligand in complex with DNA gyrase and DNA revealed that the halogen atom in the para position of the phenyl right hand side (RHS) moiety is able to establish strong symmetrical bifurcated halogen bonds with the enzyme; these are responsible for the excellent enzyme inhibitory potency and antibacterial activity of these NBTIs. To further assess the possibility of any alternative interactions (e.g., hydrogen-bonding and/or hydrophobic interactions), we introduced various non-halogen groups at the p-position of the phenyl RHS moiety. Considering the hydrophobic nature of amino acid residues delineating the NBTI's binding pocket in bacterial topoisomerases, we demonstrated that designed NBTIs cannot establish any hydrogen-bonding interactions with the enzyme; hydrophobic interactions are feasible in all respects, while halogen-bonding interactions are apparently the most preferred.

Development of the 99mTc-Labelled SST2 Antagonist TECANT-1 for a First-in-Man Multicentre Clinical Study.

Broad availability and cost-effectiveness of 99Mo/99mTc generators worldwide support the use, and thus the development, of novel 99mTc-labelled radiopharmaceuticals. In recent years, preclinical and clinical developments for neuroendocrine neoplasms patient management focused on somatostatin receptor subtype 2 (SST2) antagonists, mainly due to their superiority in SST2-tumour targeting and improved diagnostic sensitivity over agonists. The goal of this work was to provide a reliable method for facile preparation of a 99mTc-labelled SST2 antagonist, [99mTc]Tc-TECANT-1, in a hospital radiopharmacy setting, suitable for a multi-centre clinical trial. To ensure successful and reproducible on-site preparation of the radiopharmaceutical for human use shortly before administration, a freeze-dried three-vial kit was developed. The final composition of the kit was established based on the radiolabelling results obtained during the optimisation process, in which variables such as precursor content, pH and buffer, as well as kit formulations, were tested. Finally, the prepared GMP-grade batches met all predefined specification parameters together with long-term kit stability and stability of the product [99mTc]Tc-TECANT-1. Furthermore, the selected precursor content complies with micro-dosing, based on an extended single-dose toxicity study, where histopathology NOEL was established at 0.5 mg/kg BW, being more than 1000 times higher than the planned human dose of 20 µg. In conclusion, [99mTc]Tc-TECANT-1 is suitable to be advanced into a first-in-human clinical trial.

Radiolabelled CCK2 R Antagonists Containing PEG Linkers: Design, Synthesis and Evaluation.

The cholecystokinin-2/gastrin receptor (CCK2 R) is considered a suitable target for the development of radiolabelled antagonists, due to its overexpression in various tumours, but no such compounds are available in clinical use. Therefore, we designed novel 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-conjugated ligands based on CCK2 R antagonist Z360/nastorazepide. As a proof of concept that CCK2 R antagonistic activity can be retained by extending the Z360/nastorazepide structure using suitable linker, we present herein three compounds containing various PEG linkers synthesised on solid phase and in solution. The antagonistic properties were measured in a functional assay in the A431-CCK2 R cell line (in the presence of agonist G17), with IC50 values of 3.31, 4.11 and 10.4 nM for compounds containing PEG4 , PEG6 and PEG12 , respectively. All compounds were successfully radiolabelled with indium-111, lutetium-177 and gallium-68 (incorporation of radiometal >95 %). The gallium-68-labelled compounds were stable for up to 2 h (PBS, 37 °C). log D7.4 values were determined for indium-111- and gallium-68-labelled compounds, showing improved hydrophilicity compared to the reference compound.

CCK2R antagonists: from SAR to clinical trials.

The widespread involvement of the cholecystokinin-2/gastrin receptor (CCK2R) in multiple (patho)physiological processes has propelled extensive searches for nonpeptide small-molecule CCK2R antagonists. For the past three decades, considerable research has yielded numerous chemically heterogeneous compounds. None of these entered into the clinic, mainly because of inadequate biological effects. However, it appears that the ultimate goal of a clinically useful CCK2R antagonist is now just around the corner, with the most promising compounds, netazepide and nastorazepide, now in Phase II clinical trials. Here, we illustrate the structure-activity relationships (SARs) of stablished CCK2R antagonists of various structural classes, and the most recent proof-of-concept studies where new applicabilities of CCK2R antagonists as visualizing agents are presented.

Selection of the First 99mTc-Labelled Somatostatin Receptor Subtype 2 Antagonist for Clinical Translation-Preclinical Assessment of Two Optimized Candidates.

Recently, radiolabelled antagonists targeting somatostatin receptors subtype 2 (SST2) in neuroendocrine neoplasms demonstrated certain superior properties over agonists. Within the ERA-PerMED project "TECANT" two 99mTc-Tetramine (N4)-derivatized SST2 antagonists (TECANT-1 and TECANT-2) were studied for the selection of the best candidate for clinical translation. Receptor-affinity, internalization and dissociation studies were performed in human embryonic kidney-293 (HEK293) cells transfected with the human SST2 (HEK-SST2). Log D, protein binding and stability in human serum were assessed. Biodistribution and SPECT/CT studies were carried out in nude mice bearing HEK-SST2 xenografts, together with dosimetric estimations from mouse-to-man. [99mTc]Tc-TECANT-1 showed higher hydrophilicity and lower protein binding than [99mTc]-TECANT-2, while stability was comparable. Both radiotracers revealed similar binding affinity, while [99mTc]Tc-TECANT-1 had higher cellular uptake (>50%, at 2 h/37 °C) and lower dissociation rate (<30%, at 2 h/37 °C). In vivo, [99mTc]Tc-TECANT-1 showed lower blood values, kidney and muscles uptake, whereas tumour uptake was comparable to [99mTc]Tc-TECANT-2. SPECT/CT imaging confirmed the biodistribution results, providing the best tumour-to-background image contrast for [99mTc]Tc-TECANT-1 at 4 h post-injection (p.i.). The estimated radiation dose amounted to approximately 6 µSv/MBq for both radiotracers. This preclinical study provided the basis of selection of [99mTc]Tc-TECANT-1 for clinical translation of the first 99mTc-based SST2 antagonist.

Cyclohexyl amide-based novel bacterial topoisomerase inhibitors with prospective GyrA-binding fragments.

Aim: Novel bacterial topoisomerase inhibitors (NBTIs) are a promising class of bacterial topoisomerase II inhibitors that are gaining more and more importance mainly because of their excellent antibacterial activity, as well as their lack of cross-resistance to quinolones. Results: Described here is the synthesis and biological evaluation of a tiny series of new virtually assembled NBTIs containing synthetically feasible right-hand side fragments capable of binding the GyrA subunit of the bacterial DNA gyrase-DNA complex. Conclusion: NBTI variants with incorporated 1-phenylpyrazole right-hand side moiety show suitable antibacterial activity against Gram-positive Staphylococcus aureus, with confirmed selectivity over the human topoisomerase IIα enzyme.