Abstract
Measuring interactions between a drug candidate and its specific target constitutes a parameter of upmost importance in drug discovery process. Because of its robustness and very high sensitivity, radio-isotopic labeling represents up to now the reference method for these pharmacological studies. However, the use of radioactivity confers several constraints about security, health hazards and environmental issues involved by radioactive waste storage. Regarding such limitations, non-radioactive technologies have been developed principally based on fluorescence detection. But the cost of such assay as well as the fact that incorporation of a bulky fluorescent block can affect the affinity for small ligand limit their use. In this context, we aim to develop a new methodology to quantify receptor-ligands interactions. Mass spectrometry (MS) presents very interesting features in terms of detection sensitivity and specificity, making this technique a challenging analytical tool to replace radioactivity and fluorescence measurements commonly used in pharmacology. Among all MS technologies, the capacity of inductively coupled plasma-mass spectrometry (ICP-MS) to provide metallic and hetero elements absolute quantification, whatever the nature of the sample medium, prompted us to investigate this technique in combination with an appropriate labeling of the molecule of interest. Selenium was selected as a good compromise between ICP-MS response and chemical tagging ability through the creation of covalent bonds using conventional organic sulfur chemistry without disturbance of the affinity toward targeted receptor. Proof of concept was illustrated on the vasopressin receptor (V1A), a GPCR involved in vasoconstriction and emotional behavior and implying peptide as native ligand. Different strategies were applied to design selenium labeled peptides relying in either conventional amino acid substitution by corresponding selenium containing residue into peptide sequence such as cysteine (Cys) replacement by selenocysteine (Sec) as well as proline (Pro) by selenazolidine (Sez), or N-terminal peptide derivatization with a selenium containing small organic entity. ICP-MS analytical methodology was carefully investigated to provide sensitive and reliable selenium signal measurement. High inorganic salts contents of pharmacological buffer along with polyatomic interferences from plasma interfering selenium detection necessitate chromatographic separation and collision reaction cell equipment before ICP-MS detection. The pharmacological protocol was also adapted to the analytical requirements, in particular quantity of cells and sample handling. Robustness of the designed competitive binding assay was evaluated through the affinity constant (Ki) measurement of several known V1A-R ligands exhibiting either high or poor affinity for the receptor. Experimental values were strongly correlated to literature data, enabling to validate the proof of concept of such methodology and to propose a suitable alternative to radioactive labeling.