Clickable photoaffinity ligands for the human serotonin transporter based on the selective serotonin reuptake inhibitor (S)-citalopram

Abstract

To date, the development of photoaffinity ligands targeting the human serotonin transporter (hSERT), a key protein involved in disease states such as depression and anxiety, have been radioisotope-based (i.e., 3H or 125I). This letter instead highlights three derivatives of the selective serotonin reuptake inhibitor (SSRI) (S)-citalopram that were rationally designed and synthesized to contain a photoreactive benzophenone or an aryl azide for protein target capture via photoaffinity labeling and a terminal alkyne or an aliphatic azide for click chemistry-based proteomics. Specifically, clickable benzophenone-based (S)-citalopram photoprobe 6 (hSERT Ki = 0.16 nM) displayed 11-fold higher binding affinity at hSERT when compared to (S)-citalopram (hSERT Ki = 1.77 nM), and was subsequently shown to successfully undergo tandem photoaffinity labeling-biorthogonal conjugation using purified hSERT. Given clickable photoprobes can be used for various applications depending on which reporter is attached by click chemistry subsequent to photoaffinity labeling, photoprobe 6 is expected to find value in structure-function studies and other research applications involving hSERT (e.g., imaging).

Development of Clickable Photoaffinity Ligands for Metabotropic Glutamate Receptor 2 Based on Two Positive Allosteric Modulator Chemotypes

“…We chose to pursue a racemic probe due to expediency, ease of synthesis, and lower cost, with the intention to synthesize more challenging enantiomerically pure photoprobes in the event that pharmacology and labeling studies were promising. We note that this is a common approach in the field, 48 and supported by the well-validated pharmacology of BINA ((±)-12). Pharmacological evaluation of (±)-14 showed that, similar to (±)-13, this compound displayed an increase in mGlu 2 agonist and potentiator potency relative to BINA (i.e., 4-and 5-fold, respectively) with equivalent maximal responses for agonism and potentiation (Table 4).…”

Characterizing the lipid-protein interface of the human serotonin transporter by crosslinking mass spectrometry

“…After proteolytic removal of the N-terminal FLAG tag, eluted purified hSERT in mixed micelles containing mixed phospholipids and cholesterol at a concentration of ~ 1.65 mg/ml, (with more than 10% of the lipid mass being cholesterol) was reconstituted into small unilamellar vesicles after detergent removal. In analogous studies using this overexpressing inducible cell line, purified hSERT retained nanomolar binding to SSRI analogs during purification (36) and Castellano et al (manuscript in preparation). Experimental and control liposomes were spiked to contain ~2.4 mM cholesterol with and without 7.3 µM azi-cholesterol, respectively, in addition to other mixed lipids.…”

Direct visualization of an antidepressant analog using surface-enhanced Raman scattering in the brain

“…In an attempt to distinguish SERT-bound Alk-S-Cit from those not bound, we synthesized a previously reported compound, coined benzophenone-alkyne-tagged S-Cit (Benz-Alk-S-Cit) for convenience (ref. 41 and Supplemental Figures 7-10). Benz-Alk-S-Cit possesses the backbone structure of both S-Cit and an alkynyl group but shows a very low binding affinity toward SERT.…”