N desmethyltamoxifen

For the in vitro assays, tamoxifen, N-desmethyltamoxifen, 4-hydroxytamoxifen, and antimicrobials were used as standard laboratory powders and were obtained from Sigma–Aldrich (Madrid, Spain), and endoxifen was purchased from MedChemExpress (Sollentuna, Sweden). tamoxifen is water-insoluble, and to restore the standard laboratory powders, a corn oil/ethanol mixture was used. The mixture was vortexed and placed in a vacuum centrifuge for 30 min at 37 °C to evaporate the ethanol. The three tamoxifen metabolites are soluble in water. For the in vitro and in vivo studies, a mix of the three tamoxifen metabolites (metabolite mix, 33.3% each) was used, as well as N-desmethyltamoxifen alone. For the efficacy studies, clinical formulations were used: colistimethate sodium (CMS; Promixin, Bresso, Italy) and tigecycline (TIG; Normon, Madrid, Spain).

All SERMs were obtained from the following commercial sources. tamoxifen and Y-134 were purchased from Tocris Bioscience (Bristol, United Kingdom). N-desmethyl tamoxifen, 4-hydroxy tamoxifen, endoxifen, SO₄-tamoxifen, toremifene, 4-hydroxy toremifene, OSP, RAL, lasofoxifene, NAF, and BAZ were all obtained from Sigma Aldrich (St. Louis, MO, USA). Acolbifene was procured from AdooQ Bioscience (Irvine, CA, USA).
AM-630, AM-251, DAMGO, and WIN-55,212-2 were purchased from Tocris Bioscience. CP-55,940 was obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). [³⁵S]GTPγS (1250 Ci/mmol) was procured from American Radiolabeled Chemicals (St. Louis, MO, USA) and [³H]CP-55,940 (131.4 Ci/mmol) was purchased from PerkinElmer (Waltham, MA, USA).
All other reagents were purchased from Fisher Scientific Inc. (Pittsburgh, PA, USA). All compounds were dissolved in 100% DMSO to produce a stock concentration of 10 mM.