Geranylgeraniol

Pitavastatin (Livalo, Adooq), zoledronic acid and risedronate (Selleck), and GGTI-2133, Tipifarnib, farnesol, geranylgeraniol and mevalonate (Sigma-Aldrich) were prepared as 20 mM solutions in DMSO except zoledronic acid which was dissolved in H₂O.

CrtE prenyltransferase activity was determined in vitro as described previously (Jones et al., 2013 (link)). The incubation mixture contained, in a total volume of 200 μL, 50 mM Tris–HCl (pH 7.5), 5 mM MgCl₂, 1 mM DTT, 46 μM [1-¹⁴C] IPP (1.85 GBq mmol^–1), 0.5% (v/v) Triton X-100 and purified protein, and 10 μM DMAPP. Samples were incubated for 1 h at 30°C and reactions stopped by cooling on ice and the addition of 200 μL of 1 M NaCl. Samples were extracted in 2 mL of water-saturated butanol overnight at -20°C. Butanol extractable products were dephosphorylated by adding potato acid phosphatase (1 unit) (Sigma-Aldrich, United Kingdom) to 1 mL of extract and incubating in 0.2% (v/v) Triton X-100 and 4 mL of methanol in a total volume of 10 mL at 37°C overnight on a shaking platform water bath. Samples were extracted in 10 mL of 10:90 (v/v) diethyl ether/petroleum ether (boiling point 40–60°C) and the organic phase dried under nitrogen. Reaction products (geraniol, farnesol, and geranylgeraniol) were identified by thin-layer chromatography (TLC) using reversed-phase C18 F254S plates (Merck, United Kingdom), with a mobile phase of 19:1 (v/v) acetone/water. The radio-labeled products were identified by autoradiography, and alcohol standards (geraniol, farnesol, geranylgeraniol, and solanesol) (Sigma-Aldrich, United Kingdom) were visualized by exposure to iodine vapor.