Journal: Chembiochem

Article Title: Nitroreductase‐Mediated Release of Inhibitors of Lysine‐Specific Demethylase 1 (LSD1) from Prodrugs in Transfected Acute Myeloid Leukaemia Cells †

doi: 10.1002/cbic.202000138

Figure Lengend Snippet: Scheme of in vitro analysis of prodrug activation by NTR and subsequent fragmentation. A) Prodrug activation releases LSD1 inhibitor 1 a , which was further quantitatively derivatised to fluorophore 3 . i) FMOC−Cl, NaHCO 3 , H 2 O/ACN, pH 9.0. B) The activation of the prodrugs was visualised by fluorescent measurement of NADH consumption during the enzymatic reduction. As two reduction steps are necessary to generate the hydroxylamine, which can further fragment, 100 % prodrug activation was equated with the consumption of two equivalents of NADH. C) The fragmentation to 1 a was evaluated by derivatisation to 3 , which was quantified by HPLC analysis. The area under the curve was used to calculate the actual concentration of 3 , resulting in the degree of fragmentation displayed in the graph. For the activated fraction of nitrobenzyl‐containing prodrugs 1 b , 1 c and 1 d (measured in (B)), a quantitative fragmentation to 1 a was not observed. In comparison, the heteroaromatic prodrugs 1 e and 1 f fragmented quantitatively. D) Representation of the order of reactivity of the prodrugs with the NTR. The fragmentation data from (C) were used to calculate the factors, and the commonly used nitrobenzyl derivative 1 b was used as reference.

Article Snippet: Therefore, the cytotoxic negative controls 2 c and 2 f and prodrugs 1 b , 1 c and 1 f were tested in a GSH‐assay with Ellman's reagent for thiol reactivity.

Techniques: In Vitro, Activation Assay, Concentration Assay, Comparison