PYZD-4409 inhibits the E1 enzyme. (A) Chemical structure of the E1 inhibitor PYZD-4409 and the inactive control PYZD_mut. (B) GST-tagged human E1 (0.5μM) and fluorescein-labeled ubiquitin (1μM) were coincubated with increasing concentrations of PYZD-4409 or PYZD_mut for 30 minutes and resolved on SDS-PAGE under nonreducing conditions. Formation of E1-Ub conjugates were assessed by visualization of fluorescent signals using a gel imager. (C) GST-tagged human E1 (1μM), His₆-tagged human E2 (UbcH5A; 5μM), ubiquitin (20μM), and ATP (1mM) were coincubated with or without increasing concentrations of PYZD-4409 or PYZD_mut for 30 minutes at 30°C. The reactions were then fractionated on 4% to 20% gradient SDS-PAGE followed by immunoblotting with anti-His antibodies and fluorescent dye–labeled secondary antibodies. Fluorescent signals were detected with an infrared imaging system. (D) Recombinant His-tagged human E1 (1μM) was incubated with the His-tagged human UbcH5A E2 enzyme (10μM), ubiquitin (20μM), and ATP (1mM) in the presence of increasing concentrations of PYZD-4409 for 30 minutes at 30°C. Inorganic pyrophosphate resulting from ATP hydrolysis in E1-catalyzed ubiquitin activation was quantified with the use of a fluorogenic pyrophosphate assay kit and a fluorescence microplate reader as described in “E1 enzymatic assays.” Data represent the mean percentage ± SD of E1 enzyme activity compared with buffer-treated controls (n = 3). A representative experiment is shown. (E) K562 cells were treated with PYZD-4409 (50μM) for 4 hours. Cell lysates were heated in either nonreducing or reducing SDS-PAGE sample buffer and fractionated on 10% SDS-PAGE, followed by immunoblotting with antibodies against the E2 protein cdc34.