Inhibition of firefly luciferase activity by a HIF prolyl hydroxylase inhibitor

The three hypoxia-inducible factor (HIF) prolyl-4-hydroxylase domain (PHD)
1–3 enzymes confer oxygen sen-sitivity to the HIF pathway and are novel
therapeutic targets for treatment of renal anemia. Inhibition of thePHDs
may further be beneficial in other hypoxia-associated diseases, including
ischemia and chronic in-flammation. Several pharmacologic PHD inhibitors
(PHIs) are available, but our understanding of their selectivity and its
chemical basis is limited.We here report that the PHI JNJ-42041935
(JNJ-1935) is structurally similar to the firefly luciferase substrate
D-luciferin. Our results demonstrate that JNJ-1935 is a novel inhibitor of
firefly luciferase enzymatic activity. In contrast, the PHIs FG-4592
(roxadustat) and FG-2216 (ICA,BIQ,IOX3,YM311) did not affect firefly
luciferase. The JNJ-1935 mode of inhibition is competitive with a Ki of
1.36 μM. D-luciferin did not inhibit the PHDs, despite its structural
similarity to JNJ-1935. This study provides insights into a previously
unknown JNJ-1935 off-target effect as well as into the chemical
requirements for firefly luciferase and PHD inhibitors and may inform the
development of novel compounds targeting these enzymes.