The three hypoxia-inducible factor (HIF) prolyl-4-hydroxylase domain (PHD) 1–3 enzymes confer oxygen sensitivity
to the HIF pathway and are novel therapeutic targets for treatment of renal anemia. Inhibition of the
PHDs may further be beneficial in other hypoxia-associated diseases, including ischemia and chronic inflammation.
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,
YM 311) did not affect firefly luciferase. The JNJ-1935 mode of inhibition is competitive with a Ki of 1.36 μM. Dluciferin
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.