type 1 receptor called ALK2 (ACVR1) [19–21]. In addition to
inhibiting BMP signaling, dorsomorphin also inhibits the VEGF
type 2 receptor (FLK1/KDR) and disrupts angiogenesis during
zebrafish development [22]. It is an irony that while the therapeutic
potential of dorsomorphin was tempered by its known ability to
inhibit AMPK (“off-target” effects), Compound C found its use as
an AMPK inhibitor. Very little prudence was used despite its
numerous “off-target” effects as detailed below.3 Doses of Compound C Users Need to Consider
Various concentrations of Compound C as an AMPK inhibitor has
been used by laboratories (including ours) for in vitro and in vivo
experiments, and often times, doses were chosen to fit anticipated
results. In zebrafish embryos, a concentration of 5μM for 24 h was
sufficient to cause dorsoventral patterning defects that were attrib-
uted to inhibition of BMP signaling [22]. In vitro, Compound C
inhibited BMP signaling in murine pulmonary smooth muscle cells
at an IC 50 of 0.47μM[17]. At 4μM, it completely blocked
SMAD1/5/8 phosphorylation by BMP, and because SMADs reg-
ulate transcription, treatment of human liver cell lines (HepG2/
Hep3B) with 10μM Compound C for 6 h completely blocked
BMP-induced transcription of the SMAD-responsive iron regulat-
ing hepatic hormone hepcidin [16]. Because hepcidin interacts
with the cell surface iron transport protein ferroportin to control
plasma iron levels, intravenous administration of Compound C at
10 mg/kg caused 60% increase in serum iron concentrations result-
ing in hyperferremia in mice in 24 h [16]. Because iron overload
causes various systemic pathologies, the use of Compound C in vivo
in adult rodents warrants careful consideration of this side effect of
Compound C. In another study, 5μM Compound C inhibited
angiogenesis by about 50%, and at 10μM, it completely disrupted
blood vessel formation [22]. However, given that both VEGF and
BMP signaling are involved in angiogenesis, the authors could not
reach a conclusion based on the results produced by Compound
C. Given its effect on the inhibition of angiogenesis, any in vivo
study that attempts to connect AMPK with angiogenesis by the use
of Compound C alone should be considered inconclusive.4 Compound C Kinase Profiling Data
While extensive experimental analysis showed that Compound C
inhibits BMP and VEGF signaling in zebrafish and mammalian cells
at low micromolar concentrations, the reagent found continuous
use as an AMPK inhibitor in various in vitro and in vivo studies.
This is despite additional reports about the lack of selectivity of thisCompound C: Its Use and Misuse 197