AMPK Methods and Protocols

including AMPK activators in basal and stress metabolism and in

metabolic disease [12–14]. Conflicting reports about its cellular

functions, particularly in cancer, are also intriguing, and a growing

number of AMPK activators are being developed to treat human

diseases such as cancer and diabetes. However, a significant bottle-

neck in understanding AMPK function both in vitro and particu-

larly in vivo is the absence of a specific AMPK inhibitor.

2 The AMPK Inhibitor Compound C/Dorsomorphin

Compound C (C 24 H 25 N 5 O; 6-[4-(2-piperidin-1-ylethoxy) phe-

nyl]-3-pyridin-4-ylpyrazolo [1, 5-a] pyrimidine) is the primary

reagent used as an AMPK inhibitor (Fig.1). The general cell cycle

inhibitor adenine arabinoside or Ara-A, which is highly non-specific

to AMPK, has also been used in a few studies. Compound C is

widely used in biochemical experiments in vitro and in some in vivo

contexts. The history of discovery of this small molecule is probably

not known to all Compound C users. Compound C was originally

identified as an AMPK inhibitor from a screen of a 10K library by

Merck scientists. Their main interest in a selective AMPK inhibitor

was as a research tool in the delineation of the mechanism of

metformin, and they showed selectivity vs. a few unrelated kinases

[15]. While this reagent was being used for over a decade as an

AMPK inhibitor, in a high-throughput screen, another small mole-

cule had been identified as the first selective small molecule inhibi-

tor of bone morphogenetic protein (BMP) signaling. It was named

dorsomorphin because it caused dorsoventral patterning defects in

zebrafish typically seen in BMP-pathway mutant embryos [16–18];

however, dorsomorphin is structurally identical to compound

C. Dorsomorphin and its analog LDN-193189 cause stem cell

differentiation and thus may have therapeutic implications in the

progressive muscle and bone disease called fibrodysplasia ossificans

progressiva (FOP). This is an incurable disease where muscle and

tendons are slowly replaced by bone due to a mutation in the BMP

N N

N

N

O N

Fig. 1Chemical structure of compound C/dorsomorphin

196 Biplab Dasgupta and William Seibel