Past strategies and future directions for identifying AMP-activated protein kinase (AMPK) modulators

Abstract

AMP-activated protein kinase (AMPK) is a promising therapeutic target for cancer, type II diabetes, and other illnesses characterized by abnormal energy utilization. During the last decade, numerous labs have published a range of methods for identifying novel AMPK modulators. The current understanding of AMPK structure and regulation, however, has propelled a paradigm shift in which many researchers now consider ADP to be an additional regulatory nucleotide of AMPK. How can the AMPK community apply this new understanding of AMPK signaling to translational research? Recent insights into AMPK structure, regulation, and holoenzyme-sensitive signaling may provide the hindsight needed to clearly evaluate the strengths and weaknesses of past AMPK drug discovery efforts. Improving future strategies for AMPK drug discovery will require pairing the current understanding of AMPK signaling with improved experimental designs.

Keywords: AMPK; Dephosphorylation inhibition; Drug discovery; High-throughput screening; Nucleotide analogs; Regulatory fragment.

Fig. 1

A partial list of AMPK targets. A. AMPK phosphorylates and inhibits acetyl CoA carboxylase 1 and 2 (ACC1 and ACC2), resulting in decreased fatty acid synthesis and increased fatty acid oxidation, respectively (Hardie, et al., 2012). CPT1, carnitine palmitoyltransferase 1. B. AMPK phosphorylates CREB-regulated transcription coactivator 2 (CRTC2). Phosphorylation sequesters the coactivator to the cytoplasm, ultimately down-regulating gluconeogenesis (Hardie, et al., 2012; Yoon, et al., 2010). The graphical portrayal of this pathway varies, with some authors depicting phosphorylation of CRTC2 by AMPK prior to nuclear export; others depict only cytoplasmic phosphorylation (Lee, et al., 2010; Steinberg & Kemp, 2009).

Fig. 2

Regulatory mechanisms of AMPK. The simplified cartoon shows two nucleotide-binding sites on AMPK-γ. The remaining sites (Site 2 and Site 4) are omitted for clarity (Carling, et al., 2012; Hardie, et al., 2012; Russo, et al., 2013; Sanz, et al., 2013).

Fig. 3

In vivo studies. A. The effects of pharmacological activation of AMPK have been studied in models of diabetes, obesity, and sedentary lifestyle (Carling, et al., 2012; Cool, et al., 2006; Giri, et al., 2006; Halseth, et al., 2002; Narkar, et al., 2008; Xie, et al., 2011). B. Genetic deletion of isoforms has been studied in models of energetic stress. Deleted isoforms are indicated in parentheses (Barnes, et al., 2004; Steinberg, et al., 2010; Venna, et al., 2012).

Fig. 4

Protein-sensitive fluorescent probes. MANT- and coumarin-labeled nucleotides have been used to detect binding of molecules to the AMPK regulatory region (S. E. Sinnett, J. Z. Sexton, & J. E. Brenman, 2013; Xiao, et al., 2007; Xiao, et al., 2011). The Markush structure depicts the structure of adenine nucleotides that can be labeled with MANT and coumarin moieties. Note that the MANT moiety can substitute the 2′ or 3′ ribosyl hydroxyl (Cheng, Jiang, & Hackney, 1998). The stylized inset graph shows the protein-sensitive signal of a hypothetical probe.

Fig. 5

A partial list of AMPK-γ₂ mutations observed in patients with cardiomyopathy (Burwinkel, et al., 2005; Liu, et al., 2013; Moffat & Ellen Harper, 2010). These mutations affect residues (red) that are highly conserved throughout Eukarya. CBS, cystathionine-β-synthase domains in AMPK-γ.