SYM-31-03

Structural insights into the multisite allosteric activation of AMP-activated protein kinases

CG Langendorf1, KRW Ngoei1, NXY Ling1, A Hoque1, MA Gorman2, SR Walker3, YE Bozikis3, ME Camerino3, MW Parker2, JW Scott1, R Foitzik3, K Sakamoto4, GR Steinberg5, JS Oakhill1 and BE Kemp1

  1. Protein Chemistry & Metabolism,, St. Vincents Institute of Medical Research, University of Melbourne, 41 Victoria Parade, Fitzroy VIC 3065, Australia
  2. ACRF Rational Drug Discovery Centre, St. Vincents Institute of Medical Research, University of Melbourne, 41 Victoria Parade, Fitzroy VIC 3065, Australia
  3. Department of Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia
  4. Nestle Institute of Health Sciences SA, EPFL Innovation Park, batiment G, 1015 Lausanne, Switzerland
  5. Division of Endocrinology and Metabolism, Department of Medicine, University of Medicine, McMaster University, Hamilton, Ontario, Canada

AMP-activated protein kinase (AMPK) is a metabolic stress sensing kinase responsible for regulating metabolism in response to energy supply and demand. During metabolic stress the cellular AMP/ATP ratio increases leading to activation of AMPK, which in turn switches off energy consuming anabolic pathways and switches on catabolic pathways in order to restore ATP levels. There has been keen interest in developing AMPK activating drugs for potential therapeutic use in treating the metabolic dimensions of age onset diseases including Type 2 diabetes, obesity and cardiovascular disease. The AMPK heterotrimer comprises a catalytic α-subunit, and regulatory β and γ-subunits. Two allosteric sites have been identified in AMPK, 1) the allosteric drug and metabolite binding site (ADaM) [1], formed between the small lobe of the α-subunit and the β-subunit carbohydrate binding module and 2) the tandem nucleotide sensing CBS motifs in the γ-subunit. The well-characterized small-molecule activator, A769662, has been shown to activate AMPK by binding in the ADaM site [2,3], however it is specific for only the β1-isoforms. The second-generation AMPK activator SC-4, a hetero ring-fused imidazole derivative, has a 10-fold higher affinity for β1 containing heterotrimers than A769662, more importantly SC-4 can activate AMPK heterotrimers containing either β1 or β2-isoforms. We have determined the X-ray crystal structure of the potent dual isoform AMPK activator SC-4 bound to the α2β1γ1 AMPK heterotrimer to 2.77 Å resolution and compare it to A769662 bound AMPK. Another small molecule AMPK allosteric activator, 5-(5-hydroxyl-isoxazol-3-yl)-furan-2-phosphonic acid (compound 2, C2), was identified by screening an AMP mimetic library [4]. We have determined the crystal structure of full-length AMPK (2.99 Å) complexed with C2, revealing two C2 binding sites in the γ-subunit distinct from the nucleotide sites [5]. ATP has been shown to competitively inhibit AMP allosteric activation of AMPK, however this is not observed with C2, highlighting the potential of C2 as a therapeutic at high physiological ATP concentrations. Furthermore, C2 acts synergistically with either SC-4 or A769662 to activate dephosphorylated AMPK, demonstrating dual drug therapies could be effective AMPK-targeting strategies to treat metabolic diseases.
References: 1. Langendorf, C.G. and B.E. Kemp. Cell Res, 2015. 25(1): p. 5-6. 2. Calabrese, M.F., et al. Structure, 2014. 22(8): p. 1161-72. 3. Xiao, B., et al. Nat Commun, 2013. 4: p. 3017. 4. Gomez-Galeno, J.E., et al. ACS Med Chem Lett, 2010. 1(9): p. 478-82. 5. Langendorf, C.G., et al. Nat Commun, 2016. 7: p. 10912.