Structures of AMP-activated protein kinase bound to novel pharmacological activators in phosphorylated, non-phosphorylated, and nucleotide-free states

AMP-activated protein kinase (AMPK) is an attractive therapeutic target for managing metabolic diseases. A class of pharmacological activators, including Merck 991, binds the AMPK ADaM site, which forms the interaction surface between the kinase domain (KD) of the α-subunit and the carbohydrate-binding module (CBM) of the β-subunit. Here, we report the development of two new 991-derivative compounds, R734 and R739, which potently activate AMPK in a variety of cell types, including β2-specific skeletal muscle cells. Surprisingly, we found that they have only minor effects on direct kinase activity of the recombinant α1β2γ1 isoform yet robustly enhance protection against activation loop dephosphorylation. This mode of activation is reminiscent of that of ADP, which activates AMPK by binding to the nucleotide-binding sites in the γ-subunit, more than 60 Å away from the ADaM site. To understand the mechanisms of full and partial AMPK activation, we determined the crystal structures of fully active phosphorylated AMPK α1β1γ1 bound to AMP and R734/R739 as well as partially active nonphosphorylated AMPK bound to R734 and AMP and phosphorylated AMPK bound to R734 in the absence of added nucleotides at <3-Å resolution. These structures and associated analyses identified a novel conformational state of the AMPK autoinhibitory domain associated with partial kinase activity and provide new insights into phosphorylation-dependent activation loop stabilization in AMPK.

AMPK complex at the lysosomal membrane (14) without changing the AMP:ATP ratio (15,16). Together, metformin potently activates AMPK, which mediates a subset of its effects (17,18). In addition, acute activation of AMPK in skeletal muscle, which in humans is ␤ 2 -isoform-specific, is mediated by exercise, but not by metformin, and induces robust and acute glucose uptake in rodents and primates, indicating that AMPK activation in muscle has important additional benefits (5,19,20).
The ADaM site is located at the interface between the kinase domain of the ␣-subunit and the carbohydrate-binding module (CBM) of the ␤-subunit. Phosphorylation (or a phosphomimetic mutation) of CBM Ser-108 is either required or greatly increases binding of ADaM site ligands (22,25). ADaM site binders stabilize the interface, which in turn conformationally stabilizes the highly dynamic CBM and shifts the equilibrium toward the active conformation of the kinase domain (22). Consequently, ADaM site activators, activation loop phosphorylation, and AMP-mediated direct allosteric AMPK activation can all collaborate, and ADaM site activators and AMP can partially (26) and possibly fully (25) activate AMPK in the absence of activation loop phosphorylation. To explore the mechanisms of full and partial AMPK activation, we have solved high-resolution crystal structures of phosphorylated ␣ 1 ␤ 1 ␥ 1 AMPK bound to AMP and the two new AMPK ADaM site binders R734 and R739, phosphorylated ␣ 1 ␤ 1 ␥ 1 AMPK bound to R734 in the absence of AMP, and nonphosphorylated ␣ 1 ␤ 1 ␥ 1 AMPK with a CBM phosphomimetic mutant (CBM S108D) bound to AMP and R734, which have provided unanticipated insight into the conformational flexibility of AMPK.

991-derivative compounds activate AMPK in cells
An important component of the KD-CBM interaction and its stabilization by ADaM site ligands is AMPK phosphorylation at Ser-108 in the CBM, which mediates interaction with two lysines (Lys-29 and Lys-31) in the KD (22,27,28). These interactions are stabilized by ADaM site compounds 991 and A769662, which form charge interactions with Lys-29 in ␤ 1 -containing complexes (22,27). We generated two derivatives of 991 in which we replaced the Lys-29 -binding, negatively charged carboxyl group of the 991 o-methylbenzoic acid moiety against uncharged N-hydroxylamide (R734) and the 1-methylindole heterocyclic ring against 2-methylbiphenyl (R739) (Fig. 1). These compounds activated endogenous AMPK in all cell lines tested, including ␤ 2 -specific LHCN-M2 human skeletal muscle cells, with low M AC2X values (the concentration that increases activity 2-fold; Fig. 2, A and B). Both compounds also efficiently inhibited proliferation of A549 and H1299 human lung cancer cell lines ( Fig. 2A). The ability of these compounds to activate AMPK in human skeletal muscle cells, which do not contain detectable amounts of AMPK ␤ 1 complexes (5), suggests that these compounds can activate both ␤ 1 -and ␤ 2 -containing AMPK complexes in cells.
In contrast to their variable effects on the direct kinase activity of AMPK ␣ 1 ␤ 2 ␥ 1 , both compounds robustly increased protection against activation loop dephosphorylation of both AMPK ␣ 1 ␤ 1 ␥ 1 and ␣ 1 ␤ 2 ␥ 1 (Fig. 3B). As a control, both compounds did not inhibit phosphatase activity (Fig. S2). Therefore, R739 and, to a lesser degree, R734 can functionally separate two mechanisms of AMPK activation (direct kinase activation and protection against activation loop dephosphorylation) selectively for the ␣ 1 ␤ 2 ␥ 1 isoform. Therefore, AMPK ␣ 1 ␤ 2 ␥ 1 activation by R739 in cells may occur predominantly or exclusively by increasing net activation loop phosphorylation.

AMPK in complex with novel pharmacological activators R734 and R739 strongly increase net AMPK activation loop phosphorylation in cells
To understand how R734 and R739 affect activation loop phosphorylation and downstream signaling in cells, we tested their effects on AMPK activation loop phosphorylation and phosphorylation of the AMPK targets Raptor and acetyl-CoA carboxylase (ACC) in parallel with 991 and A769662. We incubated HepG2 cells with increasing doses of the four compounds, lysed cells, and analyzed lysates by immunoblotting. As shown in Fig. 4, the increase in phosphorylation of Raptor and ACC in the presence of R734 and R739 was similar to that of 991 and stronger than that of A769662 (note that A769662 does not activate AMPK ␣ 1 ␤ 2 ␥1, which is the main AMPK isoform in HepG2 cells). In contrast, AMPK ␣ 1 /␣ 2 Thr-174/Thr-172 phosphorylation was clearly stronger in the presence of R734 and R739 than in the presence of 991 and A769662. The level of AMPK activity, measured by downstream target phosphorylation, depends on the combination of activation loop phosphorylation and direct, allosteric activation. The observation that R739 and 991 induce similar levels of Raptor and ACC phos- Comp. ID   I  s  l  l  e  c  n  i  n  o  i  t  a  v  i  t  c  a  K  P  M  A  nhibition of

Figure 2. AMPK activation in cells.
A, summary of AMPK activity data. Activation was determined by ELISA-based pACC assay. AC2X, compound concentration at which activity is 2-fold above background. 991, PF249, and GSK621 activation data were included as references. nd, not determined. B, R734 and R739 dose-response curves in pACC cell-based ELISAs. n ϭ 2; error bars, S.D.

AMPK in complex with novel pharmacological activators
phorylation but different levels of AMPK activation loop phosphorylation is therefore consistent with the inability of R739 to directly activate AMPK ␣ 1 ␤ 2 ␥ 1 .

Crystal structure determination of phosphorylated AMPK in complex with AMP and R734 or R739
To gain insight into the structural basis of full and partial AMPK activation by R734 and R739, we first cocrystallized both compounds with fully active phosphorylated AMPK ␣ 1 ␤ 1 ␥ 1 , the kinase domain-stabilizing ATP-competitive kinase inhibitor staurosporine, and AMP (Fig. S3). This allowed us to determine their structures at 2.9-and 2.7-Å resolution, respectively (Fig. 5, A and B; structure statistics in Table 1). As expected, both structures show the hallmarks of fully active AMPK, including a fully aligned regulatory spine and fully resolved activation loop (Fig. S4A), as well as formation of a helix in the CBM linker (C-interacting helix; Fig. 5, A and B) that packs against and stabilizes the regulatory ␣C helix of the kinase domain, a hallmark for AMPK activated by ADaM site agonists (22,27). Both structures are highly similar to each other (r.m.s.d. value of 0.47 Å over 845 residues) as well as to AMPK ␣ 2 ␤ 1 ␥ 1 bound to the parent compound 991 (r.m.s.d. values of 1.66 Å over 806 residues (R734) and 1.69 Å over 818 residues (R739)).
Although R734 and R739 lacked the 991 carboxyl group that forms an ionic interaction with Lys-29 in the KD, they unex-

R734 weakly synergizes with AMP for AMPK activation
Nonphosphorylated AMPK has at least 100-fold lower catalytic activity than AMPK in which activation loop Thr-174 (human AMPK ␣ 1 ; Thr-172 in human ␣ 2 and rat ␣ 1 and ␣ 2 ) is phosphorylated. Surprisingly, Scott et al. (25) demonstrated that AMPK ␣ 1 ␤ 1 ␥ 1 , which lacks activation loop phosphorylation but retains phosphorylation of Ser-108 in the CBM, can be fully activated by a combination of A769662 and AMP, implying that the AMPK kinase domain can adopt a fully active conformation in the absence of activation loop phosphorylation. 991 can also synergize with AMP to activate nonphosphorylated AMPK but only to about 10% of the activity of phosphorylated AMPK (26). In our hands, Escherichia coli-produced, nonphosphorylated AMPK ␣ 1 ␤ 1 ␥ 1 carrying a CBM S108D mutation, which partially mimics the effect of Ser-108 phosphorylation (25), was activated ϳ20-fold by A769662, ϳ3-fold by AMP, and ϳ33-fold by the combination of A769662 and AMP (Fig. 6A). R734 activated the same protein ϳ5.5-fold and  in combination with AMP ϳ10-fold (Fig. 6A), suggesting a marginal synergism that is insufficient to fully compensate for activation loop phosphorylation.

Crystal structure determination of nonphosphorylated AMPK in complex with AMP and R734
Because the requirement of activation loop phosphorylation for full AMPK activity can be partially overcome by a combination of R734 and AMP (Fig. 6A), we wanted to explore the structure of nonphosphorylated AMPK ␣ 1 ␤ 1 (S108D)␥ 1 in the presence of R734, staurosporine, and AMP. The phosphorylation-mimicking CBM S108D mutation allowed stabilization of the interaction with R734 in the absence of AMPK phosphorylation. The purified protein readily crystallized (Fig. S3) and allowed us to determine its structure at 2.65-Å resolution (Fig.  6B). Although the overall complex structure was well-defined by strong electron density, the regions of the AID and the following ␣RIM domains, particularly the residues from Lys-296 through Glu-338, show relatively weak density and high B factors. To correctly build this region of the structure, we calculated a 2F o Ϫ F c composite omit map with 3000 K simulated annealing to reduce model bias (Fig. 7, A and B, and Fig. S5, A-C). We surprisingly found that the AID conformation is largely different from those resolved in previously published AMPK structures (22,24,28)  Although the overall omit map density is relatively weak, we observed reliable main chain density of the AID ␣1 helix from Met-289 through Leu-295, of the ␣2 helix from Leu-313   In addition to the unique conformation of the AID, two other features stood out. First, despite the relatively low kinase activity, the catalytic center adopted a fully active conformation with a fully resolved activation loop (Fig. S4, B and C) and a CBM linker with resolved C-interacting helix (Fig. 6B). Second, the modified benzyl ring of R734, which is connected through a freely rotatable single bond, adopted a 180°rotated configuration in the ligand-binding pocket, pointing toward the CBM instead of the KD (Fig. S7). Although the remaining parts of R734 and R739 had strong electron density, the density of the

AMPK in complex with novel pharmacological activators
N-hydroxylamide group of the benzyl ring was relatively weak in all structures, indicating that the pocket can adopt the ring in different orientations (Fig. S7). The density indicates that in phosphorylated AMPK the orientation of the N-hydroxylamide group has a weak preference toward the KD, allowing the N-hydroxylamide to interact with the ATP-binding G-loop (Fig. 5, C and D), whereas in nonphosphorylated AMPK it preferentially pointed toward the CBM at the positively charged pocket entrance (Fig.  S7).

The autoinhibitory domain of nonphosphorylated AMPK-R734 -AMP adopts a unique ␥-subunitbound conformation
The AID of AMPK is in a dynamic equilibrium between a KD-bound (inactive, ATP-induced) state and a ␥-subunit-

AMPK in complex with novel pharmacological activators
bound (active, AMP-induced) state (28 -30). Due to its dynamic nature, the AID has relatively poor density in all structures of AMPK and could not be modeled in the majority of structures of fully active AMPK, including the structures of phosphorylated AMPK bound to R734 -AMP and R739 -AMP.
In the structure of nonphosphorylated AMPK ␣ 1 ␤ 1 (S108D)␥ 1 in the presence of R734, staurosporine, and AMP, the AID is bound to the ␥-subunit in a conformation in which it is rotated relative to the structures of the more active phosphorylated AMPK (pAMPK) complexes ␣ 2 ␤ 1 ␥ 1 -991-AMP (Protein Data Bank (PDB) code 4CFE), pAMPK ␣ 1 ␤ 2 ␥ 1 -AMP (PDB code 4RER), and AMPK ␣ 2 ␤ 1 ␥ 1 -991-AMP (PDB code 5ISO) ( Fig.  7C and Fig. S5, D-G). As a consequence, the AID helix ␣1 adopts a position that overlaps with the position of ␣3 in phosphorylated, AMP-bound AMPK (Fig. 7C and Fig. S5, D-G). In contrast to the different sets of AID residues involved in ␥-subunit binding, the AID-binding residues within the well-resolved ␥-subunit are largely identical for both conformations. Importantly, this conformation is not due to crystal packing as the AID is not involved in any substantial interactions with neighboring molecules in the crystals lattice (Fig. S6). To test whether this conformation affects the activity of AMPK, we introduced single and double mutations into Met-289 and Glu-293 of the short AID ␣1 helix. These residues are modeled at the AID/␥subunit interface in nonphosphorylated AMPK (Fig. 7A) but in phosphorylated AMPK do not interact with either ␥-subunit or KD. We then purified the nonphosphorylated WT and mutant proteins and determined their kinase activities in the presence or absence of AMP, R734, and A769662. The mutations moderately enhanced the activity of AMPK in all contexts (Fig. 7D),

AMPK in complex with novel pharmacological activators
suggesting that the AID conformation seen in the structure of nonphosphorylated AMPK is less potent in shifting the equilibrium from the KD-bound state to the ␥-subunitbound state. In contrast to activity-compromising mutations, activity increases cannot be explained by protein destabilization and are therefore highly reliable indicators for the disruption of inhibitory interactions.
Recently, Willows et al. (26) determined the structure of nonphosphorylated AMPK ␣ 2 ␤ 1 ␥ 1 in complex with 991, AMP, and staurosporine. 991 activates AMPK ␣ 2 ␤ 1 ␥ 1 in conjunction with AMP much more potently than R734 and R739 (26-fold compared with 10-fold activation of AMPK ␣ 1 ␤ 1 ␥ 1 by R734 and AMP), and the AID adopted a structure very similar to that seen in phosphorylated AMPK. Together, this suggests that the AID, besides the known active and inactive conformations, can also adopt a novel conformation that is associated with intermediate kinase activity.

Nonphosphorylated AMPK-R734 -AMP adopts a stable activation loop conformation in crystals but requires phosphorylation for stabilization in solution
Although nonphosphorylated AMPK in the presence of R734 and AMP only moderately activates AMPK, the crystal structure revealed all hallmarks of an active kinase conformation, including a completely resolved activation loop (Fig. S4, B and C), similar to nonphosphorylated AMPK ␣ 2 ␤ 1 ␥ 1 -991 (26). AMPK belongs to the family of arginine-aspartate (RD) kinases, which are frequently regulated by activation loop phosphorylation. In RD kinases, the negatively charged phosphate rearranges the activation loop by binding to a positively charged pocket in the KD, which in turn arranges the arginine (R) and adjacent aspartate (D) of the catalytic loop to position the substrate phospho-acceptor site (Fig. S4A). AMPK is unusual in that the activation loop is directly stabilized by interaction with the stable core structure of AMPK, consisting of the ␤-subunitinteracting C terminus of the ␣-subunit (␣-CTD) and the ␣/␥subunit-interacting C terminus of the ␤-subunit (␤-CTD; Fig.  8) (26,31).
The fact that the nonphosphorylated activation loop has a stable, active conformation in the crystal structures of AMPK ␣ 2 ␤ 1 ␥ 1 -991 (26) and AMPK ␣ 1 ␤ 1 ␥ 1 -R734 ( Fig. 8 and Fig. S4, B and C), even though these complexes are only partially active, suggests that they may represent a minor fraction of total conformations that is captured in the crystallization snapshot due to their reduced dynamics. Moreover, the conformation could further be influenced by binding of the ATP-competitive inhibitor staurosporine, which stabilizes AMPK in a largely closed (substrate bound-mimicking) conformation. We therefore used hydrogen/deuterium exchange MS (HDX-MS) to analyze the conformational landscape of the activation loop in solution. As shown in Fig. 9 and Figs. S8 -S10, in solution the activation loop, as well as the activation loop-interacting segment of the ␤-subunit (␤-subunit shown in Figs. S8 -S10), indeed became strongly stabilized by phosphorylation and moderately stabilized by staurosporine, clearly indicating that the crystal structure represents a minor conformation snapshot.
In addition, binding of staurosporine, which induces the closed (substrate-bound) conformation of the kinase domain, stabilizes the AID (Fig. 9D). This is consistent with the proposed inability of the AID to bind the closed kinase domain conformation (28,30,32), which further shifts the equilibrium to the more stable AMP-induced AID-␥-subunit interaction.

Nucleotide occupancy in pAMPK-R734 in the absence of added nucleotides
Several structures of holo-AMPK have been solved in the AMP-bound state, but none have been solved in the apo or

AMPK in complex with novel pharmacological activators
ATP-bound state. AMP binding at CBS3 is thought to be critical for allosteric AMPK regulation because it structurally stabilizes the interaction between the AMP-sensing ␣RIM2 and AMP-bound CBS3 (29,31). This interaction in turn shifts the AID/␣RIM1 equilibrium from the inhibitory KD-bound conformation to the ␥-subunitbound conformation as a mechanism for direct allosteric kinase activation (28 -31). To gain insight into nucleotide occupancy and the conformation of AMPK with unoccupied CBS3, we determined the crystal structure of phosphorylated AMPK ␣ 1 ␤ 1 ␥ 1 -R734 in the absence of added AMP. As shown in Fig. 10A, the overall structure is very similar to that of pAMPK-R734 -AMP. Although we see density at ␣RIM2, the density was weak. The composite omit map in Fig. 10B and Fig. S11 revealed that AMP was stably bound at CBS4 as predicted by previous biochemical analysis (33,34). We further detected very weak AMP density at CBS3 (too weak to build an AMP in the model), indicating very low occupancy, whereas no reliable density could be seen at CBS1 (Fig. 10B and Fig. S11). These results confirm the stable binding of AMP at CBS4 as well as the recent reassignment of CBS3 and CBS1 as the exchangeable higher-and lower-affinity nucleotide binding-sites, respectively (35).

An ADaM site ligand can isotype-selectively separate direct AMPK activation and protection against activation loop dephosphorylation
AMPK is a central signaling hub that phosphorylates and regulates numerous targets. Consequently, AMPK dysregulation is associated with a spectrum of metabolic diseases, spanning from diabetes and obesity to cancer, inflammation, and cardiometabolic and neurodegenerative diseases. The pleiotropic effects of AMPK's known physiological activators, AMP and ADP, have made it challenging to clearly attribute complex effects to AMPK activity states and to therapeutically regulate AMPK activity in different tissues with high selectivity. In 2006, Abbot Laboratories developed compound A769662 (36), the first example of a group of small molecules that activate AMPK with high specificity by binding the ADaM site at the KD/CBM interface (22). A limitation of this class of compounds is their preference for AMPK complexes that contain the ␤ 1 -subunit and phosphorylated CBM Ser-108.
The ADaM sites formed by ␤ 1 -and ␤ 2 -subunits differ only by two CBM residues (defined as residues within 5 Å to the KD), Thr-106 and Asn-111 in ␤ 1 complexes, which correspond to Ile-106 and Asp-111 in ␤ 2 complexes, respectively (see Fig.  S12). Consistently, the high-affinity ␤ 2 ADaM site compound SC4 forms a hydrogen bond with ␤ 2 Asp-111 but not with ␤ 1 Asn-111, providing a structural rationale for its elevated ␤ 2 selectivity (37). ␤ 2 Asp-111 and ␤ 1 Asn-111 form different polar interaction networks with phospho-Ser-108, which in turn forms strong ionic interactions with Lys-29 and Lys-31 of the KD to stabilize the ADaM site. The carboxyl group of 991 further increases stability of this network by direct interaction with KD Lys-29 (Fig. 5E), suggesting that it may contribute to isoform selectivity. In addition, removal of the carboxyl group reduces uptake by hepatocyte organic anion transport proteins and would therefore be expected to decrease the liver cell:muscle cell uptake ratio.
The 991 derivatives R734 and R739 lack the carboxyl group but still activate recombinant ␤ 1 complexes to a larger degree than ␤ 2 complexes. Strikingly, R739 failed to allosterically activate E. coli-purified AMPK ␣ 1 ␤ 2 ␥ 1 but robustly decreased activation loop dephosphorylation, similar to ADP, which binds the nucleotide-binding sites in the ␥-subunit more than 60 Å away from the ADaM site. Therefore, these two activities can be pharmacologically separated by both ADaM site-binding and by ␥-subunitbinding compounds. Direct allosteric activation stabilizes the active kinase conformation (in the case of ADaM site agonists by stabilizing the regulatory ␣C helix of the KD  (22)). In contrast, protection against activation loop dephosphorylation is likely determined by the conformation of the linker between the CBM of the ␤-subunit at the ADaM site and the C-terminal scaffolding domain of the ␤-subunit at the ␥-subunit (22,31). This suggests that R739 may be unable to induce formation of a properly positioned ␣C-stabilizing helix in the CBM linker of the ␣ 1 ␤ 2 ␥ 1 complex but may sufficiently reposition the linker to shield the activation loop against phosphatases. Details of how ligands change the conformation of the linker remain unknown in the absence of a structure of holo-AMPK in the inactive, ATP-bound state.

The AID adopts a conformation in partially active AMPK that is associated with reduced activity
Activation loop phosphorylation catalytically activates E. coli-produced AMPK ϳ100-fold (38,39), and AMP activates it an additional 2-fold (in mammalian cells up to 10-fold (7)). The combination of R734 and AMP only partially activated nonphosphorylated AMPK (ϳ10-fold), but the crystal structure of AMPK ␣ 1 ␤ 1 ␥ 1 showed the hallmarks of an active kinase, including a fully resolved activation loop. This is reminiscent of the recently reported structure of nonphosphorylated AMPK ␣ 2 ␤ 1 ␥ 1 bound to 991 and AMP (26). Although 991 activates nonphosphorylated AMPK more potently than R734, it fails to fully activate the kinase; however, the kinase domain also adopted an active conformation with fully resolved activation loop. Our HDX-MS analysis demonstrated that this conformation in AMPK ␣ 1 ␤ 1 ␥ 1 represents a minor fraction that likely crystallizes more easily due to its higher order and that, for the majority of AMPK, phosphorylation is required for full activation loop stabilization. Consistently, phosphorylation is also required for full activity of AMPK bound by 991 and AMP in mammalian cells (26).
A surprise finding was that the AID adopted a novel conformation in which its ␣1 helix, rather than its ␣3 helix, interacted with the ␥-subunit. The AID can therefore interact with at least three different surfaces within AMPK: (i) the KD as seen in the inactive, ATP-bound and nonphosphorylated conformation; (ii) the ␥-subunit via AID ␣3 in the fully active phosphorylated and AMP-bound conformation; and (iii) the ␥-subunit via AID ␣1 in this structure of partially active, nonphosphorylated and AMP-bound AMPK. To probe for the biological relevance of this conformation, we tested the effect of mutations in AID ␣3 at the AID/␥ interface on the kinase activity of largely or fully active, phosphorylated AMPK. The fact that these mutations increased the kinase activity suggests that the partially active conformation competes predominantly with the fully active conformation, not the inactive conformation.
The AID in this structure was better resolved than in the corresponding structure of fully active phosphorylated AMPK. However, the mutations designed to disrupt the AID ␣1/␥-subunit interface increased AMPK activity, implying that this conformation is more stable but less active than the competing AID conformation seen in all active AMPK structures with resolved AID. The presence of the AID either in an unresolved state or in at least three different, distinct conformations as well as the high B factor of the AID in all resolved structures illustrates the highly dynamic nature of the AID in the context of holo-AMPK, consistent with its key regulatory switch role in allosteric activation by adenine nucleotides.

Chemical synthesis
Synthesis schemes for compounds 991, R734, and R739 are provided in the supporting information.

Crystallization
Purified nonphosphorylated and phosphorylated AMPK fractions were incubated with a 3-fold molar excess of staurosporine for 3 h at 4°C, spin-concentrated to ϳ7 mg/ml, and incubated with R734 or R739 at molar ratios of protein:compound of 1:5 in the presence and absence of 5 mM AMP. Crystals of both R734-bound and R739-bound phosphorylated AMPK in complex with AMP and staurosporine were grown at ϳ20°C in sitting drops containing 0.3 l of purified protein at ϳ7 mg/ml and 0.3 l of reservoir solution consisting of 0.1 M trisodium acetate, pH 5.6, 0.2 M ammonium acetate, 15% (w/v) PEG 4000. Crystals of phosphorylated R734-bound AMPK in the absence of added nucleotides were grown at ϳ20°C in sitting drops containing 0.3 l of purified protein at ϳ7 mg/ml AMPK in complex with novel pharmacological activators and 0.3 l of reservoir solution consisting of 0.1 M triammonium citrate, pH 7.0, 12% (w/v) PEG 3350. Crystals of nonphosphorylated R734-bound AMPK in complex with AMP were grown at ϳ20°C in sitting drops containing 0.3 l of purified protein at ϳ7 mg/ml and 0.3 l of reservoir solution consisting of 0.2 M magnesium formate.

Structure determination and refinement
X-ray diffraction data were collected at 21-ID of the Life Science Collaborative Access Team at the Advanced Photon Source. The observed reflections were reduced, merged using MOSFLM, and scaled with SCALA of the CCP4 package (40). The structures were solved by molecular replacement performed using the CCP4 program Phaser (41) with the structure of AMPK in complex with 991 (22) (PDB code 4CFE) as an initial search model. The program Coot (42) was used to manually fit the protein model. Model refinement was performed with the PHENIX program package (43). The statistics of data collection and the model refinement are summarized in Table S1.

Cell-based phospho-acetyl-CoA carboxylase (pACC) ELISAs
HepG2 cells (200,000 cells/well seeded a day before and starved for 2 h in low-glucose medium), LHCN-M2 cells (20,000 cells/well differentiated for 5 days), and A549 cells (20,000 cells/well seeded a day before) in 96-well flat-bottom tissue culture plates were incubated for 2 h at 37°C in the medium containing serial dilutions of the test samples. The reaction was stopped using 100 l of 4% formaldehyde in phosphate-buffered saline (PBS). Cells were washed and incubated for 1 h at room temperature on a shaker in blocking buffer followed by overnight incubation at 4°C with 200 l of antiphospho-acetyl-CoA carboxylase (Ser-79) antibody (Cell Signaling Technology, catalog number 3661) diluted in blocking buffer (1:1000). After the incubation, the cells were washed and incubated with 200 l of horseradish peroxidase-conjugated secondary antibody diluted in blocking buffer (1:1000) for 1 h at room temperature. The stained cells were washed, and chemiluminescent reagent was added. Chemiluminescence was read using a Victor plate reader (PerkinElmer Life Sciences) within 5 min. The EC 50 determination was executed using Prism software version 7 (GraphPad, Inc.). Means of EC 50 values acquired from multiple experiments (n Ն 2) are shown in Fig. 2A.

Proliferation assay
Cells were plated in ViewPlate96 96-well plates (PerkinElmer Life Sciences) in duplicate replicates. The compound dilutions for the six-point screens (from 10 to 0.41 M, 3-fold dilution) were performed manually. Following incubation with the compound for 48 h, cells were fixed with 2.0% paraformaldehyde (Sigma-Aldrich) in PBS (Ca 2ϩ /Mg 2ϩ -free) for 30 min, washed with PBS, stained for 60 min with a 6 ng/ml solution of 4,6diamidino-2-phenylindole dihydrochloride (Molecular Probes Inc., Eugene, OR) in PBS. Cells were stored at 4°C in PBS for at least 16 h before imaging to allow stain to equilibrate. A Zeiss Axiovert S100 inverted fluorescence microscope, equipped with a Plan-NEOFLUAR 10ϫ objective (Carl Zeiss Inc., Thorn-wood, NY) and a Hamamatsu Lightningcure 200 mercury-xenon light source with an Omega Optical XF57 quad filter (Hamamatsu Photonics, Japan), was used for capturing images. Nine images per well were taken, in an adjacent grid pattern, in each well of the 96-well plates of treated tumor cells. Images were analyzed in a 12-bit format using segmentation and morphological routines contained in the Image Pro software package (Media Cybernetics Inc., Bethesda, MD). The EC 50 curve fitting was executed with MathLab software version 6.5 (Math-Works Inc., Natick, MA). For cell cycle analysis, DNA content of each nucleus in the sample images was plotted and smoothed using the Lowess method (48). Apoptosis was assessed by manual inspection of the cell cycle profile and fragmented nuclei as assessed by the imaging analysis. Endoreduplication was assessed by manual inspection based upon enlarged nuclei and the presence of the 8N population.

HDX detected by mass spectrometry
Differential HDX-MS experiments were conducted as described previously with a few modifications (44).
Peptide identification-Peptides were identified using tandem MS (MS/MS) with an Orbitrap mass spectrometer (Q Exactive, Thermo Fisher). Product ion spectra were acquired in data-dependent mode with the top five most abundant ions AMPK in complex with novel pharmacological activators selected for the product ion analysis per scan event. The MS/MS data files were submitted to Mascot (Matrix Science) for peptide identification. Peptides included in the HDX analysis peptide set had a Mascot score greater than 20, and the MS/MS spectra were verified by manual inspection. The Mascot search was repeated against a decoy (reverse) sequence, and ambiguous identifications were ruled out and not included in the HDX peptide set.
HDX-MS analysis-Protein (10 M) was incubated with the respective ligands at a 1:10 protein:ligand molar ratio for 1 h at room temperature. Next, 5 l of sample was diluted into 20 l of D 2 O buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM DTT) and incubated for various time points (0, 10, 60, 300, 900, and 3,600s) at 4°C. The deuterium exchange was then slowed by mixing with 25 l of cold (4°C) 3 M urea, 1% TFA. Quenched samples were immediately injected into the HDX platform. Upon injection, samples were passed through an immobilized pepsin column (2 mm ϫ 2 cm) at 200 l min Ϫ1 , and the digested peptides were captured on a 2-mm ϫ 1-cm C 8 trap column (Agilent) and desalted. Peptides were separated across a 2.1-mm ϫ 5-cm C 18 column (1.9 l of Hypersil Gold, Thermo Fisher) with a linear gradient of 4 -40% CH 3 CN, 0.3% formic acid over 5 min. Sample handling, protein digestion, and peptide separation were conducted at 4°C. Mass spectrometric data were acquired using an Orbitrap mass spectrometer (Exactive, Thermo Fisher). HDX analyses were performed in triplicate with single preparations of each protein-ligand complex. The intensity weighted mean m/z centroid value of each peptide envelope was calculated and subsequently converted into a percentage of deuterium incorporation. This was accomplished by determining the observed averages of the undeuterated and fully deuterated spectra using the conventional formula described elsewhere (45). Statistical significance for the differential HDX data was determined by an unpaired t test for each time point, a procedure that is integrated into the HDX Workbench software (44). Corrections for back-exchange were made on the basis of an estimated 70% deuterium recovery and accounting for the known 80% deuterium content of the deuterium exchange buffer.
Data rendering-Deuterium uptake for each peptide was calculated as the average of %D for all on-exchange time points, and the difference in average %D values between the apo and ligand-bound samples is presented as a heat map with a color code given at the bottom of each figure (warm colors for deprotection and cool colors for protection). Peptides are colored by the software automatically to display significant differences, determined either by a Ͼ5% difference (less or more protection) in average deuterium uptake between the two states or by using the results of unpaired t tests at each time point (p value Ͻ0.05 for any two time points or a p value Ͻ0.01 for any single time point). Peptides with nonsignificant changes between the two states are colored gray. The exchange at the first two residues for any given peptide is not colored. Each peptide bar in the heat map view displays the average ⌬%D values, associated standard deviation, and the charge state.