SGC-CAMKK2-1: A Chemical Probe for CAMKK2.

The serine/threonine protein kinase calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) plays critical roles in a range of biological processes. Despite its importance, only a handful of inhibitors of CAMKK2 have been disclosed. Having a selective small molecule tool to interrogate this kinase will help demonstrate that CAMKK2 inhibition can be therapeutically beneficial. Herein, we disclose SGC-CAMKK2-1, a selective chemical probe that targets CAMKK2.

A novel small molecule inhibitor of human Drp1.

Mitochondrial dynamin-related protein 1 (Drp1) is a large GTPase regulator of mitochondrial dynamics and is known to play an important role in numerous pathophysiological processes. Despite being the most widely used Drp1 inhibitor, the specificity of Mdivi-1 towards human Drp1 has not been definitively proven and there have been numerous issues reported with its use including off-target effects. In our hands Mdivi-1 showed varying binding affinities toward human Drp1, potentially impacted by compound aggregation. Herein, we sought to identify a novel small molecule inhibitor of Drp1. From an initial virtual screening, we identified DRP1i27 as a compound which directly bound to the human isoform 3 of Drp1 via surface plasmon resonance and microscale thermophoresis. Importantly, DRP1i27 was found to have a dose-dependent increase in the cellular networks of fused mitochondria but had no effect in Drp1 knock-out cells. Further analogues of this compound were identified and screened, though none displayed greater affinity to human Drp1 isoform 3 than DRP1i27. To date, this is the first small molecule inhibitor shown to directly bind to human Drp1.

Structure-function analysis of the AMPK activator SC4 and identification of a potent pan AMPK activator.

The AMP-activated protein kinase (AMPK) αßγ heterotrimer is a primary cellular energy sensor and central regulator of energy homeostasis. Activating skeletal muscle AMPK with small molecule drugs improves glucose uptake and provides an opportunity for new strategies to treat type 2 diabetes and insulin resistance, with recent genetic and pharmacological studies indicating the α2ß2γ1 isoform combination as the heterotrimer complex primarily responsible. With the goal of developing α2ß2-specific activators, here we perform structure/function analysis of the 2-hydroxybiphenyl group of SC4, an activator with tendency for α2-selectivity that is also capable of potently activating ß2 complexes. Substitution of the LHS 2-hydroxyphenyl group with polar-substituted cyclohexene-based probes resulted in two AMPK agonists, MSG010 and MSG011, which did not display α2-selectivity when screened against a panel of AMPK complexes. By radiolabel kinase assay, MSG010 and MSG011 activated α2ß2γ1 AMPK with one order of magnitude greater potency than the pan AMPK activator MK-8722. A crystal structure of MSG011 complexed to AMPK α2ß1γ1 revealed a similar binding mode to SC4 and the potential importance of an interaction between the SC4 2-hydroxyl group and α2-Lys31 for directing α2-selectivity. MSG011 induced robust AMPK signalling in mouse primary hepatocytes and commonly used cell lines, and in most cases this occurred in the absence of changes in phosphorylation of the kinase activation loop residue α-Thr172, a classical marker of AMP-induced AMPK activity. These findings will guide future design of α2ß2-selective AMPK activators, that we hypothesise may avoid off-target complications associated with indiscriminate activation of AMPK throughout the body.

Hinge Binder Scaffold Hopping Identifies Potent Calcium/Calmodulin-Dependent Protein Kinase Kinase 2 (CAMKK2) Inhibitor Chemotypes.

CAMKK2 is a serine/threonine kinase and an activator of AMPK whose dysregulation is linked with multiple diseases. Unfortunately, STO-609, the tool inhibitor commonly used to probe CAMKK2 signaling, has limitations. To identify promising scaffolds as starting points for the development of high-quality CAMKK2 chemical probes, we utilized a hinge-binding scaffold hopping strategy to design new CAMKK2 inhibitors. Starting from the potent but promiscuous disubstituted 7-azaindole GSK650934, a total of 32 compounds, composed of single-ring, 5,6-, and 6,6-fused heteroaromatic cores, were synthesized. The compound set was specifically designed to probe interactions with the kinase hinge-binding residues. Compared to GSK650394 and STO-609, 13 compounds displayed similar or better CAMKK2 inhibitory potency in vitro, while compounds 13g and 45 had improved selectivity for CAMKK2 across the kinome. Our systematic survey of hinge-binding chemotypes identified several potent and selective inhibitors of CAMKK2 to serve as starting points for medicinal chemistry programs.

Post-Translational Modifications of the Energy Guardian AMP-Activated Protein Kinase.

Physical exercise elicits physiological metabolic perturbations such as energetic and oxidative stress; however, a diverse range of cellular processes are stimulated in response to combat these challenges and maintain cellular energy homeostasis. AMP-activated protein kinase (AMPK) is a highly conserved enzyme that acts as a metabolic fuel sensor and is central to this adaptive response to exercise. The complexity of AMPK's role in modulating a range of cellular signalling cascades is well documented, yet aside from its well-characterised regulation by activation loop phosphorylation, AMPK is further subject to a multitude of additional regulatory stimuli. Therefore, in this review we comprehensively outline current knowledge around the post-translational modifications of AMPK, including novel phosphorylation sites, as well as underappreciated roles for ubiquitination, sumoylation, acetylation, methylation and oxidation. We provide insight into the physiological ramifications of these AMPK modifications, which not only affect its activity, but also subcellular localisation, nutrient interactions and protein stability. Lastly, we highlight the current knowledge gaps in this area of AMPK research and provide perspectives on how the field can apply greater rigour to the characterisation of novel AMPK regulatory modifications.

Protein kinase A negatively regulates VEGF-induced AMPK activation by phosphorylating CaMKK2 at serine 495.

Activation of AMP-activated protein kinase (AMPK) in endothelial cells by vascular endothelial growth factor (VEGF) via the Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) represents a pro-angiogenic pathway, whose regulation and function is incompletely understood. This study investigates whether the VEGF/AMPK pathway is regulated by cAMP-mediated signalling. We show that cAMP elevation in endothelial cells by forskolin, an activator of the adenylate cyclase, and/or 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterases, triggers protein kinase A (PKA)-mediated phosphorylation of CaMKK2 (serine residues S495, S511) and AMPK (S487). Phosphorylation of CaMKK2 by PKA led to an inhibition of its activity as measured in CaMKK2 immunoprecipitates of forskolin/IBMX-treated cells. This inhibition was linked to phosphorylation of S495, since it was not seen in cells expressing a non-phosphorylatable CaMKK2 S495C mutant. Phosphorylation of S511 alone in these cells was not able to inhibit CaMKK2 activity. Moreover, phosphorylation of AMPK at S487 was not sufficient to inhibit VEGF-induced AMPK activation in cells, in which PKA-mediated CaMKK2 inhibition was prevented by expression of the CaMKK2 S495C mutant. cAMP elevation in endothelial cells reduced basal and VEGF-induced acetyl-CoA carboxylase (ACC) phosphorylation at S79 even if AMPK was not inhibited. Together, this study reveals a novel regulatory mechanism of VEGF-induced AMPK activation by cAMP/PKA, which may explain, in part, inhibitory effects of PKA on angiogenic sprouting and play a role in balancing pro- and anti-angiogenic mechanisms in order to ensure functional angiogenesis.

CaMKK2 is inactivated by cAMP-PKA signaling and 14-3-3 adaptor proteins.

The calcium-calmodulin-dependent protein kinase kinase-2 (CaMKK2) is a key regulator of cellular and whole-body energy metabolism. It is known to be activated by increases in intracellular Ca2+, but the mechanisms by which it is inactivated are less clear. CaMKK2 inhibition protects against prostate cancer, hepatocellular carcinoma, and metabolic derangements induced by a high-fat diet; therefore, elucidating the intracellular mechanisms that inactivate CaMKK2 has important therapeutic implications. Here we show that stimulation of cAMP-dependent protein kinase A (PKA) signaling in cells inactivates CaMKK2 by phosphorylation of three conserved serine residues. PKA-dependent phosphorylation of Ser495 directly impairs calcium-calmodulin activation, whereas phosphorylation of Ser100 and Ser511 mediate recruitment of 14-3-3 adaptor proteins that hold CaMKK2 in the inactivated state by preventing dephosphorylation of phospho-Ser495 We also report the crystal structure of 14-3-3ζ bound to a synthetic diphosphorylated peptide that reveals how the canonical (Ser511) and noncanonical (Ser100) 14-3-3 consensus sites on CaMKK2 cooperate to bind 14-3-3 proteins. Our findings provide detailed molecular insights into how cAMP-PKA signaling inactivates CaMKK2 and reveals a pathway to inhibit CaMKK2 with potential for treating human diseases.

Long-chain fatty acyl-CoA esters regulate metabolism via allosteric control of AMPK ß1 isoforms.

Long-chain fatty acids (LCFAs) play important roles in cellular energy metabolism, acting as both an important energy source and signalling molecules1. LCFA-CoA esters promote their own oxidation by acting as allosteric inhibitors of acetyl-CoA carboxylase, which reduces the production of malonyl-CoA and relieves inhibition of carnitine palmitoyl-transferase 1, thereby promoting LCFA-CoA transport into the mitochondria for ß-oxidation2-6. Here we report a new level of regulation wherein LCFA-CoA esters per se allosterically activate AMP-activated protein kinase (AMPK) ß1-containing isoforms to increase fatty acid oxidation through phosphorylation of acetyl-CoA carboxylase. Activation of AMPK by LCFA-CoA esters requires the allosteric drug and metabolite site formed between the α-subunit kinase domain and the ß-subunit. ß1 subunit mutations that inhibit AMPK activation by the small-molecule activator A769662, which binds to the allosteric drug and metabolite site, also inhibit activation by LCFA-CoAs. Thus, LCFA-CoA metabolites act as direct endogenous AMPK ß1-selective activators and promote LCFA oxidation.

New perspectives on the role of Drp1 isoforms in regulating mitochondrial pathophysiology.

Mitochondria are dynamic organelles constantly undergoing fusion and fission. A concerted balance between the process of mitochondrial fusion and fission is required to maintain cellular health under different physiological conditions. Mutation and dysregulation of Drp1, the major driver of mitochondrial fission, has been associated with various neurological, oncological and cardiovascular disorders. Moreover, when subjected to pathological insults, mitochondria often undergo excessive fission, generating fragmented and dysfunctional mitochondria leading to cell death. Therefore, manipulating mitochondrial fission by targeting Drp1 has been an appealing therapeutic approach for cytoprotection. However, studies have been inconsistent. Studies employing Drp1 constructs representing alternate Drp1 isoforms, have demonstrated differing impacts of these isoforms on mitochondrial fission and cell death. Furthermore, there are distinct expression patterns of Drp1 isoforms in different tissues, suggesting idiosyncratic engagement in specific cellular functions. In this review, we will discuss these inherent variations among human Drp1 isoforms and how they could affect Drp1-mediated mitochondrial fission and cell death.

Functional analysis of an R311C variant of Ca2+ -calmodulin-dependent protein kinase kinase-2 (CaMKK2) found as a de novo mutation in a patient with bipolar disorder.

OBJECTIVES: Loss-of-function mutations in the gene encoding the calcium-calmodulin (Ca2+ -CaM)-dependent protein kinase kinase-2 (CaMKK2) enzyme are linked to bipolar disorder. Recently, a de novo arginine to cysteine (R311C) mutation in CaMKK2 was identified from a whole exome sequencing study of bipolar patients and their unaffected parents. The aim of the present study was to determine the functional consequences of the R311C mutation on CaMKK2 activity and regulation by Ca2+ -CaM. METHODS: The effects of the R311C mutation on CaMKK2 activity and Ca2+ -CaM activation were examined using a radiolabeled adenosine triphosphate (ATP) kinase assay. We performed immunoblot analysis to determine whether the R311C mutation impacts threonine-85 (T85) autophosphorylation, an activating phosphorylation site on CaMKK2 that has also been implicated in bipolar disorder. We also expressed the R311C mutant in CaMKK2 knockout HAP1 cells and used immunoblot analysis and an MTS reduction assay to study its effects on Ca2+ -dependent downstream signaling and cell viability, respectively. RESULTS: The R311C mutation maps to the conserved HRD motif within the catalytic loop of CaMKK2 and caused a marked reduction in kinase activity and Ca2+ -CaM activation. The R311C mutation virtually abolished T85 autophosphorylation in response to Ca2+ -CaM and exerted a dominant-negative effect in cells as it impaired the ability of wild-type CaMKK2 to initiate downstream signaling and maintain cell viability. CONCLUSIONS: The highly disruptive, loss-of-function impact of the de novo R311C mutation in human CaMKK2 provides a compelling functional rationale for being considered a potential rare monogenic cause of bipolar disorder.

mTORC1 directly inhibits AMPK to promote cell proliferation under nutrient stress.

Central to cellular metabolism and cell proliferation are highly conserved signalling pathways controlled by mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK)1,2, dysregulation of which are implicated in pathogenesis of major human diseases such as cancer and type 2 diabetes. AMPK pathways leading to reduced cell proliferation are well established and, in part, act through inhibition of TOR complex-1 (TORC1) activity. Here we demonstrate reciprocal regulation, specifically that TORC1 directly down-regulates AMPK signalling by phosphorylating the evolutionarily conserved residue Ser367 in the fission yeast AMPK catalytic subunit Ssp2, and AMPK α1Ser347/α2Ser345 in the mammalian homologs, which is associated with reduced phosphorylation of activation loop Thr172. Genetic or pharmacological inhibition of TORC1 signalling led to AMPK activation in the absence of increased AMP:ATP ratios; under nutrient stress conditions this was associated with growth limitation in both yeast and human cell cultures. Our findings reveal fundamental, bi-directional regulation between two major metabolic signalling networks and uncover new opportunity for cancer treatment strategies aimed at suppressing cell proliferation in the nutrient-poor tumor microenvironment.

In Depth Analysis of Kinase Cross Screening Data to Identify CAMKK2 Inhibitory Scaffolds.

The calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) activates CAMK1, CAMK4, AMPK, and AKT, leading to numerous physiological responses. The deregulation of CAMKK2 is linked to several diseases, suggesting the utility of CAMKK2 inhibitors for oncological, metabolic and inflammatory indications. In this work, we demonstrate that STO-609, frequently described as a selective inhibitor for CAMKK2, potently inhibits a significant number of other kinases. Through an analysis of literature and public databases, we have identified other potent CAMKK2 inhibitors and verified their activities in differential scanning fluorimetry and enzyme inhibition assays. These inhibitors are potential starting points for the development of selective CAMKK2 inhibitors and will lead to tools that delineate the roles of this kinase in disease biology.

1,2,6-Thiadiazinones as Novel Narrow Spectrum Calcium/Calmodulin-Dependent Protein Kinase Kinase 2 (CaMKK2) Inhibitors.

We demonstrate for the first time that 4H-1,2,6-thiadiazin-4-one (TDZ) can function as a chemotype for the design of ATP-competitive kinase inhibitors. Using insights from a co-crystal structure of a 3,5-bis(arylamino)-4H-1,2,6-thiadiazin-4-one bound to calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2), several analogues were identified with micromolar activity through targeted displacement of bound water molecules in the active site. Since the TDZ analogues showed reduced promiscuity compared to their 2,4-dianilinopyrimidine counter parts, they represent starting points for development of highly selective kinase inhibitors.

Structural Determinants for Small-Molecule Activation of Skeletal Muscle AMPK α2ß2γ1 by the Glucose Importagog SC4.

The AMP-activated protein kinase (AMPK) αßγ heterotrimer regulates cellular energy homeostasis with tissue-specific isoform distribution. Small-molecule activation of skeletal muscle α2ß2 AMPK complexes may prove a valuable treatment strategy for type 2 diabetes and insulin resistance. Herein, we report the small-molecule SC4 is a potent, direct AMPK activator that preferentially activates α2 complexes and stimulates skeletal muscle glucose uptake. In parallel with the term secretagog, we propose "importagog" to define a substance that induces or augments cellular uptake of another substance. Three-dimensional structures of the glucose importagog SC4 bound to activated α2ß2γ1 and α2ß1γ1 complexes reveal binding determinants, in particular a key interaction between the SC4 imidazopyridine 4'-nitrogen and ß2-Asp111, which provide a design paradigm for ß2-AMPK therapeutics. The α2ß2γ1/SC4 structure reveals an interaction between a ß2 N-terminal α helix and the α2 autoinhibitory domain. Our results provide a structure-function guide to accelerate development of potent, but importantly tissue-specific, ß2-AMPK therapeutics.

AMP-activated protein kinase selectively inhibited by the type II inhibitor SBI-0206965.

Inhibition of the metabolic regulator AMP-activated protein kinase (AMPK) is increasingly being investigated for its therapeutic potential in diseases where AMPK hyperactivity results in poor prognoses, as in established cancers and neurodegeneration. However, AMPK-inhibitory tool compounds are largely limited to compound C, which has a poor selectivity profile. Here we identify the pyrimidine derivative SBI-0206965 as a direct AMPK inhibitor. SBI-0206965 inhibits AMPK with 40-fold greater potency and markedly lower kinase promiscuity than compound C and inhibits cellular AMPK signaling. Biochemical characterization reveals that SBI-0206965 is a mixed-type inhibitor. A co-crystal structure of the AMPK kinase domain/SBI-0206965 complex shows that the drug occupies a pocket that partially overlaps the ATP active site in a type IIb inhibitor manner. SBI-0206965 has utility as a tool compound for investigating physiological roles for AMPK and provides fresh impetus to small-molecule AMPK inhibitor therapeutic development.

Visualizing AMPK Drug Binding Sites Through Crystallization of Full-Length Phosphorylated α2ß1γ1 Heterotrimer.

Here, we describe the crystallization protocol for AMPK, including protein production and purification. AMPK can be readily crystallized in the presence of PEG to give diffracting crystals to a resolution of between 2.5 and 3.5 Å using synchrotron radiation. This method allows for visualization of drugs or small molecules that bind to the ADaM site, CBS sites, ATP binding site, and the newly identified C2 binding sites in the γ-subunit via co-crystallization with phosphorylated AMPK (pT172) α2ß1γ1 isoform or α2/1ß1γ1 chimera. Drugs with binding affinities above 500 nM fail to co-crystallize with AMPK using these parameters.

The autophagy initiator ULK1 sensitizes AMPK to allosteric drugs.

AMP-activated protein kinase (AMPK) is a metabolic stress-sensing enzyme responsible for maintaining cellular energy homeostasis. Activation of AMPK by salicylate and the thienopyridone A-769662 is critically dependent on phosphorylation of Ser108 in the ß1 regulatory subunit. Here, we show a possible role for Ser108 phosphorylation in cell cycle regulation and promotion of pro-survival pathways in response to energy stress. We identify the autophagy initiator Unc-51-like kinase 1 (ULK1) as a ß1-Ser108 kinase in cells. Cellular ß1-Ser108 phosphorylation by ULK1 was dependent on AMPK ß-subunit myristoylation, metabolic stress associated with elevated AMP/ATP ratio, and the intrinsic energy sensing capacity of AMPK; features consistent with an AMP-induced myristoyl switch mechanism. We further demonstrate cellular AMPK signaling independent of activation loop Thr172 phosphorylation, providing potential insight into physiological roles for Ser108 phosphorylation. These findings uncover new mechanisms by which AMPK could potentially maintain cellular energy homeostasis independently of Thr172 phosphorylation.AMPK is involved in sensing of metabolic stress. The authors show that the autophagy initiator ULK1 phosphorylates ß1-Ser108 on the regulatory ß1-subunit, sensitizing AMPK to allosteric drugs, and activates signaling pathways that appear independent of Thr172 phosphorylation in the kinase activation loop.

Fake Inhibitors: AMPK Activation Trumps Inhibition.

Protein kinase inhibitors have become increasingly important therapeutic drugs for the treatment of human diseases; however, resistance and off-target effects can limit their use. In this issue of Cell Chemical Biology, Ross et al. (2017) reveal a novel off-target mechanism where the Src kinase inhibitor SU6656 paradoxically primes AMPK for phosphorylation and activation by the upstream kinase LKB1.

Impact of Genetic Variation on Human CaMKK2 Regulation by Ca2+-Calmodulin and Multisite Phosphorylation.

The Ca2+-calmodulin dependent protein kinase kinase-2 (CaMKK2) is a key regulator of neuronal function and whole-body energy metabolism. Elevated CaMKK2 activity is strongly associated with prostate and hepatic cancers, whereas reduced CaMKK2 activity has been linked to schizophrenia and bipolar disease in humans. Here we report the functional effects of nine rare-variant point mutations that were detected in large-scale human genetic studies and cancer tissues, all of which occur close to two regulatory phosphorylation sites and the catalytic site on human CaMKK2. Four mutations (G87R, R139W, R142W and E268K) cause a marked decrease in Ca2+-independent autonomous activity, however S137L and P138S mutants displayed increased autonomous and Ca2+-CaM stimulated activities. Furthermore, the G87R mutant is defective in Thr85-autophosphorylation dependent autonomous activity, whereas the A329T mutation rendered CaMKK2 virtually insensitive to Ca2+-CaM stimulation. The G87R and R139W mutants behave as dominant-negative inhibitors of CaMKK2 signaling in cells as they block phosphorylation of the downstream substrate AMP-activated protein kinase (AMPK) in response to ionomycin. Our study provides insight into functionally disruptive, rare-variant mutations in human CaMKK2, which have the potential to influence risk and burden of disease associated with aberrant CaMKK2 activity in human populations carrying these variants.

Structural basis of allosteric and synergistic activation of AMPK by furan-2-phosphonic derivative C2 binding.

The metabolic stress-sensing enzyme AMP-activated protein kinase (AMPK) is responsible for regulating metabolism in response to energy supply and demand. Drugs that activate AMPK may be useful in the treatment of metabolic diseases including type 2 diabetes. We have determined the crystal structure of AMPK in complex with its activator 5-(5-hydroxyl-isoxazol-3-yl)-furan-2-phosphonic acid (C2), revealing two C2-binding sites in the γ-subunit distinct from nucleotide sites. C2 acts synergistically with the drug A769662 to activate AMPK α1-containing complexes independent of upstream kinases. Our results show that dual drug therapies could be effective AMPK-targeting strategies to treat metabolic diseases.