Chemistry & Biology
Previews et al. (2014) examined the role of individual CBS domains within the g subunit. When g subunit residues Asp90 (site 1), Asp245 (site 3), and Asp317 (site 4) were individually exchanged with Ala, synergistic activation induced by the combined action of A-769662 and AMP was severely blunted. Collectively, Scott et al. (2014) report how AMPK can be activated via a purely allosteric mechanism, bypassing the requirement of a subunit Thr172 and b subunit Ser108 phosphorylation. These findings are highly relevant for the development of specific directly-acting AMPK agonists and the future direction of AMPK-based therapy in disease-focused research. This study is also critical to our understanding of how to target AMPK pharmacologically, depending on the cellular context (e.g., genetic loss of upstream kinase and expression of b1- and b2-subunits). There have been two recent reports evaluating the functional effects of A-769662 in combination with AICAR or indirect AMPK activators (metformin, phenformin, oligomycin, and hypoxia) acting via the elevation of cellular AMP levels. Consistent with enhanced AMPK activation by co-treatment, inhibition of hepatic lipogenesis (Ducommun et al., 2014) and activation of cardiac glucose
transport (Timmermans et al., 2014) were greatly improved compared with A-769662 alone. These results reinforce the view that combinatorial treatments would be of value to enhance AMPK activation. In addition, such treatments could help to reduce the amount of drugs administrated to patients and better balance tolerability and efficacy. Future research will have to elucidate the beneficial effects of metformin in combination with various AMPK activators targeting the A-769662 binding site for patients suffering with type 2 diabetes, insulin resistance, cardiovascular diseases, and also cancer.
REFERENCES Chen, L., Wang, J., Zhang, Y.Y., Yan, S.F., Neumann, D., Schlattner, U., Wang, Z.X., and Wu, J.W. (2012). Nat. Struct. Mol. Biol. 19, 716–718. Chen, L., Xin, F.J., Wang, J., Hu, J., Zhang, Y.Y., Wan, S., Cao, L.S., Lu, C., Li, P., Yan, S.F., et al. (2013). Nature 498, E8–E10.
Foretz, M., He´brard, S., Leclerc, J., Zarrinpashneh, E., Soty, M., Mithieux, G., Sakamoto, K., Andreelli, F., and Viollet, B. (2010). J. Clin. Invest. 120, 2355– 2369. Goransson, O., McBride, A., Hawley, S.A., Ross, F.A., Shpiro, N., Foretz, M., Viollet, B., Hardie, D.G., and Sakamoto, K. (2007). J. Biol. Chem. 282, 32549–32560. Oakhill, J.S., Steel, R., Chen, Z.P., Scott, J.W., Ling, N., Tam, S., and Kemp, B.E. (2011). Science 332, 1433–1435. Sanders, M.J., Ali, Z.S., Hegarty, B.D., Heath, R., Snowden, M.A., and Carling, D. (2007). J. Biol. Chem. 282, 32539–32548. Scott, J.W., van Denderen, B.J., Jorgensen, S.B., Honeyman, J.E., Steinberg, G.R., Oakhill, J.S., Iseli, T.J., Koay, A., Gooley, P.R., Stapleton, D., and Kemp, B.E. (2008). Chem. Biol. 15, 1220– 1230. Scott, J.W., Ling, N., Issa, S.M., Dite, T.A., O’Brien, M.T., Chen, Z.P., Galic, S., Langendorf, C.G., Steinberg, G.R., Kemp, B.E., and Oakhill, J.S. (2014). Chem. Biol. 21, this issue, 619–627. Timmermans, A.D., Balteau, M., Gelinas, R., Renguet, E., Ginion, A., de Meester, C., Sakamoto, K., Balligand, J.L., Bontemps, F., Vanoverschelde, J.L., et al. (2014). Am. J. Physiol. Heart Circ. Physiol. http://dx.doi.org/10.1152/ajpheart.00965.2013.
Cool, B., Zinker, B., Chiou, W., Kifle, L., Cao, N., Perham, M., Dickinson, R., Adler, A., Gagne, G., Iyengar, R., et al. (2006). Cell Metab. 3, 403–416.
Xiao, B., Sanders, M.J., Underwood, E., Heath, R., Mayer, F.V., Carmena, D., Jing, C., Walker, P.A., Eccleston, J.F., Haire, L.F., et al. (2011). Nature 472, 230–233.
Ducommun, S., Ford, R.J., Bultot, L., Deak, M., Bertrand, L., Kemp, B.E., Steinberg, G.R., and Sakamoto, K. (2014). Am. J. Physiol. Endocrinol. Metab. 306, E688–E696.
Xiao, B., Sanders, M.J., Carmena, D., Bright, N.J., Haire, L.F., Underwood, E., Patel, B.R., Heath, R.B., Walker, P.A., Hallen, S., et al. (2013). Nat. Commun. 4, 3017.
Modulating Noncatalytic Function with Kinase Inhibitors Michael P. Agius1 and Matthew B. Soellner1,* 1Departments of Medicinal Chemistry & Chemistry, University of Michigan, 930 N. University Avenue, Ann Arbor, MI 48109, USA *Correspondence:
[email protected] http://dx.doi.org/10.1016/j.chembiol.2014.05.005
In this issue of Chemistry & Biology, Hari and colleagues show that conformation-selective ATP-competitive kinase inhibitors have distinct noncatalytic effects on Erk2, including the ability to modulate protein-protein interactions outside the ATP-binding site. These findings enhance our knowledge about the diverse array of activities in which kinase inhibitors can target signaling pathways. Elucidating imatinib’s binding mode with c-Abl prompted the development of myriad kinase inhibitors that stabilize (or bind to) the inactive DFG-out conformation (Figure 1A). It was originally believed that the DFG-out conformation was nonconserved across the kinome and there-
fore explained the selectivity of imatinib. However, recent studies have shown that the inactive DFG-out conformation is not unique and inhibitors that stabilize this conformation are not inherently more selective than inhibitors that bind the active conformation.
Although there are many DFG-out inhibitors (also known as type II kinase inhibitors) known, only recently has there been an appreciation that conformation-selective inhibitors, including DFG-out inhibitors, can uniquely modulate noncatalytic functions of kinases. The first report
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Figure 1. Structural Depiction of Active versus DFG-out Kinase Conformations of c-Abl and p38a (A) Superimposed structures of c-Abl. The green-colored activation loop is DFG-in, active conformation (PDB: 2GQG). The red-colored activation loop is DFG-out, inactive conformation (PDB: 1IEP). (B) Superimposed structures of p38a. The green-colored activation loop is DFG-in, active conformation (PDB: 1ZZL). The red-colored activation loop is DFG-out, inactive conformation (PDB: 2BAJ). In the stick depiction is Phe-169 of the DFG motif and Thr-180, which is recognized by MKK6 in the active conformation. The distance Thr-180 moves in the active versus inactive conformation is 13.9 A˚.
showing differential activities between inhibitors that are compatible with an active ATP-binding site conformation and DFGout inhibitors was in 2005, wherein Sullivan and coworkers described the unique ability of DFG-out inhibitors to prevent phosphorylation of inhibitor-bound p38a by MKK6 (Sullivan et al., 2005). More recently, several groups have reported conformation-selective modulation of noncatalytic kinase activity. These studies have yielded
a diverse array of kinases that are differentially effected by conformation-selective inhibitors, including the ability to modulate the ribonuclease activity of Ire1a selectively with DFG-out inhibitors (Wang et al., 2012) and the ability of conformation-selective kinase inhibitors to perturb noncatalytic interactions of c-Src (Krishnamurty et al., 2013). In this issue of Chemistry & Biology by Hari et al. (2014), conformation-selective
Figure 2. Structural Depiction of aC-Helix in Active versus aC-Helix out Inactive Structures of Cdk2 and Erk2 (A) Superimposed structures of Cdk2 kinase domain in the presence and absence of bound cyclin A. When cyclin A is bound to Cdk2, Cdk2 is in the active aC-helix in conformation (aC-helix, green; PDB: 1OKV). For clarity, cyclin A is hidden. In the absence of cyclin A, Cdk2 is in the inactive aC-helix out conformation (aC-helix, red; PDB: 1PW2). (B) Superimposed structures of Erk2 bound to putative aC-helix out inhibitor (PDB: 4N4S) and Cdk2 bound to cyclin A (PDB: 1OKV). The L16 segment of Erk2 (dark red) occupies a similar binding side that cyclin A (pink) occupies when bound to Cdk2. Both structural features aid in ‘‘locking’’ the respective kinase in the aC-helix in, active conformation.
570 Chemistry & Biology 21, May 22, 2014 ª2014 Elsevier Ltd All rights reserved
inhibitors of Erk2 are shown to have differential functions depending on the kinase conformation stabilized. The study of Erk2 is significant because, unlike most previous examples, Erk2 does not have regulatory domains, which is a common site of noncatalytic activity. In addition, a substantial number of noncatalytic functions for the kinase domain of Erk2 have been reported (Rodrı´guez and Crespo 2011). Using detailed biochemical and cellular experiments, the authors show that DFG-out inhibitors can uniquely (compared to active-conformation inhibitors) reduce Erk2 phosphorylation by MEK2. In addition, the authors demonstrate that targeting the inactive DFG-out conformation modulated Erk20 s ability to allosterically activate DUSP6 phosphatase. Both active and DFG-out stabilizing inhibitors are able to inhibit the catalytic activity of Erk2, but only DFG-out stabilizing inhibitors have the allosteric effects on Erk2. This hypothesis is due to the large change in conformation of the kinase activation loop (A-loop), which is a known consequence of a kinase in the DFG-out conformation. This hypothesis is consistent with studies demonstrating that MKK6 is unable to phosphorylate Thr180 on p38a when p38a is bound by DFG-out inhibitors (Figure 1B). A second inactive conformation that is frequently found in both apo and ligandbound kinases is called the aC-helix out conformation. Maly and coworkers have previously shown that ATP-competitive inhibitors of c-Src kinase that stabilize c-Src in the aC-helix out conformation (also referred to as the Src/Cdk-like inactive conformation) lead to a closing of the kinase that reduces accessibility to c-Src SH3 domain (Krishnamurty et al., 2013). This prior work demonstrates that, like DFG-out inhibitors, compounds that stabilize the aC-helix out conformation can modulate noncatalytic activity of kinases. In this report, Hari and colleagues identify a potent inhibitor of Erk2 that has structural features similar to known aC-helix out inhibitors of c-Src. Despite predicting that the Erk2 inhibitor would stabilize the aC-helix out conformation, no modulation of noncatalytic activity was observed. Upon structural analysis, the designed aC-helix out inhibitor of Erk2 does not cause outward rotation of the aC-helix, a classic feature of this inactive conformation (Figure 2).
Chemistry & Biology
Previews The inability of Erk2 to adopt the canonical aC-helix out conformation is likely due to the presence of a unique insertion domain found in MAPKs (L16 segment) that can prevent movement of the aC-helix. The L16 segment is found to occupy similar space as cyclin A binding of Cdk2, a binding event that prevents Cdk2 from adopting the aC-helix out conformation (Schulze-Gahmen et al., 1996) (Figure 2). Similar to the ability of Syk kinase to bind DFG-out ligands in an active conformation (e.g., imatinib), these results highlight the importance of structurally characterizing conformationally selective inhibitors with their target kinase. This work, together with previous reports, highlights the large range of allo-
steric effects that some kinase inhibitors can modulate. These allosteric effects are neglected in most reports of kinase inhibitor development; however, the ability of select inhibitors to modulate noncatalytic function can have profound pharmacological consequences when applying these kinase inhibitors to cellular systems. A picture is emerging that DFG-out inhibitors (and some aC-helix out inhibitors) have the ability to disrupt noncatalytic kinase function, while active conformation inhibitors do not. These new findings increase the complexity of predicting outcomes for kinase inhibitors, but, nevertheless, studies similar to this work are essential to reveal the vast array of activities that can emerge from kinase inhibitors.
REFERENCES Hari, S.B., Merritt, E.A., and Maly, D.J. (2014). Chem Biol. 21, this issue, 628–635. Krishnamurty, R., Brigham, J.L., Leonard, S.E., Ranjitkar, P., Larson, E.T., Dale, E.J., Merritt, E.A., and Maly, D.J. (2013). Nat. Chem. Biol. 9, 43–50. Rodrı´guez, J., and Crespo, P. (2011). Sci. Signal. 4, re3. Schulze-Gahmen, U., De Bondt, H.L., and Kim, S.H. (1996). J. Med. Chem. 39, 4540–4546. Sullivan, J.E., Holdgate, G.A., Campbell, D., Timms, D., Gerhardt, S., Breed, J., Breeze, A.L., Bermingham, A., Pauptit, R.A., Norman, R.A., et al. (2005). Biochemistry 44, 16475–16490. Wang, L., Perera, B.G., Hari, S.B., Bhhatarai, B., Backes, B.J., Seeliger, M.A., Schu¨rer, S.C., Oakes, S.A., Papa, F.R., and Maly, D.J. (2012). Nat. Chem. Biol. 8, 982–989.
Polarity Factors Play a Role in Antibiotic Resistance Aretha Fiebig1,* 1Department of Biochemistry and Molecular Biology, 929 E. 57th Street, University of Chicago, Chicago, IL 60637, USA *Correspondence:
[email protected] http://dx.doi.org/10.1016/j.chembiol.2014.05.001
In this issue of Chemistry & Biology, Kirkpatrick and Viollier describe a new twist in the relationship between bacterial cell development and antibiotic resistance. They reveal that TipN, which orchestrates development at cell poles, is required to tolerate induced expression of an antibiotic efflux pump. The utility of antibiotics as therapeutics lies in their selective inhibition of prokaryotic pathways. With the increasing prevalence of antibiotic resistance, identification of novel prokaryotic targets for the development of new classes of antibiotics is essential. The article by Kirkpatrick and Viollier (2014) in this issue of Chemistry & Biology suggests that proteins involved in bacterial cell polarity determination could serve as targets for combination therapeutic agents that enhance the efficacy of current antibiotics, even in strains that are resistant. The model a-proteobacterium Caulobacter crescentus is resistant to the quinolone antibiotic Nalidixic acid (Nal) owing to a polymorphism in DNA gyrase, the target of Nal. Kirkpatrick and Viollier (2014) show that disrup-
tion of a cell polarity factor, TipN, which marks the new cell pole and the site of flagellar assembly (Huitema et al., 2006; Lam et al., 2006), sensitizes Caulobacter to Nal and restores antibiotic toxicity by a novel mechanism. Bacterial resistance to antibiotics typically arises by one of three mechanisms: (1) alteration of the drug target such that the drug can no longer bind, (2) modification or degradation of the drug itself, or (3) acquisition or enhanced expression of efflux pumps that expel the drug from the cell. The first two mechanisms can confer resistance to one or a few very closely related antibiotics. Efflux systems, on the other hand, can transport a large repertoire of chemically unrelated molecules. Thus induction of
such systems presents a mechanism by which bacteria acquire resistance to broad groups of antibiotics (Putman et al., 2000). The resistance-nodulation-division (RND) transporters represent an important family of efflux systems that are capable of transferring molecules directly from the cytoplasm or the periplasm to the exterior of the cell. These tripartite systems, typified by AcrAB-TolC, have components in the inner membrane (AcrB), the periplasm (AcrA), and the outer membrane (TolC) that together form a continuous vehicle to export molecules from the cell (Fernando and Kumar, 2013). Expression of these systems is often regulated by a TetR-like transcriptional repressor that controls transcription
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