AMP-activated protein kinase activator A-769662 is an inhibitor of the Na+-K+-ATPase
ABSTRACT Muscle contraction and metabolic stress are potent activators of AMP-activated protein kinase (AMPK). AMPK restores energy balance by activating processes that produce energy while inhibiting those that consume energy. The role of AMPK in the regulation of active ion transport is unclear. Our aim was to determine the effect of the AMPK activator A-769662 on Na(+)-K(+)-ATPase function in skeletal muscle cells. Short-term incubation of differentiated rat L6 myotubes with 100 microM A-769662 increased AMPK and acetyl-CoA carboxylase (ACC) phosphorylation in parallel with decreased Na(+)-K(+)-ATPase alpha(1)-subunit abundance at the plasma membrane and ouabain-sensitive (86)Rb(+) uptake. Notably, the effect of A-769662 on Na(+)-K(+)-ATPase was similar in muscle cells that do not express AMPK alpha(1)- and alpha(2)-catalytic subunits. A-769662 directly inhibits the alpha(1)-isoform of the Na(+)-K(+)-ATPase, purified from rat and human kidney cells in vitro with IC(50) 57 microM and 220 microM, respectively. Inhibition of the Na(+)-K(+)-ATPase by 100 microM ouabain decreases sodium pump activity and cell surface abundance, similar to the effect of A-769662, without affecting AMPK and ACC phosphorylation. In conclusion, the AMPK activator A-769662 inhibits Na(+)-K(+)-ATPase activity and decreases the sodium pump cell surface abundance in L6 skeletal muscle cells. The effect of A-769662 on sodium pump is due to direct inhibition of the Na(+)-K(+)-ATPase activity, rather than AMPK activation. This AMPK-independent effect on Na(+)-K(+)-ATPase calls into question the use of A-769662 as a specific AMPK activator for metabolic studies.
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- "Pharmacologic studies have also shown that A-769662 has good specificity with respect to other protein kinases  . There is limited evidence to suggest the possibility of off-target effects, including inhibition of proteasomal  and sodium potassium ATPase  activities. In this regard, our observation that A- 769662 had no effect in AMPK-inactivated KD hearts is a critical finding and points to the AMPK-dependence of this agent. "
ABSTRACT: AMP-activated protein kinase (AMPK) is a stress signaling enzyme that orchestrates the regulation of energy-generating and -consuming pathways. Intrinsic AMPK activation protects the heart against ischemic injury and apoptosis, but whether pharmacologic AMPK stimulation mitigates ischemia-reperfusion damage is unknown. The aims of this study were to determine whether direct stimulation of AMPK using a small molecule activator, A-769662, attenuates myocardial ischemia-reperfusion injury and to examine its cardioprotective mechanisms. Isolated mouse hearts pre-treated with A-769662 had better recovery of left ventricular contractile function (55% vs. 29% of baseline rate-pressure product; p=0.03) and less myocardial necrosis (56% reduction in infarct size; p<0.01) during post-ischemic reperfusion compared to control hearts. Pre-treatment with A-769662 in vivo attenuated infarct size in C57Bl/6 mice undergoing left coronary artery occlusion and reperfusion compared to vehicle (36% vs. 18%, p=0.025). Mouse hearts with genetically inactivated AMPK were not protected by A-769662, indicating the specificity of this compound. Pre-treatment with A-769662 increased the phosphorylation and inactivation of eukaryotic elongation factor 2 (eEF2), preserved energy charge during ischemia, delayed the development of ischemic contracture, and reduced myocardial apoptosis and necrosis. A-769662 also augmented endothelial nitric oxide synthase (eNOS) activation during ischemia, which partially attenuated myocardial stunning, but did not prevent necrosis. AMPK is a therapeutic target that can be stimulated by a direct-acting small molecule in order to prevent injury during ischemia-reperfusion. The use of AMPK activators may represent a novel strategy to protect the heart and other solid organs against ischemia.Journal of Molecular and Cellular Cardiology 03/2011; 51(1):24-32. DOI:10.1016/j.yjmcc.2011.03.003 · 5.22 Impact Factor
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ABSTRACT: Tuberculosis (Tb) continues to be one of the world's greatest challenges in the public health arena. The current treatment for Tb entails a long duration of therapy making adherence to the whole course difficult. This has given rise to drug resistant strains of Mycobacterium tuberculosis which are posing a significant threat to Tb control strategies. To counteract this problem, there is an urgent need to develop alternative anti-tuberculous drugs which target processes that are critical for the growth and/or survival of this microbe. To identify such targets in M. tuberculosis, I used comparative genomics and mutagenesis data to identify conserved essential genes as viable targets for the development of broad-spectrum antibiotics. In addition, I validated the essentiality of three cell division genes in Mycobacterium smegmatis using conditional antisense RNA expression under different culture conditions. Furthermore, I performed high-throughput screens (HTS) using a differential susceptibility assay against one of the validated targets to identify its cognate inhibitor(s). Lastly, I developed a novel biochemical assay of the target to validate the specificity of the inhibitors identified in the HTS and evaluated the potency of the inhibitors against M. tuberculosis. This study identified 261 conserved putative essential genes as broad-spectrum targets. I hypothesized that antisense RNA expression of such genes will lead to its down-regulation and thereby affect the viability of the cells if these genes are essential. I also hypothesized that an essential gene will be required under all culture conditions. One gene, parA, demonstrated that it was essential under various culture conditions. This gene encodes for a protein which contain the conserved Walker A motif thus I theorized that it may posses ATPase activity. The results illustrated that the M. tuberculosis ParA protein possesses ATPase activity. This biochemical activity was used to validate two specific inhibitors of ParA, phenoxybenzamine and octoclothepin, which were identified in the cell-based HTS. Kinetic studies suggest that phenoxybenzamine is a mixed inhibitor while octoclothepin is a competitive inhibitor of ParA. This data is also supported by in silico docking. Both these compounds show low minimum inhibitory concentrations in M. smegmatis under nitrogen starvation conditions. In summary, this thesis illustrates that ParA is a viable target for anti-tubercular drugs. It demonstrates that ParA is an ATPase which has the potential to bind competitive and non-competitive inhibitors that can be exploited to target cell division in M. tuberculosis. Finally, this study presents phenoxybenzamine and octoclothepin as inhibitors of ParA. In conclusion, these compounds can either be developed to increase potency or be used as reference structures to screen for more potent inhibitors of the enzyme.