AMP-activated protein kinase can be allosterically activated by ADP but AMP remains the key activating ligand.

The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status. When activated by increases in ADP:ATP and/or AMP:ATP ratios (signalling energy deficit), AMPK acts to restore energy balance. Binding of AMP to one or more of three CBS repeats (CBS1, CBS3, CBS4) on the AMPK-γ subunit activates the kinase complex by three complementary mechanisms: (i) promoting α-subunit Thr172 phosphorylation by the upstream kinase LKB1; (ii) protecting against Thr172 dephosphorylation; (iii) allosteric activation. Surprisingly, binding of ADP has been reported to mimic the first two effects, but not the third. We now show that at physiologically relevant concentrations of Mg.ATP2- (above those used in the standard assay) ADP binding does cause allosteric activation. However, ADP causes only a modest activation because (unlike AMP), at concentrations just above those where activation becomes evident, ADP starts to cause competitive inhibition at the catalytic site. Our results cast doubt on the physiological relevance of the effects of ADP and suggest that AMP is the primary activator in vivo. We have also made mutations to hydrophobic residues involved in binding adenine nucleotides at each of the three γ subunit CBS repeats of the human α2ß2γ1 complex and examined their effects on regulation by AMP and ADP. Mutation of the CBS3 site has the largest effects on all three mechanisms of AMP activation, especially at lower ATP concentrations, while mutation of CBS4 reduces the sensitivity to AMP. All three sites appear to be required for allosteric activation by ADP.

Review of the role of additional treatments including oseltamivir, oral steroids, macrolides, and vitamin supplementation for children with severe pneumonia in low- and middle-income countries.

Background: Pneumonia is a major cause of death in children aged under five years. As children with severe pneumonia have the highest risk of morbidity and mortality, previous studies have evaluated the additional benefit of adjunctive treatments such as oseltamivir, oral steroids, macrolides, and vitamin supplementation that can be added to standard antibiotic management to improve clinical outcomes. The study reviewed the evidence for the role of these additional treatments for children with severe pneumonia in low- and middle-income countries (LMICs). Methods: Four electronic databases were searched for English-language articles between 2000 to 2020. Systematic reviews (SRs) with meta-analyses, comparative cohort studies, and randomised controlled trials (RCTs) from LMICs that reported clinical outcomes for children with severe pneumonia aged between one month to 9 years who received adjunct treatment in addition to standard care were included. Risk of bias of included SRs was assessed using AMSTAR 2, and of individual studies using the Effective Public Health Practice Project (EPHPP) quality assessment tool for quantitative studies. Results: Overall, the search identified 2147 articles, 32 of which were eligible, including 7 SRs and 25 RCTs. These studies evaluated zinc (4 SRs, 17 RCTs), Vitamin D (1 SR, 4 RCTs), Vitamin A (3 SRs, 1 RCT), Vitamin C (1 SR, 2 RCTs) and micronutrients (1 RCT). Most studies reported clinical outcomes of time to improvement, length of stay, and treatment failure (including mortality). No studies of oseltamivir, steroids, or macrolides fulfilling the inclusion criteria were identified. For zinc, pooled analyses from SRs showed no evidence of benefit. Similarly, a Cochrane review and one RCT found that Vitamin A did not improve clinical outcomes. For Vitamin D, an RCT evaluating a single high dose of 100 000 international units (IU) of vitamin D found a reduction in time to improvement, with 38%-40% documented vitamin D deficiency at baseline. However, two other studies of 1000 IU daily did not show any effect, but vitamin D status was not measured. For vitamin C, two studies found a reduction in time to symptom resolution in those with severe disease, with one reporting a shorter length of hospital stay. However, both studies were of weak quality. Most studies excluded malnourished children, and studies which included these children did not report specifically on the effect of micronutrients. Conclusions: This review found that adjunctive zinc and vitamin A, in addition to standard care, does not improve clinical outcomes in children with severe pneumonia in LMICs (strong evidence). However, a reduction in time to symptom resolution was reported with high dose vitamin D supplementation in children with documented vitamin D deficiency (strong evidence from one study) and vitamin C (weak evidence), although further research is needed, especially in underweight children.

Mechanism of Activation of AMPK by Cordycepin.

Cordycepin (3'-deoxyadenosine) is a major bioactive agent in Cordyceps militaris, a fungus used in traditional Chinese medicine. It has been proposed to have many beneficial metabolic effects by activating AMP-activated protein kinase (AMPK), but the mechanism of activation remained uncertain. We report that cordycepin enters cells via adenosine transporters and is converted by cellular metabolism into mono-, di-, and triphosphates, which at high cordycepin concentrations can almost replace cellular adenine nucleotides. AMPK activation by cordycepin in intact cells correlates with the content of cordycepin monophosphate and not other cordycepin or adenine nucleotides. Genetic knockout of AMPK sensitizes cells to the cytotoxic effects of cordycepin. In cell-free assays, cordycepin monophosphate mimics all three effects of AMP on AMPK, while activation in cells is blocked by a γ-subunit mutation that prevents activation by AMP. Thus, cordycepin is a pro-drug that activates AMPK by being converted by cellular metabolism into the AMP analog cordycepin monophosphate.