Aldehyde-mediated inhibition of asparagine biosynthesis has implications for diabetes and alcoholism

Patients with alcoholism and type 2 diabetes manifest altered metabolism, including elevated aldehyde levels and unusually low asparagine levels. We show that asparagine synthetase B (ASNS), the only human asparagine-forming enzyme, is inhibited by disease-relevant reactive aldehydes, including formaldehyde and acetaldehyde. Cellular studies show non-cytotoxic amounts of reactive aldehydes induce a decrease in asparagine levels. Biochemical analyses reveal inhibition results from reaction of the aldehydes with the catalytically important N-terminal cysteine of ASNS. The combined cellular and biochemical results suggest a possible mechanism underlying the low asparagine levels in alcoholism and diabetes. The results will stimulate research on the biological consequences of the reactions of aldehydes with nucleophilic residues

Altered glycolysis triggers impaired mitochondrial metabolism and mTORC1 activation in diabetic β-cells

Chronic hyperglycaemia causes a dramatic decrease in mitochondrial metabolism and insulin content in pancreatic β-cells. This underlies the progressive decline in β-cell function in diabetes. However, the molecular mechanisms by which hyperglycaemia produces these effects remain unresolved. Using isolated islets and INS-1 cells, we show here that one or more glycolytic metabolites downstream of phosphofructokinase and upstream of GAPDH mediates the effects of chronic hyperglycemia. This metabolite stimulates marked upregulation of mTORC1 and concomitant downregulation of AMPK. Increased mTORC1 activity causes inhibition of pyruvate dehydrogenase which reduces pyruvate entry into the tricarboxylic acid cycle and partially accounts for the hyperglycaemia-induced reduction in oxidative phosphorylation and insulin secretion. In addition, hyperglycaemia (or diabetes) dramatically inhibits GAPDH activity, thereby impairing glucose metabolism. Our data also reveal that restricting glucose metabolism during hyperglycaemia prevents these changes and thus may be of therapeutic benefit. In summary, we have identified a pathway by which chronic hyperglycaemia reduces β-cell function

Repurposing rapid diagnostic tests to detect falsified vaccines in supply chains

Substandard (including degraded) and falsified (SF) vaccines are a relatively neglected issue with serious global implications for public health. This has been highlighted during the rapid and widespread rollout of COVID-19 vaccines. There has been increasing interest in devices to screen for SF non-vaccine medicines including tablets and capsules to empower inspectors and standardise surveillance. However, there has been very limited published research focussed on repurposing or developing new devices for screening for SF vaccines. To our knowledge, rapid diagnostic tests (RDTs) have not been used for this purpose and have important potential for detecting falsified vaccines. We performed a proof-in-principle study to investigate their diagnostic accuracy using a diverse range of RDT-vaccine/falsified vaccine surrogate pairs. In an initial assessment, we demonstrated the utility of four RDTs in detecting seven vaccines. Subsequently, the four RDTs were evaluated by three blinded assessors with seven vaccines and four falsified vaccines surrogates. The results provide preliminary data that RDTs could be used by multiple international organisations, national medicines regulators and vaccine manufacturers/distributors to screen for falsified vaccines in supply chains, aligned with the WHO global ‘Prevent, Detect and Respond’ strategy

Tibetan PHD2, an allele with loss-of-function properties

Tibetans have adapted to the chronic hypoxia of high altitude and display a distinctive suite of physiologic adaptations, including augmented hypoxic ventilatory response and resistance to pulmonary hypertension. Genome-wide studies have consistently identified compelling genetic signatures of natural selection in two genes of the Hypoxia Inducible Factor pathway, PHD2 and HIF2A. The product of the former induces the degradation of the product of the latter. Key issues regarding Tibetan PHD2 are whether it is a gain-of-function or loss-of-function allele, and how it might contribute to high-altitude adaptation. Tibetan PHD2 possesses two amino acid changes, D4E and C127S. We previously showed that in vitro, Tibetan PHD2 is defective in its interaction with p23, a cochaperone of the HSP90 pathway, and we proposed that Tibetan PHD2 is a loss-of-function allele. Here, we report that additional PHD2 mutations at or near Asp-4 or Cys-127 impair interaction with p23 in vitro. We find that mice with the Tibetan Phd2 allele display augmented hypoxic ventilatory response, supporting this loss-of-function proposal. This is phenocopied by mice with a mutation in p23 that abrogates the PHD2:p23 interaction. Hif2a haploinsufficiency, but not the Tibetan Phd2 allele, ameliorates hypoxia-induced increases in right ventricular systolic pressure. The Tibetan Phd2 allele is not associated with hemoglobin levels in mice. We propose that Tibetans possess genetic alterations that both activate and inhibit selective outputs of the HIF pathway to facilitate successful adaptation to the chronic hypoxia of high altitude

Innovative method for rapid detection of falsified COVID-19 vaccines through unopened vials using handheld Spatially Offset Raman Spectroscopy (SORS)

Preventing, detecting, and responding to substandard and falsified vaccines is of critical importance for ensuring the safety, efficacy, and public trust in vaccines. This is of heightened importance in context of public health crisis, such as the COVID-19 pandemic, in which extreme world-wide shortages of vaccines provided a fertile ground for exploitation by falsifiers. Here, a proof-of-concept study explored the feasibility of using a handheld Spatially Offset Raman Spectroscopy (SORS) device to authenticate COVID-19 vaccines through rapid analysis of unopened vaccine vials. The results show that SORS can verify the chemical identity of dominant excipients non-invasively through vaccine vial walls. The ability of SORS to identify potentially falsified COVID-19 vaccines was demonstrated by measurement of surrogates for falsified vaccines contained in vaccine vials. In all cases studied, the SORS technique was able to differentiate between surrogate samples from the genuine COVISHIELD™ vaccine. The genuine vaccines tested included samples from six batches across two manufacturing sites to account for any potential variations between batches or manufacturing sites. Batch and manufacturing site variations were insignificant. In conjunction with existing security features, for example on labels and packaging, SORS provided an intrinsic molecular fingerprint of the dominant excipients of the vaccines. The technique could be extended to other COVID-19 and non-COVID-19 vaccines, as well as other liquid medicines. As handheld and portable SORS devices are commercially available and widely used for other purposes, such as airport security, they are rapidly deployable non-invasive screening tools for vaccine authentication.</p

Towards the Synthesis and Delivery of Halogenated MCT1 Substrates

International audienc

Towards the Synthesis and Delivery of Halogenated MCT1 Substrates

International audienc

Roles of metal ions in the selective inhibition of oncogenic variants of isocitrate dehydrogenase 1

Cancer linked isocitrate dehydrogenase (IDH) 1 variants, notably R132H IDH1, manifest a ‘gain-of-function’ to reduce 2-oxoglutarate to 2-hydroxyglutarate. High-throughput screens have enabled clinically useful R132H IDH1 inhibitors, mostly allosteric binders at the dimer interface. We report investigations on roles of divalent metal ions in IDH substrate and inhibitor binding that rationalise this observation. Mg2+/Mn2+ ions enhance substrate binding to wt IDH1 and R132H IDH1, but with the former manifesting lower Mg2+/Mn2+ KMs. The isocitrate-Mg2+ complex is the preferred wt IDH1 substrate; with R132H IDH1, separate and weaker binding of 2-oxoglutarate and Mg2+ is preferred. Binding of R132H IDH1 inhibitors at the dimer interface weakens binding of active site Mg2+ complexes; their potency is affected by the Mg2+ concentration. Inhibitor selectivity for R132H IDH1 over wt IDH1 substantially arises from different stabilities of wt and R132H IDH1 substrate-Mg2+ complexes. The results reveal the importance of substrate-metal ion complexes in wt and R132H IDH1 catalysis and the basis for selective R132H IDH1 inhibition. Further studies on roles of metal ion complexes in TCA cycle and related metabolism, including from an evolutionary perspective, are of interest

Roles of metal ions in the selective inhibition of oncogenic variants of isocitrate dehydrogenase 1

Cancer linked isocitrate dehydrogenase (IDH) 1 variants, notably R132H IDH1, manifest a ‘gain-of-function’ to reduce 2-oxoglutarate to 2-hydroxyglutarate. High-throughput screens have enabled clinically useful R132H IDH1 inhibitors, mostly allosteric binders at the dimer interface. We report investigations on roles of divalent metal ions in IDH substrate and inhibitor binding that rationalise this observation. Mg2+/Mn2+ ions enhance substrate binding to wt IDH1 and R132H IDH1, but with the former manifesting lower Mg2+/Mn2+ KMs. The isocitrate-Mg2+ complex is the preferred wt IDH1 substrate; with R132H IDH1, separate and weaker binding of 2-oxoglutarate and Mg2+ is preferred. Binding of R132H IDH1 inhibitors at the dimer interface weakens binding of active site Mg2+ complexes; their potency is affected by the Mg2+ concentration. Inhibitor selectivity for R132H IDH1 over wt IDH1 substantially arises from different stabilities of wt and R132H IDH1 substrate-Mg2+ complexes. The results reveal the importance of substrate-metal ion complexes in wt and R132H IDH1 catalysis and the basis for selective R132H IDH1 inhibition. Further studies on roles of metal ion complexes in TCA cycle and related metabolism, including from an evolutionary perspective, are of interest