A high-throughput mass spectrometry based Mpro assay identifies penicillin esters as new SARS-CoV-2 Mpro inhibitors.

Abstract:Efficient photosynthesis depends on balancing the rate of light-driven electron transport occurring in the photosystem I (PSI) and photosystem II (PSII) reaction centers of plant chloroplast thylakoid membranes. Balance is achieved through a process called state transitions which, via redox control of the phosphorylation state of light harvesting antenna complex II (LHCII), increases its energy transfer towards PSI (State II) when PSII is overexcited, and vice versa (State I). In addition to LHCII, PSI is also served by four light harvesting antenna complex I (LHCI) subunits, Lhca1, 2, 3 and 4. Here we demonstrate that despite unchanged levels of LHCII phosphorylation, absence of specific Lhca subunits in the Lhca1, 2, 3 and 4 mutants reduces state transitions in Arabidopsis. The severest phenotype is observed in Lhca4, with a 69% reduction compared to the wild-type. Surprisingly, the amounts of the PSI-LHCI-LHCII supercomplex isolated by native-PAGE from digitonin-solubilized thylakoids were similar in the wild-type and Lhca mutants. Fluorescence excitation spectroscopy revealed that in the wildtype this PSI-LHCI-LHCII supercomplex is supplemented by energy transfer from additional LHCII trimers in State II, whose binding is sensitive to digitonin, and which are absent in Lhca4. The grana margins of the thylakoid membrane were found to be the primary site of interaction between this 'extra' LHCII and the PSI-LHCI-LHCII supercomplex in State II. The results suggest that the LHCI complexes mediate energetic interactions between the 'extra' LHCII and PSI in the intact membrane.3

SummaryThe identification of mutated metabolic enzymes in hereditary cancer syndromes has established a direct link between metabolic dysregulation and cancer. Mutations in the Krebs cycle enzyme, fumarate hydratase (FH), predispose affected individuals to leiomyomas, renal cysts, and cancers, though the respective pathogenic roles of mitochondrial and cytosolic FH isoforms remain undefined. On the basis of comprehensive metabolomic analyses, we demonstrate that FH1-deficient cells and tissues exhibit defects in the urea cycle/arginine metabolism. Remarkably, transgenic re-expression of cytosolic FH ameliorated both renal cyst development and urea cycle defects associated with renal-specific FH1 deletion in mice. Furthermore, acute arginine depletion significantly reduced the viability of FH1-deficient cells in comparison to controls. Our findings highlight the importance of extramitochondrial metabolic pathways in FH-associated oncogenesis and the urea cycle/arginine metabolism as a potential therapeutic target.

FTO catalyzes the Fe­(II) and 2-oxoglutarate (2OG)-dependent modification of nucleic acids, including the demethylation of N 6-methyladenosine (m6A) in mRNA. FTO is a proposed target for anti-cancer therapy. Using information from crystal structures of FTO in complex with 2OG and substrate mimics, we designed and synthesized two series of FTO inhibitors, which were characterized by turnover and binding assays, and by X-ray crystallography with FTO and the related bacterial enzyme AlkB. A potent inhibitor employing binding interactions spanning the FTO 2OG and substrate binding sites was identified. Selectivity over other clinically targeted 2OG oxygenases was demonstrated, including with respect to the hypoxia-inducible factor prolyl and asparaginyl hydroxylases (PHD2 and FIH) and selected JmjC histone demethylases (KDMs). The results illustrate how structure-based design can enable the identification of potent and selective 2OG oxygenase inhibitors and will be useful for the development of FTO inhibitors for use in vivo.

Galactooligosaccharides (GOS) are nondigestible dietary fibers which have a beneficial effect on human health by promoting the growth of probiotic bacteria in the gut. In addition, other health benefits have been reported from oligosaccharides consumption such as stimulation of intestinal mobility, colon cancer prevention, mineral absorption as well as protection against certain pathogenic bacterial infections. The goal of this research was to develop an efficient biotransformation system using a consortium of microbes for the production of ≥85% pure GOS and reusing the cell biomass in repeated cycles of biotransformation. Production of GOS by lactose transgalactosylation using whole cells of Sporobolomyces singularis MTCC 5491 as a source of β-galactosidase and monosaccharides utilization by yeast isolate (NUTIDY007) were studied. For increasing the purity of GOS, growth and bioconversion parameters on the transgalactosylation by the whole cells were investigated. Further, continuous production of GOS was studied in a reactor with microfiltration membrane system. A maximum GOS purity of 42% was achieved using single culture of S. singularis. Under optimized conditions, single culture of S. singularis produced a maximum of 56% pure GOS. Addition of second culture to the reaction mixture for utilization of glucose significantly increased the GOS purity from 56% to ≥85%. The product consisted of tri- to penta-galactooligosaccharides. Trisaccharides were the main component of the reaction mixture. A maximum productivity of 10.9 g/L/hr was obtained under the optimum conditions.