Structure-Based Design of Selective Fat Mass and Obesity Associated Protein (FTO) Inhibitors.

FTO catalyzes the Fe(II) and 2-oxoglutarate (2OG)-dependent modification of nucleic acids, including the demethylation of N6-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.

Mass spectrometry reveals potential of ß-lactams as SARS-CoV-2 Mpro inhibitors.

The main viral protease (Mpro) of SARS-CoV-2 is a nucleophilic cysteine hydrolase and a current target for anti-viral chemotherapy. We describe a high-throughput solid phase extraction coupled to mass spectrometry Mpro assay. The results reveal some ß-lactams, including penicillin esters, are active site reacting Mpro inhibitors, thus highlighting the potential of acylating agents for Mpro inhibition.

An intact light harvesting complex I antenna system is required for complete state transitions in Arabidopsis.

Efficient photosynthesis depends on maintaining balance between the rate of light-driven electron transport occurring in photosystem I (PSI) and photosystem II (PSII), located in the chloroplast thylakoid membranes. Balance is achieved through a process of 'state transitions' that increases energy transfer towards PSI when PSII is overexcited (state II), and towards PSII when PSI is overexcited (state I). This is achieved through redox control of the phosphorylation state of light-harvesting antenna complex II (LHCII). PSI is served by both LHCII and 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 reduces state transitions in Arabidopsis. The severest phenotype-observed in a mutant lacking Lhca4 (ΔLhca4)-displayed a 69% reduction compared with the wild type. Yet, surprisingly, the amounts of the PSI-LHCI-LHCII supercomplex isolated by blue native polyacrylamide gel electrophoresis (BN-PAGE) from digitonin-solubilized thylakoids were similar in the wild type and ΔLhca mutants. Fluorescence excitation spectroscopy revealed that in the wild type 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 ΔLhca4. 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 LHCII and PSI in the intact membrane.

A role for cytosolic fumarate hydratase in urea cycle metabolism and renal neoplasia.

The 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.