Biology subjects

Fujimori, D. G.

Publications and source records attributed to Fujimori, D. G..

4 recordsLinked to original sources

bioRxiv2026

Structural modification of oxazolidinone antibiotics alters nascent peptide stalling preference and peptide trajectory through the ribosome

The oxazolidinone antibiotic linezolid binds to the peptidyl transferase center of the ribosome, where it inhibits a subset of peptide bond formation events. This context-specificity of translation inhibition is dictated by the nature of the amino acid at the penultimate position of the nascent peptide. It remains unknown whether this is a general feature of oxazolidinones and whether it can be modulated by their structural alterations. Here, we show that the oxazolidinone tedizolid also inhibits translation in a context-specific manner, but with dramatically altered selectivity, favoring Ile, His, and Gln as the penultimate residues. Delpazolid, which shares the C5 hydroxymethyl moiety with tedizolid, shows a similar preference. Structural analysis of the ribosome with tedizolid and a stalled nascent peptide showed a compacted, helical conformation of the nascent chain induced by the drug. Our findings reveal that stalling preferences of oxazolidinones can be modulated by structural modifications within this antibiotic class.

biochemistry↗

bioRxiv2025

An intrinsically disordered region of histone demethylase KDM5A activates catalysis through interactions with the nucleosomal acidic patch and DNA

Lysine demethylase 5A (KDM5A) plays a key role in the regulation of chromatin accessibility by catalyzing the removal of trimethyl marks on histone H3K4 (H3K4me3). KDM5A is also an oncogenic driver, with overexpression of KDM5A observed in various cancers, including breast, lung, and ovarian cancer. Past studies have characterized the functions of KDM5A domains, including KDM5A interactions with the histone H3 tail, but have yet to identify the broader mechanisms that drive KDM5A binding to the nucleosome. Through investigation of binding and catalysis on nucleosome substrates, we uncovered multivalent interactions of KDM5A with the H2A/H2B acidic patch and DNA that play crucial roles in the regulation of catalytic activity. We also identified an intrinsically disordered region (IDR) containing bifunctional arginine-rich motifs capable of binding to both the histone H2A/H2B acidic patch and nucleosomal DNA that is necessary for catalysis on nucleosome substrates. Our findings both elucidate previously unknown mechanisms that regulate KDM5A catalytic activity and reveal the ability of an IDR to engage in multiple interactions with chromatin. ARTICLE HIGHLIGHTSO_LIThe intrinsically disordered region of KDM5A binds the acidic patch and DNA. C_LIO_LIInteractions with the nucleosome are mediated by arginine-rich motifs in the IDR. C_LIO_LIThe IDR properly orients KDM5A on the nucleosome to enable catalysis. C_LI

biochemistry↗

bioRxiv2024

Context-specific inhibition of mitochondrial ribosomes by phenicol and oxazolidinone antibiotics

The antibiotics chloramphenicol (CHL) and oxazolidinones including linezolid (LZD) are known to inhibit mitochondrial translation. This can result in serious, potentially deadly, side effects when used therapeutically. Although the mechanism by which CHL and LZD inhibit bacterial ribosomes has been elucidated in detail, their mechanism of action against mitochondrial ribosomes has yet to be explored. CHL and oxazolidinones bind to the ribosomal peptidyl transfer center (PTC) of the bacterial ribosome and prevent incorporation of incoming amino acids under specific sequence contexts, causing ribosomes to stall only at certain sequences. Through mitoribosome profiling, we show that inhibition of mitochondrial ribosomes is similarly context-specific - CHL and LZD lead to mitoribosome stalling primarily when there is an alanine, serine, or threonine in the penultimate position of the nascent peptide chain. We further validate context-specific stalling through in vitro translation assays. A high resolution cryo-EM structure of LZD bound to the PTC of the human mitoribosome shows extensive similarity to the mode of bacterial inhibition and also suggests potential avenues for altering selectivity. Our findings could help inform the rational development of future, less mitotoxic, antibiotics, which are critically needed in the current era of increasing antimicrobial resistance.

biochemistry↗

bioRxiv2023

Structure-based discovery of inhibitors of the SARS-CoV-2 Nsp14 N7-methyltransferase

An under-explored target for SARS-CoV-2 is non-structural protein 14 (Nsp14), a crucial enzyme for viral replication that catalyzes the methylation of N7-guanosine of the viral RNA at 5'-end; this enables the virus to evade the host immune response by mimicking the eukaryotic post-transcriptional modification mechanism. We sought new inhibitors of the S-adenosyl methionine (SAM)-dependent methyltransferase (MTase) activity of Nsp14 with three large library docking strategies. First, up to 1.1 billion make-on-demand ("tangible") lead-like molecules were docked against the enzymes SAM site, seeking reversible inhibitors. On de novo synthesis and testing, three inhibitors emerged with IC50 values ranging from 6 to 43 M, each with novel chemotypes. Structure-guided optimization and in vitro characterization supported their non-covalent mechanism. In a second strategy, docking a library of 16 million tangible fragments revealed nine new inhibitors with IC50 values ranging from 12 to 341 M and ligand efficiencies from 0.29 to 0.42. In a third strategy, a newly created library of 25 million tangible, virtual electrophiles were docked to covalently modify Cys387 in the SAM binding site. Seven inhibitors emerged with IC50 values ranging from 3.2 to 39 M, the most potent being a reversible aldehyde. Initial optimization of a second series yielded a 7 M acrylamide inhibitor. Three inhibitors characteristic of the new series were tested for selectivity against 30 human protein and RNA MTases, with one showing partial selectivity and one showing high selectivity. Overall, 32 inhibitors encompassing eleven chemotypes had IC50 values <50 M and 5 inhibitors in four chemotypes had IC50 values <10 M. These molecules are among the first non-SAM-like inhibitors of Nsp14, providing multiple starting points for optimizing towards antiviral activity.

biophysics↗