Biology subjects

Alumasa, J. N.

Publications and source records attributed to Alumasa, J. N..

7 recordsLinked to original sources

bioRxiv2026

Isoindole-benzohydrazide derivative potentiates colistin activity to overcome resistance conferred by MCR-1 variants in Escherichia coli

Polymyxin antibiotic resistance mediated by the mobilized colistin resistance (MCR) enzyme threatens the last-resort treatment of deadly multidrug-resistant pathogens, including Acinetobacter baumannii, and calls for urgent mitigation efforts. We used a multifaceted approach involving computational and wet-lab techniques to identify an isoindole-benzohydrazide derivative, JNAL-042, which potentiates colistin activity in an Escherichia coli MCR-resistance phenotype screening model. JNAL-042 lacked intrinsic antibiotic activity but potentiated that of polymyxin antibiotics at 12-24 mug/ml, as demonstrated by bacterial growth susceptibility screens. Cell viability counts and time-kill assays corroborated these findings, demonstrating complete abolition of MCR-mediated resistance by the antibiotic-adjuvant combination at concentrations mirroring those of empty vector controls. Post-induction treatment with this combination similarly prevented the propagation of the resistance phenotype, albeit at a higher antibiotic dose. This landmark study presents JNAL-042 as a promising antibiotic adjuvant to combat MCR-mediated resistance and offers a viable solution to preserve last-resort polymyxin antibiotics.

microbiology↗

bioRxiv2026

Structural Basis for C8 methylation of 23S ribosomal RNA by Cfr

Cfr methylates C8 of adenosine 2503 (A2503) in 23S ribosomal RNA (rRNA) and will also methylate C2 of A2503 after methylating C8. C8 methylation confers resistance to more than five classes of clinically used antibiotics, highlighting it as a worrisome mechanism of antibiotic resistance. Here, we report the structure of Cfr, determined by cryogenic electron microscopy (Cryo-EM). Despite its small size ([~]36 kDa), we exploit a transient protein-RNA crosslink that forms during catalysis, which requires Cys105 to resolve. Using a Cfr Cys105Ala variant and an 87-nucleotide strand of rRNA, we isolate the crosslinked species and determine its structure to 3.0 [A] resolution. Notably, the 87-mer rRNA adopts an L-shaped conformation characteristic of tRNAs, rather than the conformation it assumes in the ribosome. One Sentence SummaryCryo-EM structure of Cfr, a radical S-adenosylmethionine methylase that confers antibiotic resistance

biochemistry↗

bioRxiv2025

Antibiotic-adjuvants abolish resistance conferred by the Staphylococcus aureus erythromycin resistance methyltransferase in an Escherichia coli model

Enzyme-mediated resistance is among the main strategies bacteria use to evade antibiotic action. S-adenosylmethionine-dependent erythromycin resistance methyltransferases catalyze the methylation of 23S ribosomal RNA in bacteria, causing resistance to macrolides, lincosamides, and streptogramin type-B antibiotics. Given the diversity and number of identified variants of these enzymes, it is vital to devise ways of inhibiting their activity to rescue affected antibiotics. Here, we use computer-aided solvent mapping and virtual screening techniques to identify inhibitors of Erms displaying promising adjuvant properties. We further demonstrate that an E. coli model expressing a recombinant S. aureus ErmC (SaErmC) variant causes substantial resistance to representative macrolide and lincosamide antibiotics. Assessment of test compounds using this resistance model revealed candidates displaying promising adjuvant activity when combined with erythromycin or clindamycin. Antibiotic combinations with a principal candidate oxadiazole, JNAL-016, completely blocked SaErmC-mediated resistance against erythromycin, resulting in an antibiotic-sensitive phenotype in broth microdilution screening assays. This compound also suppressed ErmC activity, allowing erythromycin to regain its bactericidal properties when assessed in actively growing cultures using time-kill assays. JNAL-016 displayed a noncompetitive mode of inhibition against SaErmC activity in vitro and bound the purified enzyme with high affinity (Kd = 1.8 {+/-} 0.7 M) based on microscale thermophoresis data. Competition experiments suggested that JNAL-016 competes with SAM for its binding pocket on the enzyme, and this compound exhibited no toxicity against human embryonic kidney cells. These findings establish a practical strategy for targeting Erm-mediated resistance, which could lead to a viable adjuvant-based therapy against bacterial pathogens that weaponize variants of this class of methyltransferases.

microbiology↗

bioRxiv2025

A trans-translation inhibitor kills Mycobacterium tuberculosis by targeting ribosomal protein bL12

New antibiotics with novel mechanisms of action are needed to treat infections by multidrug-resistant strains of Mycobacterium tuberculosis. Here, we show that KKL-1005, an anti-tubercular triazole-based molecule, binds to ribosomal protein bL12 and specifically inhibits the trans-translation ribosome rescue pathway, a process essential for the survival of M. tuberculosis. Our data demonstrate that KKL-1005 binds to the N terminal domain of bL12, both in vitro and in bacterial cells, and specifically inhibits trans-translation and not normal translation. These results suggest that tmRNA-SmpB interacts with bL12 differently from tRNA, and raise the possibility of developing antibiotics targeting bL12.

microbiology↗

bioRxiv2024

Trans-Translation inhibitors and copper ions synergize for enhanced antibiotic activity

Trans-Translation is the most effective ribosome rescue mechanism and a compelling target for new antimicrobial agents. A recent proteomic study revealed similarities between the responses of Bacillus subtilis to the inhibitors small-molecule inhibitors oxadiazole KKL-40 and tetrazole KKL-55 and divalent cation ionophores, indicating the disturbance of metal homeostasis as potential secondary mechanism of action. Here, we report increased copper levels in KKL-40 and KKL-55-treated B. subtilis. Both inhibitors form copper complexes that enter large unilamellar vesicles. Copper supplementation enhanced the antibacterial activity against B. subtilis by simultaneously increasing inhibitor and copper uptake. The co-treatment of B. subtilis with trans-translation inhibitors and copper concentrations normally benign for trans-translation-competent cells, caused an immediate stalling of growth and translation, as observed at higher KKL-40 and KKL-55 concentrations without copper supplementation. Proteome analysis showed that during translation stalling cells were unable to mount an effective copper toxicity response. Taken together, the synergetic uptake of KKL-40 and KKL-55 with copper leads to a quick-onset translation stalling, preventing B. subtilis from counteracting the toxic effects of rapid copper influx. Significance statementThe challenge of antimicrobial resistance is growing, necessitating an exploration of novel antibiotic targets. Among these, trans-translation has attracted considerable attention due to its ubiquitous presence in bacteria as well as its role in virulence and pathogenesis. Several inhibitors of trans-translation have been identified in a target-based screening using a whole-cell assay. However, recent proteomic profiling studies suggested that the tested trans-translation inhibitors might have an additional mode of action. In this work, we shed light on their previously undiscovered copper ionophore activity and explore the consequences of co-treating B. subtilis with KKL-40 or KKL-55 and CuCl2. This co-treatment results in a rapid antibiotic influx, and, consequently to the stalling of ribosomes, translation, and bacterial growth. Simultaneously, massive amounts of copper accumulate in the cells, the toxic effects of which require a copper stress response to mitigate. However, such a response is averted by the stalled translation. Dual mechanism antibacterial agents are attractive because they are typically associated with slow emergence of resistance. A deep understanding of the complex interplay of KKL-40 and KKL-55 with metal ions will help to fully exploit trans-translation as an antibacterial target and to develop KKL-40 and KKL-55-based antibiotics into novel treatments for bacterial infections.

microbiology↗

bioRxiv2023

Antibiotic that inhibits trans-translation blocks binding of EF-Tu to tmRNA but not to tRNA

Trans-Translation is conserved throughout bacteria and is essential in many species. High-throughput screening identified a tetrazole-based trans-translation inhibitor, KKL-55, that has broad-spectrum antibiotic activity. A biotinylated version of KKL-55 pulled down Elongation Factor Thermo-unstable (EF-Tu) from bacterial lysates. Purified EF-Tu bound KKL-55 in vitro with a Kd = 2 {micro}M, confirming a high-affinity interaction. An X-ray crystal structure showed KKL-55 binds in domain 3 of EF-Tu, and mutation of residues in the binding pocket abolished KKL-55 binding. RNA binding assays in vitro showed that KKL-55 inhibits binding between EF-Tu and tmRNA, but not between EF-Tu and tRNA. These data demonstrate a new mechanism for inhibition of EF-Tu function and suggest that this specific inhibition of EF-Tu*tmRNA binding is a viable target for antibiotic development. IMPORTANCEEF-Tu is a universally conserved translation factor that mediates productive interactions between tRNAs and the ribosome. In bacteria, EF-Tu also delivers tmRNA-SmpB to the ribosome during trans-translation. We report the first small molecule, KKL-55, that specifically inhibits EF-Tu activity in trans-translation without affecting its activity in normal translation. KKL-55 has broad-spectrum antibiotic activity, suggesting that compounds targeted to the tmRNA-binding interface of EF-Tu could be developed into new antibiotics to treat drug-resistant infections.

biochemistry↗

bioRxiv2020

Ribosome rescue inhibitors clear Neisseria gonorrhoeae in vivo using a new mechanism

The trans-translation pathway for rescuing stalled ribosomes is conserved and essential in bacterial pathogens but has no mammalian homolog, making it an ideal target for new antibiotics. We previously reported the discovery of a family of acylaminooxadiazoles that selectively inhibit trans-translation, resulting in broad-spectrum antibiotic activity. Optimization of the pharmacokinetic and antibiotic properties of the acylaminooxadiazoles produced MBX-4132, which cleared multiple-drug resistant Neisseria gonorrhoeae infection in mice after a single oral dose. Cryo-EM studies of non-stop ribosomes showed that acylaminooxadiazoles bind to a unique site near the peptidyl-transfer center and significantly alter the conformation of ribosomal protein L27, suggesting a novel mechanism for specific inhibition of trans-translation by these molecules. One Sentence SummaryRibosome rescue inhibitors reveal a new conformation of the ribosome and kill drug-resistant Neisseria gonorrhoeae in vivo.

microbiology↗