Infections caused by antibiotic-resistant bacteria (ARB) are one of the major global health challenges of our time. In addition to developing new antibiotics to combat ARB, sensitizing ARB, or pursuing alternatives to existing antibiotics are promising options to counter antibiotic resistance. This review compiles the most promising anti-ARB strategies currently under development. These strategies include the following: (i) discovery of novel antibiotics by modification of existing antibiotics, screening of small-molecule libraries, or exploration of peculiar places; (ii) improvement in the efficacy of existing antibiotics through metabolic stimulation or by loading a novel, more efficient delivery systems; (iii) development of alternatives to conventional antibiotics such as bacteriophages and their encoded endolysins, anti-biofilm drugs, probiotics, nanomaterials, vaccines, and antibody therapies. Clinical or preclinical studies show that these treatments possess great potential against ARB. Some anti-ARB products are expected to become commercially available in the near future.

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Current Promising Strategies against Antibiotic-Resistant Bacterial Infections

A class of piperazine hybridized coumarin indolylcyanoenones was exploited as new structural antibacterial frameworks to combat intractable bacterial resistance. Bioactive assessment discovered that 4-chlorobenzyl derivative 11f showed a prominent inhibition on Pseudomonas aeruginosa ATCC 27853 with a low MIC of 1 μg/mL, which was four-fold more effective than norfloxacin. Importantly, the highly active 11f with inconspicuous hemolysis towards human red blood cells displayed quite low proneness to trigger bacterial resistance. Preliminary explorations on its antibacterial behavior disclosed that 11f possessed the ability to destroy bacterial cell membrane, leading to increased permeability of inner and outer membranes, the depolarization and fracture of membrane, and the effusion of intracellular components. Furthermore, bacterial oxidative stress and metabolic turbulence aroused by 11f also accelerated bacterial apoptosis. In particular, 11f could not only effectively inset into DNA, but also bind with DNA gyrase through forming supramolecular complex, thereby affecting the biological function of DNA. The above findings of new piperazine hybridized coumarin indolylcyanoenones provided an inspired possibility for the treatment of resistant bacterial infections.

https://www.mdpi.com/1420-3049/28/6/2511/pdf?version=1678371025

Synthesis and Biological Evaluation of Piperazine Hybridized Coumarin Indolylcyanoenones with Antibacterial Potential

Aromatic residues form a significant part of the protein core, where they make tight interactions with multiple surrounding side chains. Despite the dense packing of internal side chains, the aromatic rings of phenylalanine and tyrosine residues undergo 180° rotations, or flips, which are mediated by transient and large-scale “breathing” motions that generate sufficient void volume around the aromatic ring. Forty years after the seminal work by Wagner and Wüthrich, NMR studies of aromatic ring flips are now undergoing a renaissance as a powerful means of probing fundamental dynamic properties of proteins. Recent developments of improved NMR methods and isotope labeling schemes have enabled a number of advances in addressing the mechanisms and energetics of aromatic ring flips. The nature of the transition states associated with ring flips can be described by thermodynamic activation parameters, including the activation enthalpy, activation entropy, activation volume, and also the isothermal volume compressibility of activation. Consequently, it is of great interest to study how ring flip rate constants and activation parameters might vary with protein structure and external conditions like temperature and pressure. The field is beginning to gather such data for aromatic residues in a variety of environments, ranging from surface exposed to buried. In the future, the combination of solution and solid-state NMR spectroscopy together with molecular dynamics simulations and other computational approaches is likely to provide detailed information about the coupled dynamics of aromatic rings and neighboring residues. In this Perspective, we highlight recent developments and provide an outlook toward the future.

https://pubs.acs.org/doi/pdf/10.1021/acs.jpcb.2c07258

NMR Studies of Aromatic Ring Flips to Probe Conformational Fluctuations in Proteins

Antimicrobial resistance is a major obstacle for the treatment of infectious diseases and currently represents one of the most significant threats to global health. Staphylococcus aureus remains a formidable human pathogen with high mortality rates associated with severe systemic infections. S. aureus has become notorious as a multidrug resistant bacterium, which when combined with its extensive arsenal of virulence factors that exacerbate disease, culminates in an incredibly challenging pathogen to treat clinically. Compounding this major health issue is the lack of antibiotic discovery and development, with only two new classes of antibiotics approved for clinical use in the last 20 years. Combined efforts from the scientific community have reacted to the threat of dwindling treatment options to combat S. aureus disease in several innovative and exciting developments. This review describes current and future antimicrobial strategies aimed at treating staphylococcal colonization and/or disease, examining therapies that show significant promise at the preclinical development stage to approaches that are currently being investigated in clinical trials.

https://onlinelibrary.wiley.com/doi/pdfdirect/10.1111/1751-7915.14268

Novel antimicrobial strategies to treat multi‐drug resistant Staphylococcus aureus infections

The brevicidine and laterocidine family of lipopeptide antibiotics exhibit strong activity against multidrug-resistant Gram-negative bacteria, while showing low propensity to induce resistance. Both peptides feature a branched lipid tail on the N-terminal residue, which for brevicidine is chiral. Here, we report the synthesis and biological evaluation of a library of brevicidine and laterocidine analogues wherein the N-terminal lipid is replaced with linear achiral fatty acids. Optimal lipid chain lengths were determined and new analogues with strong activity against colistin-resistant E. coli produced.

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Synthesis and structure–activity relationship studies of N-terminal analogues of the lipopeptide antibiotics brevicidine and laterocidine

Abstract Antibiotics that use novel mechanisms are needed to combat antimicrobial resistance 1–3 . Teixobactin 4 represents a new class of antibiotics with a unique chemical scaffold and lack of detectable resistance. Teixobactin targets lipid II, a precursor of peptidoglycan 5 . Here we unravel the mechanism of teixobactin at the atomic level using a combination of solid-state NMR, microscopy, in vivo assays and molecular dynamics simulations. The unique enduracididine C-terminal headgroup of teixobactin specifically binds to the pyrophosphate-sugar moiety of lipid II, whereas the N terminus coordinates the pyrophosphate of another lipid II molecule. This configuration favours the formation of a β-sheet of teixobactins bound to the target, creating a supramolecular fibrillar structure. Specific binding to the conserved pyrophosphate-sugar moiety accounts for the lack of resistance to teixobactin 4 . The supramolecular structure compromises membrane integrity. Atomic force microscopy and molecular dynamics simulations show that the supramolecular structure displaces phospholipids, thinning the membrane. The long hydrophobic tails of lipid II concentrated within the supramolecular structure apparently contribute to membrane disruption. Teixobactin hijacks lipid II to help destroy the membrane. Known membrane-acting antibiotics also damage human cells, producing undesirable side effects. Teixobactin damages only membranes that contain lipid II, which is absent in eukaryotes, elegantly resolving the toxicity problem. The two-pronged action against cell wall synthesis and cytoplasmic membrane produces a highly effective compound targeting the bacterial cell envelope. Structural knowledge of the mechanism of teixobactin will enable the rational design of improved drug candidates. 

/pdf/teixobactin-kills-bacteria-by-a-two-pronged-attack-on-the-3rcj89x9.pdf

Teixobactin kills bacteria by a two-pronged attack on the cell envelope

Abstract Control of posttranscriptional mRNA decay is a crucial determinant of cell homeostasis and differentiation. mRNA lifetime is governed by cis-regulatory elements in their 3′ untranslated regions (UTR). Despite ongoing progress in the identification of cis elements we have little knowledge about the functional and structural integration of multiple elements in 3′UTR regulatory hubs and their recognition by mRNA-binding proteins (RBPs). Structural analyses are complicated by inconsistent mapping and prediction of RNA fold, by dynamics, and size. We here, for the first time, provide the secondary structure of a complete mRNA 3′UTR. We use NMR spectroscopy in a divide-and-conquer strategy complemented with SAXS, In-line probing and SHAPE-seq applied to the 3′UTR of Ox40 mRNA, which encodes a T-cell co-receptor repressed by the protein Roquin. We provide contributions of RNA elements to Roquin-binding. The protein uses its extended bi-modal ROQ domain to sequentially engage in a 2:1 stoichiometry with a 3′UTR core motif. We observe differential binding of Roquin to decay elements depending on their structural embedment. Our data underpins the importance of studying RNA regulation in a full sequence and structural context. This study serves as a paradigm for an approach in analysing structured RNA-regulatory hubs and their binding by RBPs.

NMR-derived secondary structure of the full-length Ox40 mRNA 3′UTR and its multivalent binding to the immunoregulatory RBP Roquin

Probing the dynamics of aromatic side chains provides important insights into the behavior of a protein because flips of aromatic rings in a protein's hydrophobic core report on breathing motion involving a large part of the protein. Inherently invisible to crystallography, aromatic motions have been primarily studied by solution NMR. The question how packing of proteins in crystals affects ring flips has, thus, remained largely unexplored. Here we apply magic-angle spinning NMR, advanced phenylalanine 1H-13C/2H isotope labeling and MD simulation to a protein in three different crystal packing environments to shed light onto possible impact of packing on ring flips. The flips of the two Phe residues in ubiquitin, both surface exposed, appear remarkably conserved in the different crystal forms, even though the intermolecular packing is quite different: Phe4 flips on a ca. 10-20 ns time scale, and Phe45 are broadened in all crystals, presumably due to µs motion. Our findings suggest that intramolecular influences are more important for ring flips than intermolecular (packing) effects. 

https://doi.org/10.1016/j.yjsbx.2022.100079

Aromatic ring flips in differently packed ubiquitin protein crystals from MAS NMR and MD

Probing the dynamics of aromatic side chains provides important insights into the behavior of a protein because flips of aromatic rings in a protein’s hydrophobic core report on breathing motion involving large part of the protein. Inherently invisible to crystallography, aromatic motions have been primar-ily studied by solution NMR. Here we apply magic-angle spinning NMR, advanced phenylalanine 1 H- 13 C/ 2 H isotope labeling and molecular simulations to a protein in three different crystal packing environments to shed light onto possible impact of packing on ring flips. We find that ring flips in ubiquitin are quite conserved in the different crystal forms, even though the intermolecular packing is quite different suggesting that intramolecular influences are more important than intermolecular (packing) effects.

Aromatic ring flips in differently packed protein crystals: MAS NMR and MD studies of 3 ubiquitin lattices

Rock-solid amyloid?. Aromatic residues in the core of the amyloid fibrils HELLF and HET-s show no sign of ring flips suggesting a rigid core that does not undergo "breathing motions". Aromatic residues facing outside on the other hand are highly flexible and show ring flips on various timescales. These conclusions could be drawn from dynamics measurements by solid-state MAS NMR in combination with specific labeling of phenylalanines and tyrosines as Paul Schanda et al. describe in their Research Article (e202219314). Rock-solid amyloid?. Aromatic residues in the core of the amyloid fibrils HELLF and HET-s show no sign of ring flips suggesting a rigid core that does not undergo "breathing motions". Aromatic residues facing outside on the other hand are highly flexible and show ring flips on various timescales. These conclusions could be drawn from dynamics measurements by solid-state MAS NMR in combination with specific labeling of phenylalanines and tyrosines as Paul Schanda et al. describe in their Research Article (e202219314). Coordination Compounds Nanocatalysis Host-Guest Systems Asymmetric Catalysis 

https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/anie.202304138

Lea Marie Becker

Papers

Teixobactin kills bacteria by a two-pronged attack on the cell envelope

NMR-derived secondary structure of the full-length Ox40 mRNA 3′UTR and its multivalent binding to the immunoregulatory RBP Roquin

Aromatic ring flips in differently packed ubiquitin protein crystals from MAS NMR and MD

Aromatic ring flips in differently packed protein crystals: MAS NMR and MD studies of 3 ubiquitin lattices

The Rigid Core and Flexible Surface of Amyloid Fibrils Probed by Magic‐Angle‐Spinning NMR Spectroscopy of Aromatic Residues