Gram-negative bacteria are protected by a three-layered cell envelope and must tightly coordinate the biosynthesis of phospholipids (PL), peptidoglycan (PG) and lipopolysaccharides (LPS) to maintain envelope integrity. In Escherichia coli, the inner membrane protein LapB (YciM) plays a critical dual role in LPS homeostasis by acting as a scaffold for cytoplasmic LPS and PL biosynthesis enzymes and by promoting FtsH-dependent proteolysis of the key enzyme LpxC. Because LPS and PG biosynthesis compete for the shared precursor UDP-GlcNAc, we investigated whether MurA, catalyzing the first committed step of PG biosynthesis, is an integral component of the LapB complex. Using bacterial two-hybrid analysis, pull-down assays and microscale thermophoresis, we demonstrate that MurA directly interacts with LapB, LpxA, LpxC, LpxD, and FabZ. Together, these findings support a model in which PL, PG, and LPS biosynthesis enzymes are engaged in a protein-protein interaction hub that synchronizes the production of all three layers of the Gram-negative cell envelope in E. coli.

Phosphatidylethanolamine (PE) is the second most abundant phospholipid in mammalian cells. The cone-shaped structure of PE positions this lipid to act as a cornerstone of signaling events, such as those involving protein-protein and lipid-protein interactions, as it has been shown to promote conformational changes in both protein and lipid-based membrane structures. Despite this importance, chemical tools to interrogate the biological activity and trafficking of PE lipids are limited. Herein, we report the development of ethanolamine probes functionalized with clickable tags that are capable of infiltrating native cellular pathways and subsequently producing labeled PE lipids. Derivatization of click-tagged PE products enabled fluorescence microscopy imaging of these lipids in cells. Interrogation of the efficacy of these probes for biological incorporation was conducted using mass spectrometry and thin-layer chromatography analysis to identify various species of tagged PE molecules. This approach provides an invaluable step toward metabolic labeling strategies that will aid in the tracking of PE biosynthesis and trafficking pathways in cells.

Formate dehydrogenases represent a highly attractive tool for nicotinamide cofactor regeneration to drive biocatalytic reductions and oxygenations by NADH-dependent oxidoreductases with formate as a cheap, atom-economic terminal reductant. However, despite the clear benefits over other recycling systems, native FDHs remain rather unpopular, mainly due to their intrinsic, low specific activity. While the active site architecture is generally highly conserved across many FDHs from very diverse origins, smaller patterns of dissonance in the active site's vicinity can serve as hotspots for the rational design of more effective variants. In this study, we have created a series of Candida boidinii FDH variants incorporating nonconsensus amino acids found in Saccharomyces cerevisiae FDH. A double variant (C23S/F285D) was identified that exhibits a significantly higher specific activity, an overall more desirable pH and temperature profile, and an improved stability profile.

The rapid progress of multidrug-resistant (MDR) bacterial infections and the clinical aggressiveness of triple-negative breast cancer (TNBC) possess a great problem to human beings. It necessitates the discovery and development of multifunctional therapeutic biomaterials. Herein, we report two enzymatically stable short cationic peptide hydrogelators comprising of N-terminal lysine headgroups, non-coded amino acid spacers, aromatic phenylalanine residues, and dodecylamine tails. These amphiphilic peptides are rapidly self-assembled in Tris-HCl buffer (pH 7.4) to form hydrogels with nanofibrillar network. Remarkably, both of these hydrogelators exhibit potent antibacterial activity against several Gram-positive and Gram-negative drug-resistant strains, functioning through multitiered mechanism including membrane permeabilization, trans-membrane depolarization, and intracellular reactive oxygen species (ROS) generation. Moreover, one of these peptide amphiphiles demonstrates a significant anticancer efficacy (with IC50 value of 6.9 µM) against human TNBC cells by activating the extrinsic caspase-8/caspase-3-driven apoptotic pathway and reversing epithelial-to-mesenchymal transition (EMT). Interestingly, the peptide with centrally located amino acid residue with lesser number of methylene units than that of the other peptide shows better anticancer efficacy and less antimicrobial activity. This study vividly demonstrates a tunable class of proteolytically stable amphiphilic peptide gelators with potential antimicrobial as well as anticancer activities establishing a structure-function relationship of these bioactive peptide scaffolds suggesting a versatile blueprint for peptide-based next-generation soft bioactive materials.

The COVID-19 pandemic caused by SARS-CoV-2 created a global health crisis, and the virus still circulates, mutates, and causes illness and death. Vaccination remains the primary defense, but effectiveness can be reduced in immunocompromised individuals and against new variants, and clinically approved antivirals are limited by side effects, drug interactions, and resistance. Herein, we propose a novel application of tanespimycin (17AAG), the chaperone heat shock protein (HSP)90 inhibitor, as an antiviral against the SARS-CoV-2 main protease (M[pro]). M[pro], due to its indispensable role in viral replication and absence of expression in the host, represents a key target for drug development. Through biochemical and cell-based assays, structural analysis, and resistance-mutant testing, we present data showing that 17AAG covalently binds to the active-site cysteine of M[pro], disrupts its secondary structure, and protects cells from M[pro]-induced toxicity more effectively than nirmatrelvir. We further showed that 17AAG retains its covalent binding and structure-disrupting activity against the nirmatrelvir-resistance M165I variant. Additionally, since HSP90 is important for viral protein stability, virion assembly, and modulation of host immune response, 17AAG is a promising, versatile drug candidate that could accelerate antiviral development for COVID-19.

Peptide-responsive G protein-coupled receptors (GPCRs) often recognize endogenous peptide ligands through extended receptor interfaces, providing opportunities for peptide-based ligands to engage receptor contacts that conventional small molecules struggle to access effectively. In this context, cyclic peptides are more than stabilized peptide analogs: their constrained topologies can preorganize key pharmacophoric elements, support engagement with extended orthosteric or allosteric receptor interfaces, and allow precise tuning of selectivity and signaling output. In this review, we discuss cyclic peptides targeting peptide-responsive GPCRs through three connected dimensions: natural macrocyclic ligands and scaffolds, chemical strategies for topological and functional optimization, and emerging discovery platforms for GPCR-active macrocycles. Specifically, we examine how endogenous cyclic peptides, venom-derived peptides, plant cyclotides, and microbial macrocycles provide structurally defined templates for probing GPCR recognition, and how engineering approaches, including bridge replacement, conformational locking, residue modification, and half-life extension, have expanded their utility as both bioactive ligands and molecular probes. We further highlight discovery technologies (e.g., mRNA display, phage display, cell-based screening, and structure- and computation-guided design) that are advancing the discovery and optimization of macrocycles with improved receptor specificity and pharmacological properties. Together, these advances position cyclic peptides as a topology-guided molecular strategy for dissecting peptide-GPCR recognition and developing next-generation therapeutics.

Using a set of spectroscopic experiments, we investigated the binding of Cu[2+] and Cu[+] to a comprehensive series of nucleic acid-related molecules, ranging from individual nucleobases to oligonucleotides up to six nucleotides in length. We demonstrate the selective binding of Cu[+] to cytosine- and guanine-containing oligonucleotides, as evidenced by their ability to compete with ferrozine for Cu[+]. The capacity to inhibit ascorbate oxidation, through effective sequestration catalytically active copper, increases with the number of cytosine residues in the oligonucleotides and is associated with the formation of a 1:1 Cu[+]-oligonucleotide complex accompanied by a pronounced conformational change. In contrast, guanine-rich oligonucleotides exhibit the opposite behavior: they form Cu[+] G-quadruplex structures that permit copper redox cycling. These findings highlight an unexpected binding of copper ions to nucleic acids in inert or redox-active modes and thus expand its potential roles within cells through its direct interaction with DNA or RNA.

About 50 years after their development, the benzimidazole anthelmintic drugs still occupy a key role in the treatment of parasitic diseases. Like other anti-infective drugs, their extensive use has led to resistance development and now requires new solutions to treat these diseases. Organometallic derivatization of existing drugs has become a common strategy to design new drug candidates, which could benefit from enhanced activity against resistant or sensitive strains through the incorporation of an organometallic fragment. In this study, we describe the development of a new series of organometallic derivatives of benzimidazole anthelmintics. Ten derivatives, six of which were organometallic, were synthesized and characterized. In silico and in vitro studies were performed to determine their biological activity. Therefore, they were screened against a range of nematodes and other parasites. Biological activities were found for some of the derivatives against Strongyloides ratti, Ancylostoma ceylanicum, Trichuris muris, Schistosoma mansoni, Echinococcus multilocularis, or Leishmania infantum in similar ranges as the parent anthelmintics. Even if the activity of the benzimidazole scaffold was not systematically enhanced, this strategy is a good starting point for the development of new antiparasitic compounds and for further study of the mode of action of these drugs.

Actinoplanes teichomyceticus is a well-established producer of bioactive secondary metabolites, including the glycopeptide antibiotic teicoplanin. Although its antibiotic biosynthetic capacity has been extensively investigated, its siderophore diversity and any additional biological functions of these iron-chelating metabolites remain comparatively underexplored. We identified a reproducibly bioactive, teicoplanin-independent fraction that inhibited Bacillus spizizenii. Molecular networking applied to this fraction identified hydroxamate ferrioxamine and desferrioxamine-type siderophores as the dominant metabolites, including acylated analogs detected as Al[3+]- and Fe[3+]-chelated species. Robust siderophore secretion was confirmed by the CAS assay. Notably, siderophore-enriched fractions exhibited selective antibacterial activity against Gram-positive bacteria, with minimum inhibitory concentrations of approximately 16 µg/mL against B. spizizenii and partial inhibition of Staphylococcus aureus, while no activity was observed against Escherichia coli. Synthetic C7 and C9 acyl-desferrioxamine analogs showed enhanced antibacterial activity upon Al[3+] chelation, indicating a metal-dependent bioactivity. These findings reveal an unexpected antibacterial role for ferrioxamine-type siderophores produced by A. teichomyceticus, extending their function beyond iron acquisition, possibly through a "Trojan horse" (or "Trojan metal") mechanism.

Microcin J25 (MccJ25) is of special interest due to its "lariat" topology. The lasso architecture is thought to be responsible for its remarkable thermal stability (100°C for 30 min incubation) and potency against Gram-negative bacteria (i.e., MBC of ~4.75 µM against E. coli strain DH5α). This naturally occurring lasso peptide is formed via post-translational modifications (PTMs), including leader peptide removal and macrolactam cyclization between the N-terminus and 8-Glu side chain. Since the discovery of MccJ25 in 1992, synthetic endeavors toward the total chemical synthesis of MccJ25 have remained elusive. Herein, we investigated the chemical synthesis of MccJ25 utilizing β-thiol amino acids as the chemical tools to prestaple and rigidify the peptide backbone through disulfide linkages. Although the proposed chemical strategy failed to afford the desired lasso configuration, synthetic routes toward Fmoc-SPPS compatible, stereoselective β-thiol amino acids via thiazoline derivatives were developed. In addition, chemical/biochemical literature synthesis of MccJ25 and its analogs, as well as the synthesis of β-thiol amino acids, are summarized within.

Staphylococcus aureus strains have emerged with resistance mechanisms that reduce the efficacy of last resort antibiotics and evade the immune system. One strategy to combat antimicrobial resistance is to modulate host immunity to eliminate infections more effectively. This has led to the development of immunotherapeutics consisting of vancomycin conjugated to formyl peptides (fPeps), with vancomycin targeting the cell wall and the fPeps engaging host innate immunity. Here, we used flow cytometry to quantify the binding of vancomycin=fPep conjugates to S. aureus clinical isolates. This revealed reduced binding of vancomycin=fPeps compared to vancomycin alone and quantified the interaction between the conjugates and the bacterial cell surface, which is important to quantify to then control the chemotactic gradient established by the fPep cargo. The direct antimicrobial activity of these conjugates was also reduced when compared to vancomycin, reflecting the reduced binding of these conjugates to S. aureus. This flow cytometry method allows quantification of vancomycin=fPep binding to bacteria and will assist in future studies to understand how attached fPeps and other immune signalling cargoes can stimulate innate immune cell activation leading to bacterial phagocytosis.

Design and synthesis of water-soluble π-conjugated chromophores are one of the significant challenges due to the intrinsic hydrophobicity of the conjugated frameworks. Consequently, rational molecular design to access novel water-soluble biologically relevant organic chromophores with red-to-NIR emission, large Stokes' shifts, and good fluorescent quantum yields always attracts significant attention. Here, we establish a straightforward synthetic strategy to access a novel class of water-soluble red-light-emissive (up to 645 nm) cationic dipyridyl dipyrroethene derivatives, achieved through strategic engineering of the (E)-dipyrroethene (DPE) core. The X-ray structures of cationic species 8 revealed a very interesting supramolecular nanoarchitectonics. These sets of molecules are showcasing a fluorescence in both the solid state and solution phase across a wide range of solvents, including water, with a decent fluorescent quantum yield and a large Stokes' shift. Detailed spectroscopic and computational studies support that the balanced intramolecular charge transfer (ICT) leads to a redshift in its absorption/emission. We further expanded the applicability of the water-soluble chromophore 10 as a rapid, selective, and effective fluorescent-turn-on sensor for recognition of serum albumin proteins with a strong binding constant of 2.7 × 10[6] M[-1]. This work provides a fundamental platform for designing robust, bio-responsive water-soluble π-conjugated systems with tenable structural and electronic properties.

Pyridoxal-5'-phosphate (PLP)-dependent L-arginine oxidases (PDAOs) catalyze 4-electron oxidations of L-arginine in the context of secondary metabolite biosynthesis. PDAOs are separated into two classes based on whether they hydroxylate or desaturate L-arginine. A detailed mechanistic understanding of these enzymes will extend our knowledge of natural products biosynthesis. Here, we present detailed transient kinetic analyses of representative hydroxylating and desaturating PDAOs: MppP from Streptomyces wadayamensis (SwMppP) and the Ind4 homolog from Pseudoalteromonas luteoviolacea (Plu4). While both SwMppP and Plu4 kinetics can be accurately predicted using the same kinetic model, they in fact use subtly different mechanisms. This work seeks to uncover how two enzymes that are so similar in terms of sequence, structure, and catalytic function can have different mechanisms. To this end, we determined the structure of an additional desaturating PDAO, the Ind4 homolog from Bacillus cereus (Bcer4). This brings the total number of PDAOs whose structures have been determined to four: two hydroxylases and two desaturases. Examination of the available structural data fails to point out any class-specific structural differences that could explain the mechanistic differences between the classes. The subtle mechanistic differences are likely due to differences in protein dynamics rather than structure.

To overcome the limitations of microbial infections in modern medicine, we developed compound I incorporating the amphiphile 12-hydroxy lauric acid (12-HDA) at the N-terminus linked to a heterochiral diphenylalanine motif (D- and L-Phe) at the C-terminus guided by the principle of chiral orchestration (R. Ahuja, M. Singh, and A. Dutt Konar, ACS Appl. Biomater. 2025, 8, 1108). Although preliminary minimum inhibitory concentration (MIC) data indicated encouraging antimicrobial activity against the Gram-positive pathogens Staphylococcus aureus and Bacillus subtilis, these results alone were insufficient to consider the molecule a viable therapeutic lead. Consequently, comprehensive in vitro evaluation revealed a pronounced, organism-dependent response: Compound I reduced B. subtilis colony formation by 98%, compared to only 22% for S. aureus. Flow cytometric Live/Dead analyses corroborated these findings, showing increased cellular granularity, enhanced PI uptake, and 59.6% non-viable B. subtilis cells, whereas S. aureus exhibited limited susceptibility. Motivated by this selectivity, we evaluated Compound I in a B. subtilis-infected excisional wound model, where treated animals achieved complete wound closure, robust hair regrowth, and scar-free healing within 14 days, comparable to that of the standard drug. Overall, these findings establish compound I as a strong therapeutic candidate for future wound dressing.

Cytochrome P450 2C8 (CYP2C8) is known to cause drug interactions via pharmacokinetic alterations of drugs, but it's also explicitly crucial for endogenous metabolism, like the generation of epoxyeicosatrienoic acids (EETs) from arachidonic acid. CYP2C8 expression is reported to be altered in various cancers, where CYP2C8-mediated EETs promote tumorigenesis. Conversely, thymoquinone, an extensively used complementary medicine, has emerged as a promising candidate against cancer due to its ability to curb tumorigenesis. Nevertheless, to date, limited information is available about the impact of thymoquinone on CYP2C8 inhibition. Therefore, we planned to explore the same using in silico, in vitro, and in vivo approaches. The current results reveal the followings: (a) thymoquinone could markedly inhibit CYP2C8 based on study using amodiaquine N-deethylation in human liver microsomes (HLM); (b) thymoquinone could strongly interact with the active site of the human CYP2C8 as demonstrated by molecular docking analysis; (c) thymoquinone could restrict the metabolic depletion of repaglinide (a CYP2C8 substrate) in rat liver microsomes; (d) thymoquinone altered the pharmacokinetic profile of repaglinide (a CYP2C8 substrate) in rats, resulting in repaglinide's increased systemic exposure and reduced clearance; (e) thymoquinone could retard EET's formation in HLM. Further studies are warranted to evaluate the biological significance of thymoquinone-mediated modulation of the CYP2C8/EET axis in relevant cancer models.