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Antibiotics, particularly the β-lactams, are a cornerstone of modern medicine. However, the rise of bacterial resistance to these agents, particularly through the actions of β-lactamases, poses a significant threat to our continued ability to effectively treat infections. Metallo-β-lactamases (MBLs) are of particular concern due to their ability to hydrolyze a wide range of β-lactam antibiotics including carbapenems. For this reason there is growing interest in the development of MBL inhibitors as well as novel antibiotics that can overcome MBL-mediated resistance. Here, we report the synthesis and evaluation of novel conjugates that combine a carbapenem (meropenem or ertapenem) with a recently reported MBL inhibiting indole carboxylate scaffold. These hybrids were found to display potent inhibition against MBLs including NDM-1 and IMP-1, with IC 50 values in the low nanomolar range. However, their antibacterial potency was limited. Mechanistic studies suggest that despite maintaining effective MBL inhibiting activity in live bacteria, the new carbapenem/MBL inhibitor conjugates have a reduced ability to engage with the bacterial target of the β-lactams., (© 2024 The Author(s). ChemMedChem published by Wiley-VCH GmbH.)

Hematologic cancers are among the most common cancers in adults and children. Despite significant improvements in therapies, many patients still succumb to the disease. Therefore, novel therapies are needed. The Wiskott-Aldrich syndrome protein (WASp) family regulates actin assembly in conjunction with the Arp2/3 complex, a ubiquitous nucleation factor. WASp is expressed exclusively in hematopoietic cells and exists in two allosteric conformations: autoinhibited or activated. Here, we describe the development of EG-011, a first-in-class small molecule activator of the autoinhibited form of WASp. EG-011 possesses in vitro and in vivo antitumor activity as a single agent in lymphoma, leukemia, and multiple myeloma, including models of secondary resistance to PI3K, BTK, and proteasome inhibitors. The in vitro activity was confirmed in a lymphoma xenograft. Actin polymerization and WASp binding were demonstrated using multiple techniques. Transcriptome analysis highlighted homology with drugs inducing actin polymerization.

The escalating threat posed by antibiotic resistance is a global concern and underscores the need for new antibiotics. In this context, the recent discovery of evybactin, a nonribosomal depsipeptide antibiotic that selectively and potently inhibits the growth of M. tuberculosis, is particularly noteworthy. Here, we present the first total synthesis of this natural product, along with a revision of its assigned structure. Our studies revealed a disparity between the structure originally proposed for evybactin and its actual configuration. Specifically, the 3-methylhistidine residue present in the evybactin core macrocycle was found to be of the d-configuration rather than the previously assigned l-His(Me). Having addressed this, we further optimized our solid-phase synthetic route to provide access to evybactin on a multi-hundred-milligram scale. Access to such quantities will allow for more comprehensive studies with this promising antibiotic., (© 2024 The Author(s). Chemistry - A European Journal published by Wiley-VCH GmbH.)

The synthetic small molecule DCAP is a chemically well-characterized compound with antibiotic activity against Gram-positive and Gram-negative bacteria, including drug-resistant pathogens. Until now, its mechanism of action was proposed to rely exclusively on targeting the bacterial membrane, thereby causing membrane depolarization, and increasing membrane permeability (Eun et al. 2012, J. Am. Chem. Soc. 134 (28), 11322-11325; Hurley et al. 2015, ACS Med. Chem. Lett. 6, 466-471). Here, we show that the antibiotic activity of DCAP results from a dual mode of action that is more targeted and multifaceted than previously anticipated. Using microbiological and biochemical assays in combination with fluorescence microscopy, we provide evidence that DCAP interacts with undecaprenyl pyrophosphate-coupled cell envelope precursors, thereby blocking peptidoglycan biosynthesis and impairing cell division site organization. Our work discloses a concise model for the mode of action of DCAP which involves the binding to a specific target molecule to exert pleiotropic effects on cell wall biosynthetic and divisome machineries.

Antimicrobial peptides (AMPs) are a promising alternative to antibiotics in the fight against multi-drug resistant and immune system-evading bacterial infections. Protegrins are porcine cathelicidins which have been identified in porcine leukocytes. Protegrin-1 is the best characterized family member and has broad antibacterial activity by interacting and permeabilizing bacterial membranes. Many host defense peptides (HDPs) like LL-37 or chicken cathelicidin 2 (CATH-2) have also been shown to have protective biological functions during infections. In this regard, it is interesting to study if Protegrin-1 has the immune modulating potential to suppress unnecessary immune activation by neutralizing endotoxins or by influencing the macrophage functionality in addition to its direct antimicrobial properties. This study showed that Protegrin-1 neutralized lipopolysaccharide- (LPS) and bacteria-induced activation of RAW macrophages by binding and preventing LPS from cell surface attachment. Furthermore, the peptide treatment not only inhibited bacterial phagocytosis by murine and porcine macrophages but also interfered with cell surface and intracellular bacterial survival. Lastly, Protegrin-1 pre-treatment was shown to inhibit the amastigote survival in Leishmania infected macrophages. These experiments describe an extended potential of Protegrin-1's protective role during microbial infections and add to the research towards clinical application of cationic AMPs., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Bacteria have evolved resistance to nearly all known antibacterials, emphasizing the need to identify antibiotics that operate via novel mechanisms. Here we report a class of allosteric inhibitors of DNA gyrase with antibacterial activity against fluoroquinolone-resistant clinical isolates of Escherichia coli. Screening of a small-molecule library revealed an initial isoquinoline sulfonamide hit, which was optimized via medicinal chemistry efforts to afford the more potent antibacterial LEI-800. Target identification studies, including whole-genome sequencing of in vitro selected mutants with resistance to isoquinoline sulfonamides, unanimously pointed to the DNA gyrase complex, an essential bacterial topoisomerase and an established antibacterial target. Using single-particle cryogenic electron microscopy, we determined the structure of the gyrase-LEI-800-DNA complex. The compound occupies an allosteric, hydrophobic pocket in the GyrA subunit and has a mode of action that is distinct from the clinically used fluoroquinolones or any other gyrase inhibitor reported to date. LEI-800 provides a chemotype suitable for development to counter the increasingly widespread bacterial resistance to fluoroquinolones., (© 2024. The Author(s).)

Bacteria are capable of remarkable adaptations to their environment, including undesirable bacterial resistance to antibacterial agents. One of the most serious cases is an infection caused by multidrug-resistant Staphylococcus aureus , which has unfortunately also spread outside hospitals. Therefore, the development of new effective antibacterial agents is extremely important to solve the increasing problem of bacterial resistance. The bacteriolytic enzyme autolysin E (AtlE) is a promising new drug target as it plays a key role in the degradation of peptidoglycan in the bacterial cell wall. Consequently, disruption of function can have an immense impact on bacterial growth and survival. An in silico and in vitro evaluation of iminosugar derivatives as potent inhibitors of S. aureus (AtlE) was performed. Three promising hit compounds ( 1 , 3 and 8 ) were identified as AtlE binders in the micromolar range as measured by surface plasmon resonance. The most potent compound among the SPR response curve hits was 1 , with a K D of 19 μM. The K D value for compound 8 was 88 μM, while compound 3 had a K D value of 410 μM.

Gram-positive bacterial infections present a major clinical challenge, with methicillin- and vancomycin-resistant strains continuing to be a cause for concern. In recent years, semisynthetic vancomycin derivatives have been developed to overcome this problem as exemplified by the clinically used telavancin, which exhibits increased antibacterial potency but has also raised toxicity concerns. Thus, glycopeptide antibiotics with enhanced antibacterial activities and improved safety profiles are still necessary. We describe the development of a class of highly potent semisynthetic glycopeptide antibiotics, the guanidino lipoglycopeptides, which contain a positively charged guanidino moiety bearing a variable lipid group. These glycopeptides exhibited enhanced in vitro activity against a panel of Gram-positive bacteria including clinically relevant methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant strains, showed minimal toxicity toward eukaryotic cells, and had a low propensity for resistance selection. Mechanistically, guanidino lipoglycopeptides engaged with bacterial cell wall precursor lipid II with a higher binding affinity than vancomycin. Binding to both wild-type d-Ala-d-Ala lipid II and the vancomycin-resistant d-Ala-d-Lac variant was confirmed, providing insight into the enhanced activity of guanidino lipoglycopeptides against vancomycin-resistant isolates. The in vivo efficacy of guanidino lipoglycopeptide EVG7 was evaluated in a S. aureus murine thigh infection model and a 7-day sepsis survival study, both of which demonstrated superiority to vancomycin. Moreover, the minimal to mild kidney effects at supratherapeutic doses of EVG7 indicate an improved therapeutic safety profile compared with vancomycin. These findings position guanidino lipoglycopeptides as candidates for further development as antibacterial agents for the treatment of clinically relevant multidrug-resistant Gram-positive infections.

Bacitracin is a macrocyclic peptide antibiotic that is widely used as a topical treatment for infections caused by gram-positive bacteria. Mechanistically, bacitracin targets bacteria by specifically binding to the phospholipid undecaprenyl pyrophosphate (C 55 PP), which plays a key role in the bacterial lipid II cycle. Recent crystallographic studies have shown that when bound to C 55 PP, bacitracin adopts a highly ordered amphipathic conformation. In doing so, all hydrophobic side chains align on one face of the bacitracin-C 55 PP complex, presumably interacting with the bacterial cell membrane. These insights led us to undertake structure-activity investigations into the individual contribution of the nonpolar amino acids found in bacitracin. To achieve this we designed, synthesized, and evaluated a series of bacitracin analogues, a number of which were found to exhibit significantly enhanced antibacterial activity against clinically relevant, drug-resistant pathogens. As for the natural product, these next-generation bacitracins were found to form stable complexes with C 55 PP. The structure-activity insights thus obtained serve to inform the design of C 55 PP-targeting antibiotics, a key and underexploited antibacterial strategy., Competing Interests: Competing interests statement:The authors declare no competing interest.

Antimicrobial resistance is a leading cause of mortality, calling for the development of new antibiotics. The fungal antibiotic plectasin is a eukaryotic host defence peptide that blocks bacterial cell wall synthesis. Here, using a combination of solid-state nuclear magnetic resonance, atomic force microscopy and activity assays, we show that plectasin uses a calcium-sensitive supramolecular killing mechanism. Efficient and selective binding of the target lipid II, a cell wall precursor with an irreplaceable pyrophosphate, is achieved by the oligomerization of plectasin into dense supra-structures that only form on bacterial membranes that comprise lipid II. Oligomerization and target binding of plectasin are interdependent and are enhanced by the coordination of calcium ions to plectasin's prominent anionic patch, causing allosteric changes that markedly improve the activity of the antibiotic. Structural knowledge of how host defence peptides impair cell wall synthesis will likely enable the development of superior drug candidates., (© 2024. The Author(s).)

Background: Osteosarcoma (OS) is a primary bone malignancy that mostly affects young people, characterized by high metastatic potential, and a marked chemoresistance that is responsible for disease relapse in most patients. Therefore, it is necessary to identify novel molecules to setup targeted strategies to improve the clinical outcome. The enzyme nicotinamide N-methyltransferase (NNMT) catalyses the N-methylation of nicotinamide and other analogs, playing a crucial role in the biotransformation of drugs and xenobiotics. NNMT overexpression was reported in a wide variety of cancers, and several studies demonstrated that is able to promote cell proliferation, migration and resistance to chemotherapy. The aim of this study was to explore the potential involvement of NNMT in OS., Methods: Immunohistochemical analyses have been performed to evaluate NNMT expression in selected OS and healthy bone tissue samples. Subsequently, OS cell lines have been transfected with vectors targeting NNMT mRNA (shRNAs) and the impact of this downregulation on migration, cell proliferation, and response to chemotherapeutic treatment was also analysed by wound healing, MTT, SRB and Trypan blue assays, respectively., Results: Results showed that OS samples display a significantly higher NNMT expression compared with healthy tissue. Preliminary results suggest that NNMT silencing in OS cell lines is associated to a decrease of cell proliferation and migration, as well as to enhanced sensitivity to chemotherapy. Data obtained showed that NNMT may represent an interesting marker for OS detection and a promising target for effective anti-cancer therapy., (© 2024 Stichting European Society for Clinical Investigation Journal Foundation. Published by John Wiley & Sons Ltd.)

The prevalence of multidrug-resistant (MDR) pathogens combined with a decline in antibiotic discovery presents a major challenge for health care. To refill the discovery pipeline, we need to find new ways to uncover new chemical entities. Here, we report the global genome mining-guided discovery of new lipopeptide antibiotics tridecaptin A 5 and tridecaptin D, which exhibit unusual bioactivities within their class. The change in the antibacterial spectrum of Oct-TriA 5 was explained solely by a Phe to Trp substitution as compared to Oct-TriA 1 , while Oct-TriD contained 6 substitutions. Metabolomic analysis of producer Paenibacillus sp. JJ-21 validated the predicted amino acid sequence of tridecaptin A 5 . Screening of tridecaptin analogues substituted at position 9 identified Oct-His9 as a potent congener with exceptional efficacy against Pseudomonas aeruginosa and reduced hemolytic and cytotoxic properties. Our work highlights the promise of tridecaptin analogues to combat MDR pathogens.

Merkel cell carcinoma (MCC) is an aggressive skin cancer, with a propensity for early metastasis. Therefore, early diagnosis and the identification of novel targets become fundamental. The enzyme nicotinamide N-methyltransferase (NNMT) catalyzes the reaction of N-methylation of nicotinamide and other analogous compounds. Although NNMT overexpression was reported in many malignancies, the significance of its dysregulation in cancer cell phenotype was partly clarified. Several works demonstrated that NNMT promotes cancer cell proliferation, migration, and chemoresistance. In this study, we investigated the possible involvement of this enzyme in MCC. Preliminary immunohistochemical analyses were performed to evaluate NNMT expression in MCC tissue specimens. To explore the enzyme function in tumor cell metabolism, MCC cell lines have been transfected with plasmids encoding for short hairpin RNAs (shRNAs) targeting NNMT mRNA. Preliminary immunohistochemical analyses showed elevated NNMT expression in MCC tissue specimens. The effect of enzyme downregulation on cell proliferation, migration, and chemosensitivity was then evaluated through MTT, trypan blue, and wound healing assays. Data obtained clearly demonstrated that NNMT knockdown is associated with a decrease of cell proliferation, viability, and migration, as well as with enhanced sensitivity to treatment with chemotherapeutic drugs. Taken together, these results suggest that NNMT could represent an interesting MCC biomarker and a promising target for targeted anti-cancer therapy., (© 2024. The Author(s).)

The polymyxins are nonribosomal lipopeptides produced by Paenibacillus polymyxa and are potent antibiotics with activity specifically directed against Gram-negative bacteria. While the clinical use of polymyxins has historically been limited due to their toxicity, their use is on the rise given the lack of alternative treatment options for infections due to multidrug resistant Gram-negative pathogens. The Gram-negative specificity of the polymyxins is due to their ability to target lipid A, the membrane embedded LPS anchor that decorates the cell surface of Gram-negative bacteria. Notably, the mechanisms responsible for polymyxin toxicity, and in particular their nephrotoxicity, are only partially understood with most insights coming from studies carried out in the past decade. In parallel, many synthetic and semisynthetic polymyxin analogues have been developed in recent years in an attempt to mitigate the nephrotoxicity of the natural products. Despite these efforts, to date, no polymyxin analogues have gained clinical approval. This may soon change, however, as at the moment there are three novel polymyxin analogues in clinical trials. In this context, this review provides an update of the most recent insights with regard to the structure-activity relationships and nephrotoxicity of new polymyxin variants reported since 2010. We also discuss advances in the synthetic methods used to generate new polymyxin analogues, both via total synthesis and semisynthesis.

Epilancin 15X is a lantibiotic that has an antimicrobial activity in the nanomolar concentration range towards Staphylococcus simulans. Such low MICs usually imply that these peptides employ a mechanism of action (MoA) involving high affinity targets. Here we studied this MoA by using epilancin 15X's ability to dissipate the membrane potential of intact S. simulans cells. These membrane depolarization assays showed that treatment of the bacteria by antibiotics known to affect the bacterial cell wall synthesis pathway decreased the membrane depolarization effects of epilancin 15X. Disruption of the Lipid II cycle in intact bacteria using several methods led to a decrease in the activity of epilancin 15X. Antagonism-based experiments on 96-well plate and agar diffusion plate pointed towards a possible interaction between epilancin 15X and Lipid II and this was confirmed by Circular Dichroism (CD) based experiments. However, this interaction did not lead to a detectable effect on either carboxyfluorescein (CF) leakage or proton permeability. All experiments point to the involvement of a phosphodiester-containing target within a polyisoprene-based biosynthesis pathway, yet the exact identity of the target remains obscure so far., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Importance: Microorganisms that live on or inside plants can influence plant growth and health. Among the plant-associated bacteria, streptomycetes play an important role in defense against plant diseases, but the underlying mechanisms are not well understood. Here, we demonstrate that the plant hormones jasmonic acid (JA) and methyl jasmonate directly affect the life cycle of streptomycetes by modulating antibiotic synthesis and promoting faster development. Moreover, the plant hormones specifically stimulate the synthesis of the polyketide antibiotic actinorhodin in Streptomyces coelicolor . JA is then modified in the cell by amino acid conjugation, thereby quenching toxicity. Collectively, these results provide new insight into the impact of a key plant hormone on diverse phenotypic responses of streptomycetes., Competing Interests: The authors declare no conflict of interest.

The development of new treatment options for bacterial infections requires access to new targets for antibiotics and antivirulence strategies. Chemoproteomic approaches are powerful tools for profiling and identifying novel druggable target candidates, but their functions often remain uncharacterized. Previously, we used activity-based protein profiling in the opportunistic pathogen Staphylococcus aureus to identify active serine hydrolases termed fluorophosphonate-binding hydrolases (Fph). Here, we provide the first characterization of S. aureus FphH, a conserved, putative carboxylesterase (referred to as yva K in Bacillus subtilis ) at the molecular and cellular level. First, phenotypic characterization of fph H-deficient transposon mutants revealed phenotypes during growth under nutrient deprivation, biofilm formation, and intracellular survival. Biochemical and structural investigations revealed that FphH acts as an esterase and lipase based on a fold well suited to act on a small to long hydrophobic unbranched lipid group within its substrate and can be inhibited by active site-targeting oxadiazoles. Prompted by a previous observation that fph H expression was upregulated in response to fusidic acid, we found that FphH can deacetylate this ribosome-targeting antibiotic, but the lack of FphH function did not infer major changes in antibiotic susceptibility. In conclusion, our results indicate a functional role of this hydrolase in S. aureus stress responses, and hypothetical functions connecting FphH with components of the ribosome rescue system that are conserved in the same gene cluster across Bacillales are discussed. Our atomic characterization of FphH will facilitate the development of specific FphH inhibitors and probes to elucidate its physiological role and validity as a drug target.

Targeting the PI3K-AKT-mTOR pathway is a promising therapeutic strategy for breast cancer treatment. However, low response rates and development of resistance to PI3K-AKT-mTOR inhibitors remain major clinical challenges. Here, we show that MYC activation drives resistance to mTOR inhibitors (mTORi) in breast cancer. Multiomic profiling of mouse invasive lobular carcinoma (ILC) tumors revealed recurrent Myc amplifications in tumors that acquired resistance to the mTORi AZD8055. MYC activation was associated with biological processes linked to mTORi response and counteracted mTORi-induced translation inhibition by promoting translation of ribosomal proteins. In vitro and in vivo induction of MYC conferred mTORi resistance in mouse and human breast cancer models. Conversely, AZD8055-resistant ILC cells depended on MYC, as demonstrated by the synergistic effects of mTORi and MYCi combination treatment. Notably, MYC status was significantly associated with poor response to everolimus therapy in metastatic breast cancer patients. Thus, MYC is a clinically relevant driver of mTORi resistance that may stratify breast cancer patients for mTOR-targeted therapies., (© 2023 Bhin et al.)

Developments in computational omics technologies have provided new means to access the hidden diversity of natural products, unearthing new potential for drug discovery. In parallel, artificial intelligence approaches such as machine learning have led to exciting developments in the computational drug design field, facilitating biological activity prediction and de novo drug design for molecular targets of interest. Here, we describe current and future synergies between these developments to effectively identify drug candidates from the plethora of molecules produced by nature. We also discuss how to address key challenges in realizing the potential of these synergies, such as the need for high-quality datasets to train deep learning algorithms and appropriate strategies for algorithm validation., (© 2023. Springer Nature Limited.)

The growing incidence of infections caused by multi-drug resistant Gram-negative bacteria has led to an increased use of last-resort antibiotics such as the polymyxins. Polymyxin therapy is limited by toxicity concerns, most notably nephrotoxicity. Recently we reported the development of a novel class of semisynthetic polymyxins with reduced toxicity wherein the N-terminal lipid and diaminobutyric acid residue are replaced by a cysteine-linked lipid featuring a reductively labile disulfide bond. In the present study we further explored the potential of this approach by also varying the amino acid residue directly adjacent to the polymyxin macrocycle. This led to the identification of new semisynthetic polymyxins that maintain the potent antibacterial activity of the clinically used polymyxin B while exhibiting a further reduction in toxicity toward human proximal tubule epithelial cells. Furthermore, these new polymyxins were found to effectively synergize with novobiocin, rifampicin, and erythromycin against mcr -positive, polymyxin resistant E. coli ., Competing Interests: The authors declare no competing financial interest., (This journal is © The Royal Society of Chemistry.)

Since Fleming's discovery of penicillin nearly a century ago, a bounty of natural product antibiotics have been discovered, many of which continue to be of clinical importance today. The structural diversity encountered among nature's repertoire of antibiotics is mirrored by the varying mechanisms of action by which they selectively target and kill bacterial cells. The ability for bacteria to construct and maintain a strong cell wall is essential for their robust growth and survival under a range of conditions. However, the need to maintain the cell wall also presents a vulnerability that is exploited by many natural antibiotics. Bacterial cell wall biosynthesis involves both the construction of complex membrane-bound precursor molecules and their subsequent crosslinking by dedicated enzymes. Interestingly, many naturally occurring antibiotics function not by directly inhibiting the enzymes associated with cell wall biosynthesis, but rather by binding tightly to their membrane-bound substrates. Such substrate sequestration mechanisms are comparatively rare outside of the antibiotics space with most small-molecule drug discovery programs instead aimed at developing inhibitors of target enzymes. In this feature article we provide the reader with an overview of the unique and ever increasing family of natural product antibiotics known to specifically function by binding to membrane-anchored bacterial cell wall precursors. In doing so, we highlight both our own contributions to the field as well as those made by other researchers engaged in exploring the potential offered by antibiotics that target bacterial cell wall precursors.

There is an increasing interest in the application of metal-organic cages (MOCs) in a biomedicinal context, as they can offer non-classical distribution in organisms compared to molecular substrates, while revealing novel cytotoxicity mechanisms. Unfortunately, many MOCs are not sufficiently stable under in vivo conditions, making it difficult to study their structure-activity relationships in living cells. As such, it is currently unclear whether MOC cytotoxicity stems from supramolecular features or their decomposition products. Herein, we describe the toxicity and photophysical properties of highly-stable rhodamine functionalized platinum-based Pt 2 L 4 nanospheres as well as their building blocks under in vitro and in vivo conditions. We show that in both zebrafish and human cancer cell lines, the Pt 2 L 4 nanospheres demonstrate reduced cytotoxicity and altered biodistribution within the body of zebrafish embryos compared to the building blocks. We anticipate that the composition-dependent biodistribution of Pt 2 L 4 spheres together with their cytotoxic and photophysical properties provides the fundament for MOC application in cancer therapy., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Ductal carcinoma in situ (DCIS) is a non-obligate precursor of invasive breast cancer (IBC). Due to a lack of biomarkers able to distinguish high- from low-risk cases, DCIS is treated similar to early IBC even though the minority of untreated cases eventually become invasive. Here, we characterized 115 patient-derived mouse-intraductal (MIND) DCIS models reflecting the full spectrum of DCIS observed in patients. Utilizing the possibility to follow the natural progression of DCIS combined with omics and imaging data, we reveal multiple prognostic factors for high-risk DCIS including high grade, HER2 amplification, expansive 3D growth, and high burden of copy number aberrations. In addition, sequential transplantation of xenografts showed minimal phenotypic and genotypic changes over time, indicating that invasive behavior is an intrinsic phenotype of DCIS and supporting a multiclonal evolution model. Moreover, this study provides a collection of 19 distributable DCIS-MIND models spanning all molecular subtypes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Brevicidine and laterocidine are macrocyclic lipodepsipeptides with selective activity against Gram-negative bacteria, including colistin-resistant strains. Previously, the macrocyclic core of these peptides was thought essential for antibacterial activity. In this study, we show that C-terminal amidation of linear brevicidine and laterocidine scaffolds, and substitution of the native Thr9, yields linear analogues that retain the potent antibacterial activity and low hemolysis of the parent compounds. Furthermore, an alanine scan of both peptides revealed that the aromatic and basic amino acids within the common central scaffold are essential for antibacterial activity. This linearization strategy for modification of cyclic peptides is a highly effective way to reduce the time and cost of peptide synthesis and may be applicable to other non-ribosomal antibacterial peptides.

Antibiotic resistance is reaching alarming levels, demanding for the discovery and development of antibiotics with novel chemistry and mechanisms of action. The recently discovered antibiotic cacaoidin combines the characteristic lanthionine residue of lanthipeptides and the linaridin-specific N-terminal dimethylation in an unprecedented N-dimethyl lanthionine ring, being therefore designated as the first class V lanthipeptide (lanthidin). Further notable features include the high D-amino acid content and a unique disaccharide substitution attached to the tyrosine residue. Cacaoidin shows antimicrobial activity against gram-positive pathogens and was shown to interfere with peptidoglycan biosynthesis. Initial investigations indicated an interaction with the peptidoglycan precursor lipid II PGN as described for several lanthipeptides. Using a combination of biochemical and molecular interaction studies we provide evidence that cacaoidin is the first natural product demonstrated to exhibit a dual mode of action combining binding to lipid II PGN and direct inhibition of cell wall transglycosylases., Competing Interests: The authors declare no competing interests., (© 2023 The Authors.)

Peptide antibiotics have gathered attention given the urgent need to discover antimicrobials with new mechanisms of action. Their extended role as immunomodulators makes them interesting candidates for the development of compounds with dual mode of action. The objective of this study was to test the anti-inflammatory capacity of a recently reported chimeric peptidomimetic antibiotic (CPA) composed of polymyxin B nonapeptide (PMBN) and a macrocyclic β-hairpin motif (MHM). We investigated the potential of CPA to inhibit lipopolysaccharide (LPS)-induced activation of RAW264.7 macrophages. In addition, we elucidated which structural motif was responsible for this activity by testing CPA, its building blocks, and their parent compounds separately. CPA showed excellent LPS neutralizing activity for both smooth and rough LPSs. At nanomolar concentrations, CPA completely inhibited LPS-induced nitric oxide, TNF-α, and IL-10 secretion. Murepavadin, MHM, and PMBN were incapable of neutralizing LPS in this assay, while PMB was less active compared to CPA. Isothermal titration calorimetry showed strong binding between the CPA and LPS with similar binding characteristics also found for the other compounds, indicating that binding does not necessarily correlate with neutralization of LPS. Finally, we showed that CPA-killed bacteria caused significantly less macrophage activation than bacteria killed with gentamicin, heat, or any of the other compounds. This indicates that the combined killing activity and LPS neutralization of CPA can prevent unwanted inflammation, which could be a major advantage over conventional antibiotics. Our data suggests that immunomodulatory activity can further strengthen the therapeutic potential of peptide antibiotics and should be included in the characterization of novel compounds.

Phenotypic screening is a powerful approach to identify novel antibiotics, but elucidation of the targets responsible for the antimicrobial activity is often challenging in the case of compounds with a polypharmacological mode of action. Here, we show that activity-based protein profiling maps the target interaction landscape of a series of 1,3,4-oxadiazole-3-ones identified in a phenotypic screen to have high antibacterial potency against multidrug-resistant Staphylococcus aureus . In situ competitive and comparative chemical proteomics with a tailor-made activity-based probe, in combination with transposon and resistance studies, revealed several cysteine and serine hydrolases as relevant targets. Our data showcase oxadiazolones as a novel antibacterial chemotype with a polypharmacological mode of action, in which FabH, FphC, and AdhE play a central role.

The growing threat of drug-resistant bacteria is a global concern, highlighting the urgent need for new antibiotics and antibacterial strategies. In this light, practical synthetic access to natural product antibiotics can provide important structure-activity insights while also opening avenues for the development of novel analogues with improved properties. To this end, we report an optimised synthetic route for the preparation of the clinically used macrocyclic peptide antibiotic bacitracin. Our combined solid- and solution-phase approach addresses the problematic, and previously unreported, formation of undesired epimers associated with the stereochemically fragile N-terminal thiazoline moiety. A number of bacitracin analogues were also prepared wherein the thiazoline motif was replaced by other known zinc-binding moieties and their antibacterial activities evaluated., (© 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

With increasing rates of resistance toward commonly used antibiotics, especially among Gram-negative bacteria, there is renewed interested in polymyxins. Polymyxins are lipopeptide antibiotics with potent anti-Gram-negative activity and are generally believed to target lipid A, the lipopolysaccharide (LPS) anchor found in the outer membrane of Gram-negative bacteria. To characterize the stereochemical aspects of their mechanism(s) of action, we synthesized the full enantiomers of polymyxin B and the polymyxin B nonapeptide (PMBN). Both compounds were compared with the natural compounds in biological and biophysical assays, revealing strongly reduced antibacterial activity for the enantiomeric species. The enantiomeric compounds also exhibit reduced LPS binding, lower outer membrane (OM) permeabilization, and loss of synergetic potential. These findings provide new insights into the stereochemical requirements underlying the mechanisms of action of polymyxin B and PMBN.

Polymyxins are a class of lipopeptide anti-infective agents with potent and specific activity against Gram-negative bacteria. While toxicity concerns associated with polymyxin B and E (colistin) have historically limited their clinical application, today they are increasingly used as last-resort antibiotics given the rise of multidrug-resistant Gram-negative pathogens. The adverse side effects of polymyxins are well known, particularly as related to their nephrotoxicity. Here, we describe the synthesis and evaluation of a novel series of polymyxin analogues, aimed at reducing their nephrotoxic effects. Using a semisynthetic approach, we explored modifications of the exocyclic part of the polymyxin scaffold, namely, the terminal amino acid and lipophilic tail. By incorporating a reductively labile disulfide linkage in the lipid tail, we obtained novel polymyxins that exhibit potent antibacterial activity on par with polymyxin B but with reduced toxicity toward human renal proximal tubular epithelial cells.

In the hunt for new antibiotics with activity against Gram-negative pathogens, the outer membrane β-barrel assembly machine (BAM) complex has become an increasingly interesting target. The recently reported BAM complex inhibitor, MRL-494, was discovered via a screening campaign for molecules that target the outer membrane. Notably, MRL-494 was reported to be an unintended byproduct generated during the synthesis of an unrelated compound, and as such no synthesis of the compound was disclosed. We here present a convenient and reliable route for the synthesis of MRL-494 that scales well. The antibacterial activity measured for synthesized MRL-494 matches that reported in the literature. Furthermore, MRL-494 was found to exhibit potent synergistic activity with rifampicin against Gram-negative bacteria, including E. coli , K. pneumoniae , A. baumannii , and P. aeruginosa . MRL-494 was also found to cause outer membrane disruption and induction of the Rcs stress response pathway. In addition, we undertook a focused structure-activity study specifically aimed at elucidating the roles played by the two guanidine moieties contained within the structure of MRL-494.

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., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

New approaches to target antibacterial agents toward Gram-negative bacteria are key, given the rise of antibiotic resistance. Since the discovery of polymyxin B nonapeptide as a potent Gram-negative outer membrane (OM)-permeabilizing synergist in the early 1980s, a vast amount of literature on such synergists has been published. This Review addresses a range of peptide-based and small organic compounds that disrupt the OM to elicit a synergistic effect with antibiotics that are otherwise inactive toward Gram-negative bacteria, with synergy defined as a fractional inhibitory concentration index (FICI) of <0.5. Another requirement for the inclusion of the synergists here covered is their potentiation of a specific set of clinically used antibiotics: erythromycin, rifampicin, novobiocin, or vancomycin. In addition, we have focused on those synergists with reported activity against Gram-negative members of the ESKAPE family of pathogens namely, Escherichia coli , Pseudomonas aeruginosa , Klebsiella pneumoniae , and/or Acinetobacter baumannii . In cases where the FICI values were not directly reported in the primary literature but could be calculated from the published data, we have done so, allowing for more direct comparison of potency with other synergists. We also address the hemolytic activity of the various OM-disrupting synergists reported in the literature, an effect that is often downplayed but is of key importance in assessing the selectivity of such compounds for Gram-negative bacteria.

The EPIC consortium brings together experts from a wide range of fields that include clinical, molecular and basic microbiology, infectious diseases, computational biology and chemistry, drug discovery and design, bioinformatics, biochemistry, biophysics, pharmacology, toxicology, veterinary sciences, environmental sciences, and epidemiology. The main question to be answered by the EPIC alliance is the following: "What is the best approach for data mining on carbapenemase inhibitors and how to translate this data into experiments?" From this forum, we propose that the scientific community think up new strategies to be followed for the discovery of new carbapenemase inhibitors, so that this process is efficient and capable of providing results in the shortest possible time and within acceptable time and economic costs.

The accelerated appearance of drug-resistant bacteria poses an ever-growing threat to modern medicine's capacity to fight infectious diseases. Gram-positive species such as methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcus pneumoniae continue to contribute significantly to the global burden of antimicrobial resistance. For decades, the treatment of serious Gram-positive infections relied upon the glycopeptide family of antibiotics, typified by vancomycin, as a last line of defense. With the emergence of vancomycin resistance, the semisynthetic glycopeptides telavancin, dalbavancin, and oritavancin were developed. The clinical use of these compounds is somewhat limited due to toxicity concerns and their unusual pharmacokinetics, highlighting the importance of developing next-generation semisynthetic glycopeptides with enhanced antibacterial activities and improved safety profiles. This Review provides an updated overview of recent advancements made in the development of novel semisynthetic glycopeptides, spanning the period from 2014 to today. A wide range of approaches are covered, encompassing innovative strategies that have delivered semisynthetic glycopeptides with potent activities against Gram-positive bacteria, including drug-resistant strains. We also address recent efforts aimed at developing targeted therapies and advances made in extending the activity of the glycopeptides toward Gram-negative organisms.

Although nanopores can be used for single-molecule sequencing of nucleic acids using low-cost portable devices, the characterization of proteins and their modifications has yet to be established. Here, we show that hydrophilic or glycosylated peptides translocate too quickly across FraC nanopores to be recognized. However, high ionic strengths (i.e., 3 M LiCl) and low pH (i.e., pH 3) together with using a nanopore with a phenylalanine at its constriction allows the recognition of hydrophilic peptides, and to distinguish between mono- and diglycosylated peptides. Using these conditions, we devise a nanopore method to detect, characterize, and quantify post-translational modifications in generic proteins, which is one of the pressing challenges in proteomic analysis.

Coactivator-associated arginine methyltransferase 1 (CARM1) is a member of the family of protein arginine methyltransferases. CARM1 catalyzes methyl group transfer from the cofactor S -adenosyl-l-methionine (AdoMet) to both histone and nonhistone protein substrates. CARM1 is involved in a range of cellular processes, mainly involving RNA transcription and gene regulation. As the aberrant expression of CARM1 has been linked to tumorigenesis, the enzyme is a potential therapeutic target, leading to the development of inhibitors and tool compounds engaging with CARM1. To evaluate the effects of these compounds on the activity of CARM1, sensitive and specific analytical methods are needed. While different methods are currently available to assess the activity of methyltransferases, these assays mainly focus on either the measurement of the cofactor product S -adenosyl-l-homocysteine (AdoHcy) or employ radioactive or expensive reagents, each with their own advantages and limitations. To complement the tools currently available for the analysis of CARM1 activity, we here describe the development of a convenient assay employing peptide substrates derived from poly(A)-binding protein 1 (PABP1). This operationally straightforward liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach allows for the direct detection of substrate methylation with minimal workup. The method was validated, and its value in characterizing CARM1 activity and inhibition was demonstrated through a comparative analysis involving a set of established small molecules and peptide-based CARM1 inhibitors.

Endogenous Staphylococcus aureus sortase A (SrtA) covalently incorporates cell wall anchored proteins equipped with a SrtA recognition motif (LPXTG) via a lipid II-dependent pathway into the staphylococcal peptidoglycan layer. Previously, we found that the endogenous S. aureus SrtA is able to recognize and process a variety of exogenously added synthetic SrtA substrates, including K(FITC)LPMTG-amide and K(FITC)-K-vancomycin-LPMTG-amide. These synthetic substrates are covalently incorporated into the bacterial peptidoglycan (PG) of S. aureus with varying efficiencies. In this study, we examined if native and synthetic substrates are processed by SrtA via the same pathway. Therefore, the effect of the lipid II inhibiting antibiotic bacitracin on the incorporation of native and synthetic SrtA substrates was assessed. Treatment of S. aureus with bacitracin resulted in a decreased incorporation of protein A in the bacterial cell wall, whereas incorporation of exogenous synthetic substrates was increased. These results suggest that natural and exogenous synthetic substrates are processed by S. aureus via different pathways.

Brevicidine and laterocidine are two recently discovered lipopeptide antibiotics with promising antibacterial activity. Possessing a macrocyclic core, multiple positive charges, and a lipidated N-terminus, these lipopeptides exhibit potent and selective activity against Gram-negative pathogens, including polymyxin-resistant isolates. Given the low amounts of brevicidine and laterocidine accessible by fermentation of the producing microorganisms, synthetic routes to these lipopeptides present an attractive alternative. We here report the convenient solid-phase syntheses of both brevicidine and laterocidine and confirm their potent anti-Gram-negative activities. The synthetic routes developed also provide convenient access to novel structural analogues of both brevicidine and laterocidine that display improved hydrolytic stability while maintaining potent antibacterial activity in both in vitro assays and in vivo infection models., Competing Interests: A United Kingdom priority patent application has been jointly filed by Queen's University Belfast and Leiden University, covering the synthetic routes used for the preparation of brevicidine, laterocidine, as well as all novel analogues for use in the treatment of bacterial infection., (This journal is © The Royal Society of Chemistry.)

The continued rise of antibiotic resistance is a global concern that threatens to undermine many aspects of modern medical practice. Key to addressing this threat is the discovery and development of new antibiotics that operate by unexploited modes of action. The so-called calcium-dependent lipopeptide antibiotics (CDAs) are an important emerging class of natural products that provides a source of new antibiotic agents rich in structural and mechanistic diversity. Notable in this regard is the subset of CDAs comprising the laspartomycins and amphomycins/friulimicins that specifically target the bacterial cell wall precursor undecaprenyl phosphate (C 55 -P). In this study we describe the design and synthesis of new C 55 -P-targeting CDAs with structural features drawn from both the laspartomycin and amphomycin/friulimicin classes. Assessment of these lipopeptides revealed previously unknown and surprisingly subtle structural features that are required for antibacterial activity. High-resolution crystal structures further indicate that the amphomycin/friulimicin-like lipopeptides adopt a unique crystal packing that governs their interaction with C 55 -P and provides an explanation for their antibacterial effect. In addition, live-cell microscopy studies provide further insights into the biological activity of the C 55 -P targeting CDAs highlighting their unique mechanism of action relative to the clinically used CDA daptomycin., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

The dynamic interplay of post-translational modifications (PTMs) in chromatin provides a communication system for the regulation of gene expression. An increasing number of studies have highlighted the role that such crosstalk between PTMs plays in chromatin recognition. In this study, (bio)chemical and structural approaches were applied to specifically probe the impact of acetylation of Lys 18 in the histone H3 tail peptide on peptide recognition by the protein methyltransferase coactivator-associated arginine methyltransferase 1 (CARM1). Peptidomimetics that recapitulate the transition state of protein arginine N-methyltransferases, were designed based on the H3 peptide wherein the target Arg 17 was flanked by either a free or an acetylated lysine. Structural studies with these peptidomimetics and the catalytic domain of CARM1 provide new insights into the binding of the H3 peptide within the enzyme active site. While the co-crystal structures reveal that lysine acetylation results in minor conformational differences for both CARM1 and the H3 peptide, acetylation of Lys 18 does lead to additional interactions (Van der Waals and hydrogen bonding) and likely reduces the cost of desolvation upon binding, resulting in increased affinity. Informed by these findings a series of smaller peptidomimetics were also prepared and found to maintain potent and selective CARM1 inhibition. These findings provide new insights both into the mechanism of crosstalk between arginine methylation and lysine acetylation as well as towards the development of peptidomimetic CARM1 inhibitors., (© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Pentamidine, an FDA-approved antiparasitic drug, was recently identified as an outer membrane disrupting synergist that potentiates erythromycin, rifampicin, and novobiocin against Gram-negative bacteria. The same study also described a preliminary structure-activity relationship using commercially available pentamidine analogues. We here report the design, synthesis, and evaluation of a broader panel of bis-amidines inspired by pentamidine. The present study both validates the previously observed synergistic activity reported for pentamidine, while further assessing the capacity for structurally similar bis-amidines to also potentiate Gram-positive specific antibiotics against Gram-negative pathogens. Among the bis-amidines prepared, a number of them were found to exhibit synergistic activity greater than pentamidine. These synergists were shown to effectively potentiate the activity of Gram-positive specific antibiotics against multiple Gram-negative pathogens such as Acinetobacter baumannii , Klebsiella pneumoniae , Pseudomonas aeruginosa, and Escherichia coli , including polymyxin- and carbapenem-resistant strains.

Nicotinamide N-methyltransferase (NNMT) methylates nicotinamide (NA) to generate 1-methyl nicotinamide. Since its discovery 70 years ago, the appreciation of the role of NNMT in human health has evolved from serving only metabolic functions to also being a driving force in diseases, including a variety of cancers. Despite the increasing evidence indicating NNMT as a viable therapeutic target, the development of cell-active inhibitors against this enzyme is lacking. In this review, we provide an overview of the current status of NNMT inhibitor development, relevant in vitro and in vivo studies, and a discussion of the challenges faced in the development of NNMT inhibitors., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

A recently discovered bisubstrate inhibitor of Nicotinamide N -methyltransferase (NNMT) was found to be highly potent in biochemical assays with a single digit nanomolar IC 50 value but lacking in cellular activity. We, here, report a prodrug strategy designed to translate the observed potent biochemical inhibitory activity of this inhibitor into strong cellular activity. This prodrug strategy relies on the temporary protection of the amine and carboxylic acid moieties of the highly polar amino acid side chain present in the bisubstrate inhibitor. The modification of the carboxylic acid into a range of esters in the absence or presence of a trimethyl-lock (TML) amine protecting group yielded a range of candidate prodrugs. Based on the stability in an aqueous buffer, and the confirmed esterase-dependent conversion to the parent compound, the isopropyl ester was selected as the preferred acid prodrug. The isopropyl ester and isopropyl ester-TML prodrugs exhibit improved cell permeability, which also translates to significantly enhanced cellular activity as established using assays designed to measure the enzymatic activity of NNMT in live cells.

Multidrug-resistant bacteria pose a serious global health threat as antibiotics are increasingly losing their clinical efficacy. A molecular level understanding of the mechanism of action of antimicrobials plays a key role in developing new agents to combat the threat of antimicrobial resistance. Daptomycin, the only clinically used calcium-dependent lipopeptide antibiotic, selectively disrupts Gram-positive bacterial membranes to illicit its bactericidal effect. In this study, we use isothermal titration calorimetry to further characterize the structural features of the target bacterial phospholipids that drive daptomycin binding. Our studies reveal that daptomycin shows a clear preference for the phosphoglycerol headgroup. Furthermore, unlike other calcium-dependent lipopeptide antibiotics, calcium binding by daptomycin is strongly dependent on the presence of phosphatidylglycerol. These investigations provide new insights into daptomycin's phospholipid specificity and calcium binding behavior.

Vancomycin functions by binding to lipid II, the penultimate bacterial cell wall building block used by both Gram-positive and Gram-negative species. However, vancomycin is generally only able to exert its antimicrobial effect against Gram-positive strains as it cannot pass the outer membrane (OM) of Gram-negative bacteria. To address this challenge, we here describe efforts to conjugate vancomycin to the OM disrupting polymyxin E nonapeptide (PMEN) to yield the hybrid "vancomyxins". In designing these hybrid antibiotics, different spacers and conjugation sites were explored for connecting vancomycin and PMEN. The vancomyxins show improved activity against Gram-negative strains compared with the activity of vancomycin or vancomycin supplemented with PMEN separately. In addition, the vancomyxins maintain the antimicrobial effect of vancomycin against Gram-positive strains and, in some cases, show enhanced activity against vancomycin-resistant strains. The hybrid antibiotics described here have reduced nephrotoxicity when compared with clinically used polymyxin antibiotics. This study demonstrates that covalent conjugation to an OM disruptor contributes to sensitizing Gram-negative strains to vancomycin while retaining anti-Gram-positive activity.

Nicotinamide N -methyltransferase (NNMT) methylates nicotinamide (vitamin B3) to generate 1-methylnicotinamide (MNA). NNMT overexpression has been linked to a variety of diseases, most prominently human cancers, indicating its potential as a therapeutic target. The development of small-molecule NNMT inhibitors has gained interest in recent years, with the most potent inhibitors sharing structural features based on elements of the nicotinamide substrate and the S -adenosyl-l-methionine (SAM) cofactor. We here report the development of new bisubstrate inhibitors that include electron-deficient aromatic groups to mimic the nicotinamide moiety. In addition, a trans -alkene linker was found to be optimal for connecting the substrate and cofactor mimics in these inhibitors. The most potent NNMT inhibitor identified exhibits an IC 50 value of 3.7 nM, placing it among the most active NNMT inhibitors reported to date. Complementary analytical techniques, modeling studies, and cell-based assays provide insights into the binding mode, affinity, and selectivity of these inhibitors.

Nicotinamide N -methyltransferase (NNMT) methylates nicotinamide to form 1-methylnicotinamide (MNA) using S -adenosyl-l-methionine (SAM) as the methyl donor. The complexity of the role of NNMT in healthy and disease states is slowly being elucidated and provides an indication that NNMT may be an interesting therapeutic target for a variety of diseases including cancer, diabetes, and obesity. Most inhibitors of NNMT described to date are structurally related to one or both of its substrates. In the search for structurally diverse NNMT inhibitors, an mRNA display screening technique was used to identify macrocyclic peptides which bind to NNMT. Several of the cyclic peptides identified in this manner show potent inhibition of NNMT with IC 50 values as low as 229 nM. The peptides were also found to downregulate MNA production in cellular assays. Interestingly, substrate competition experiments reveal that these cyclic peptide inhibitors are noncompetitive with either SAM or NA indicating they may be the first allosteric inhibitors reported for NNMT., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

The continuing rise of antibiotic resistance, particularly among Gram-negative pathogens, threatens to undermine many aspects of modern medical practice. To address this threat, novel antibiotics that utilize unexploited bacterial targets are urgently needed. Over the past decade, a number of studies have highlighted the antibacterial potential of macrocyclic peptides that target Gram-negative outer membrane proteins (OMPs). Recently, it was reported that the antibacterial activities of OMP-targeting macrocyclic peptidomimetics of the antimicrobial peptide protegrin-1 are dramatically enhanced upon linking to polymyxin E nonapeptide (PMEN). In this study, we describe a convergent, chemoenzymatic route for the convenient preparation of such conjugates. Specifically, we investigated the use of both amide bond formation and azide-alkyne ligation for connecting an OMP-targeting macrocyclic peptide to a PMEN building block (obtained by enzymatic degradation of polymyxin E). The conjugates obtained via both approaches display potent antibacterial activity against a range of Gram-negative pathogens including multi-drug-resistant isolates.