Discovery and Derivatization of Tridecaptin Antibiotics with Altered Host Specificity and Enhanced Bioactivity.

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 A5 and tridecaptin D, which exhibit unusual bioactivities within their class. The change in the antibacterial spectrum of Oct-TriA5 was explained solely by a Phe to Trp substitution as compared to Oct-TriA1, while Oct-TriD contained 6 substitutions. Metabolomic analysis of producer Paenibacillus sp. JJ-21 validated the predicted amino acid sequence of tridecaptin A5. 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.

Knockdown of nicotinamide N-methyltransferase suppresses proliferation, migration, and chemoresistance of Merkel cell carcinoma cells in vitro.

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.

Recent Advances in the Development of Polymyxin Antibiotics: 2010-2023.

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.

The enigmatic mode of action of the lantibiotic epilancin 15X.

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.

Semisynthetic polymyxins with potent antibacterial activity and reduced kidney cell toxicity.

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.

The plant stress hormone jasmonic acid evokes defensive responses in streptomycetes.

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.

Biochemical and Cellular Characterization of the Function of Fluorophosphonate-Binding Hydrolase H (FphH) in Staphylococcus aureus Support a Role in Bacterial Stress Response.

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 yvaK in Bacillus subtilis) at the molecular and cellular level. First, phenotypic characterization of fphH-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 fphH 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.

Artificial intelligence for natural product drug discovery.

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.

MYC is a clinically significant driver of mTOR inhibitor resistance in breast cancer.

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.

In vivo biodistribution of kinetically stable Pt2L4 nanospheres that show anti-cancer activity.

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 Pt2L4 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 Pt2L4 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 Pt2L4 spheres together with their cytotoxic and photophysical properties provides the fundament for MOC application in cancer therapy.

Targeting membrane-bound bacterial cell wall precursors: a tried and true antibiotic strategy in nature and the clinic.

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.

Linearization of the Brevicidine and Laterocidine Lipopeptides Yields Analogues That Retain Full Antibacterial Activity.

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.

A living biobank of patient-derived ductal carcinoma in situ mouse-intraductal xenografts identifies risk factors for invasive progression.

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.

Dual targeting of the class V lanthipeptide antibiotic cacaoidin.

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 IIPGN 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 IIPGN and direct inhibition of cell wall transglycosylases.

Total Synthesis and Structure Assignment of the Relacidine Lipopeptide Antibiotics and Preparation of Analogues with Enhanced Stability.

The unabated rise of antibiotic resistance has raised the specter of a post-antibiotic era and underscored the importance of developing new classes of antibiotics. The relacidines are a recently discovered group of nonribosomal lipopeptide antibiotics that show promising activity against Gram-negative pathogens and share structural similarities with brevicidine and laterocidine. While the first reports of the relacidines indicated that they possess a C-terminal five-amino acid macrolactone, an N-terminal lipid tail, and an overall positive charge, no stereochemical configuration was assigned, thereby precluding a full structure determination. To address this issue, we here report a bioinformatics guided total synthesis of relacidine A and B and show that the authentic natural products match our predicted and synthesized structures. Following on this, we also synthesized an analogue of relacidine A wherein the ester linkage of the macrolactone was replaced by the corresponding amide. This analogue was found to possess enhanced hydrolytic stability while maintaining the antibacterial activity of the natural product in both in vitro and in vivo efficacy studies.

Chimeric Peptidomimetic Antibiotic Efficiently Neutralizes Lipopolysaccharides (LPS) and Bacteria-Induced Activation of RAW Macrophages.

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 ß-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.

Chemical Proteomics Reveals Antibiotic Targets of Oxadiazolones in MRSA.

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.

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

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.

Polymyxin Stereochemistry and Its Role in Antibacterial Activity and Outer Membrane Disruption.

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.

Synthesis and Structure-Activity Studies of ß-Barrel Assembly Machine Complex Inhibitor MRL-494.

In the hunt for new antibiotics with activity against Gram-negative pathogens, the outer membrane ß-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.