Full Profiling of GE81112A, an Underexplored Tetrapeptide Antibiotic with Activity against Gram-Negative Pathogens.

After the first total synthesis combined with structure revision, we performed thorough in vitro and in vivo profiling of the underexplored tetrapeptide GE81112A. From the determination of the biological activity spectrum and physicochemical and early absorption-distribution-metabolism-excretion-toxicity (eADMET) properties, as well as in vivo data regarding tolerability and pharmacokinetics (PK) in mice and efficacy in an Escherichia coli-induced septicemia model, we were able to identify the critical and limiting parameters of the original hit compound. Thus, the generated data will serve as the basis for further compound optimization programs and developability assessments to identify candidates for preclinical/clinical development derived from GE81112A as the lead structure. IMPORTANCE The spread of antimicrobial resistance (AMR) is becoming a more and more important global threat to human health. With regard to current medical needs, penetration into the site of infection represents the major challenge in the treatment of infections caused by Gram-positive bacteria. Considering infections associated with Gram-negative bacteria, resistance is a major issue. Obviously, novel scaffolds for the design of new antibacterials in this arena are urgently needed to overcome this crisis. Such a novel potential lead structure is represented by the GE81112 compounds, which inhibit protein synthesis by interacting with the small 30S ribosomal subunit using a binding site distinct from that of other known ribosome-targeting antibiotics. Therefore, the tetrapeptide antibiotic GE81112A was chosen for further exploration as a potential lead for the development of antibiotics with a new mode of action against Gram-negative bacteria.

Total Synthesis of GE81112A: An Orthoester-Based Approach.

The GE81112 series, consisting of three naturally occurring tetrapeptides and synthetic derivatives, is evaluated as a potential lead structure for the development of a new antibacterial drug. Although the first total synthesis of GE81112A reported by our group provided sufficient amounts of material for an initial in depth biological profiling of the compound, improvements of the routes toward the key building blocks were needed for further upscaling and structure-activity relationship studies. The major challenges identified were poor stereoselectivity in the synthesis of the C-terminal ß-hydroxy histidine intermediate and a concise access to all four isomers of the 3-hydroxy pipecolic acid. Herein, we report a second-generation synthesis of GE81112A, which is also applicable to access further representatives of this series. Based on Lajoie's ortho-ester-protected serine aldehydes as key building blocks, the described route provides both a satisfactory improvement in stereoselectivity of the ß-hydroxy histidine intermediate synthesis and a stereoselective approach toward both orthogonally protected cis and trans-3-hydroxy pipecolic acid.

Fighting antibiotic resistance-strategies and (pre)clinical developments to find new antibacterials.

Antibacterial resistance is one of the greatest threats to human health. The development of new therapeutics against bacterial pathogens has slowed drastically since the approvals of the first antibiotics in the early and mid-20th century. Most of the currently investigated drug leads are modifications of approved antibacterials, many of which are derived from natural products. In this review, we highlight the challenges, advancements and current standing of the clinical and preclinical antibacterial research pipeline. Additionally, we present novel strategies for rejuvenating the discovery process and advocate for renewed and enthusiastic investment in the antibacterial discovery pipeline.

Isolation, characterization, and bioactivity profiling of oxazoline containing madurastatin siderophores from Actinomadura sp.

Nine new hydroxyphenyloxazolines, madurastatin B4, C2, D3 and D4, E1 and E2, F1 as well as G1 and G2 (8-16), along with two new enantiomers of madurastatin D1 (ent-6) and D2 (ent-7) and two known congeners, madurastatin B1 (2) and C1 (5), were isolated from the liquid culture of Actinomadura sp. ST100801 based on the initial activity against Escherichia coli screened in bicarbonate-supplemented Mueller Hinton II medium and identification via molecular networking. Structure elucidation was achieved by comprehensive 1D and 2D NMR as well as MS/MS fragmentation analyses. Their absolute configuration was determined by Marfey's analysis. Complemented with functionalized hydroxyphenyloxazolines (2, 4, 17-18) obtained by total synthesis, the isolated compounds were evaluated for antibacterial activities revealing MICs down to 4 µg ml-1 against Moraxella catarrhalis. Therefore, this study enlarges the family of madurastatin siderophores.

Natural products in antibiotic development: is the success story over?

Natural product (NP)-based antibiotics have been exploited for more than eighty years and continue saving uncountable lives every year. However, antimicrobial R&D is inadequate to counteract antimicrobial resistance. The majority of marketed antibiotics are inspired by NP classes that were discovered more than 50 years ago. With the advent of advanced genomic approaches, cultivation methods, and modern analytical techniques, NP discovery holds promise that there are way more powerful antibiotic scaffolds to be discovered. However, the currently lean antibiotic R&D pipeline shows a clear trend away from NP-based programs and innovative compounds are also rare in early stages. Within this review, we give an overview of the current NP antibiotic development pipeline, elaborate constraints the field is facing, and suggest measures to streamline NP-based antibiotic discovery. It is unlikely that NPs have lost significance, but reinforcement of discovery will require more targeted efforts and support to revitalize this established source.

Genomic and Chemical Decryption of the Bacteroidetes Phylum for Its Potential to Biosynthesize Natural Products.

With progress in genome sequencing and data sharing, 1,000s of bacterial genomes are publicly available. Genome mining-using bioinformatics tools in terms of biosynthetic gene cluster (BGC) identification, analysis, and rating-has become a key technology to explore the capabilities for natural product (NP) biosynthesis. Comprehensively, analyzing the genetic potential of the phylum Bacteroidetes revealed Chitinophaga as the most talented genus in terms of BGC abundance and diversity. Guided by the computational predictions, we conducted a metabolomics and bioactivity driven NP discovery program on 25 Chitinophaga strains. High numbers of strain-specific metabolite buckets confirmed the upfront predicted biosynthetic potential and revealed a tremendous uncharted chemical space. Mining this data set, we isolated the new iron chelating nonribosomally synthesized cyclic tetradeca- and pentadecalipodepsipeptide antibiotics chitinopeptins with activity against Candida, produced by C. eiseniae DSM 22224 and C. flava KCTC 62435, respectively. IMPORTANCE The development of pipelines for anti-infectives to be applied in plant, animal, and human health management are dried up. However, the resistance development against compounds in use calls for new lead structures. To fill this gap and to enhance the probability of success for the discovery of new bioactive natural products, microbial taxa currently underinvestigated must be mined. This study investigates the potential within the bacterial phylum Bacteroidetes. A combination of omics-technologies revealed taxonomical hot spots for specialized metabolites. Genome- and metabolome-based analyses showed that the phylum covers a new chemical space compared with classic natural product producers. Members of the Bacteroidetes may thus present a promising bioresource for future screening and isolation campaigns.

Identification, Characterization, and Synthesis of Natural Parasitic Cysteine Protease Inhibitors: Pentacitidins Are More Potent Falcitidin Analogues.

Protease inhibitors represent a promising therapeutic option for the treatment of parasitic diseases such as malaria and human African trypanosomiasis. Falcitidin was the first member of a new class of inhibitors of falcipain-2, a cysteine protease of the malaria parasite Plasmodium falciparum. Using a metabolomics dataset of 25 Chitinophaga strains for molecular networking enabled identification of over 30 natural analogues of falcitidin. Based on MS/MS spectra, they vary in their amino acid chain length, sequence, acyl residue, and C-terminal functionalization; therefore, they were grouped into the four falcitidin peptide families A-D. The isolation, characterization, and absolute structure elucidation of two falcitidin-related pentapeptide aldehyde analogues by extensive MS/MS spectrometry and NMR spectroscopy in combination with advanced Marfey's analysis was in agreement with the in silico analysis of the corresponding biosynthetic gene cluster. Total synthesis of chosen pentapeptide analogues followed by in vitro testing against a panel of proteases revealed selective parasitic cysteine protease inhibition and, additionally, low-micromolar inhibition of α-chymotrypsin. The pentapeptides investigated here showed superior inhibitory activity compared to falcitidin.

Trichoderma-Derived Pentapeptides from the Infected Nest Mycobiome of the Subterranean Termite Coptotermes testaceus.

Termites live in a dynamic environment where colony health is strongly influenced by surrounding microbes. However, little is known about the mycobiomes of lower termites and their nests, and how these change in response to disease. Here we compared the individual and nest mycobiomes of a healthy subterranean termite colony (Coptotermes testaceus) to one infected and ultimately eradicated by a fungal pathogen. We identified Trichoderma species in the materials of both nests, but they were also abundant in the infected termites. Methanolic extracts of Trichoderma sp. FHG000531, isolated from the infected nest, were screened for secondary metabolites by UHPLC-HR MS/MS-guided molecular networking. We identified many bioactive compounds with potential roles in the eradication of the infected colony, as well as a cluster of six unknown peptides. The novel peptide FE011 was isolated and characterized by NMR spectroscopy. The function of this novel peptide family as well as the role of Trichoderma species in dying termite colonies therefore requires further investigation.

The Discovery and Structure-Activity Evaluation of (+)-Floyocidin B and Synthetic Analogs.

Tuberculosis represents one of the ten most common courses of death worldwide and the emergence of multidrug-resistant M. tuberculosis makes the discovery of novel anti-tuberculosis active structures an urgent priority. Here, we show that (+)-floyocidin B representing the first example of a novel dihydroisoquinoline class of fungus-derived natural products, displays promising antitubercular hit properties. (+)-Floyocidin B was identified by activity-guided extract screening and its structure was unambiguously determined by total synthesis. The absolute configuration was deduced from a key synthesis intermediate by single crystal X-ray diffraction analysis. A hit series was generated by the isolation of further natural congeners and the synthesis of analogs of (+)-floyocidin B. Extensive biological and physicochemical profiling of this series revealed first structure-activity relationships and set the basis for further optimization and development of this novel antitubercular scaffold.

Combination of high-throughput microfluidics and FACS technologies to leverage the numbers game in natural product discovery.

High-throughput platforms facilitating screening campaigns of environmental samples are needed to discover new products of natural origin counteracting the spreading of antimicrobial resistances constantly threatening human and agricultural health. We applied a combination of droplet microfluidics and fluorescence-activated cell sorting (FACS)-based technologies to access and assess a microbial environmental sample. The cultivation performance of our microfluidics workflow was evaluated in respect to the utilized cultivation media by Illumina amplicon sequencing of a pool of millions of droplets, respectively. This enabled the rational selection of a growth medium supporting the isolation of microbial diversity from soil (five phyla affiliated to 57 genera) including a member of the acidobacterial subgroup 1 (genus Edaphobacter). In a second phase, the entire diversity covered by 1071 cultures was used for an arrayed bioprospecting campaign, resulting in > 6000 extracts tested against human pathogens and agricultural pests. After redundancy curation by using a combinatorial chemical and genomic fingerprinting approach, we assigned the causative agents present in the extracts. Utilizing UHPLC-QTOF-MS/MS-guided fractionation and microplate-based screening assays in combination with molecular networking the production of bioactive ionophorous macrotetrolides, phospholipids, the cyclic lipopetides massetolides E, F, H and serratamolide A and many derivatives thereof was shown.

Novel Glycerophospholipid, Lipo- and N-acyl Amino Acids from Bacteroidetes: Isolation, Structure Elucidation and Bioactivity.

The 'core' metabolome of the Bacteroidetes genus Chitinophaga was recently discovered to consist of only seven metabolites. A structural relationship in terms of shared lipid moieties among four of them was postulated. Here, structure elucidation and characterization via ultra-high resolution mass spectrometry (UHR-MS) and nuclear magnetic resonance (NMR) spectroscopy of those four lipids (two lipoamino acids (LAAs), two lysophosphatidylethanolamines (LPEs)), as well as several other undescribed LAAs and N-acyl amino acids (NAAAs), identified during isolation were carried out. The LAAs represent closely related analogs of the literature-known LAAs, such as the glycine-serine dipeptide lipids 430 (2) and 654. Most of the here characterized LAAs (1, 5-11) are members of a so far undescribed glycine-serine-ornithine tripeptide lipid family. Moreover, this study reports three novel NAAAs (N-(5-methyl)hexanoyl tyrosine (14) and N-(7-methyl)octanoyl tyrosine (15) or phenylalanine (16)) from Olivibacter sp. FHG000416, another Bacteroidetes strain initially selected as best in-house producer for isolation of lipid 430. Antimicrobial profiling revealed most isolated LAAs (1-3) and the two LPE 'core' metabolites (12, 13) active against the Gram-negative pathogen M. catarrhalis ATCC 25238 and the Gram-positive bacterium M. luteus DSM 20030. For LAA 1, additional growth inhibition activity against B. subtilis DSM 10 was observed.

Towards the sustainable discovery and development of new antibiotics.

An ever-increasing demand for novel antimicrobials to treat life-threatening infections caused by the global spread of multidrug-resistant bacterial pathogens stands in stark contrast to the current level of investment in their development, particularly in the fields of natural-product-derived and synthetic small molecules. New agents displaying innovative chemistry and modes of action are desperately needed worldwide to tackle the public health menace posed by antimicrobial resistance. Here, our consortium presents a strategic blueprint to substantially improve our ability to discover and develop new antibiotics. We propose both short-term and long-term solutions to overcome the most urgent limitations in the various sectors of research and funding, aiming to bridge the gap between academic, industrial and political stakeholders, and to unite interdisciplinary expertise in order to efficiently fuel the translational pipeline for the benefit of future generations.

Towards the sustainable discovery and development of new antibiotics.

An ever-increasing demand for novel antimicrobials to treat life-threatening infections caused by the global spread of multidrug-resistant bacterial pathogens stands in stark contrast to the current level of investment in their development, particularly in the fields of natural-product-derived and synthetic small molecules. New agents displaying innovative chemistry and modes of action are desperately needed worldwide to tackle the public health menace posed by antimicrobial resistance. Here, our consortium presents a strategic blueprint to substantially improve our ability to discover and develop new antibiotics. We propose both short-term and long-term solutions to overcome the most urgent limitations in the various sectors of research and funding, aiming to bridge the gap between academic, industrial and political stakeholders, and to unite interdisciplinary expertise in order to efficiently fuel the translational pipeline for the benefit of future generations.

High-Throughput Cultivation for the Selective Isolation of Acidobacteria From Termite Nests.

Microbial communities in the immediate environment of socialized invertebrates can help to suppress pathogens, in part by synthesizing bioactive natural products. Here we characterized the core microbiomes of three termite species (genus Coptotermes) and their nest material to gain more insight into the diversity of termite-associated bacteria. Sampling a healthy termite colony over time implicated a consolidated and highly stable microbiome, pointing toward the fact that beneficial bacterial phyla play a major role in termite fitness. In contrast, there was a significant shift in the composition of the core microbiome in one nest during a fungal infection, affecting the abundance of well-characterized Streptomyces species (phylum Actinobacteria) as well as less-studied bacterial phyla such as Acidobacteria. High-throughput cultivation in microplates was implemented to isolate and identify these less-studied bacterial phylogenetic group. Amplicon sequencing confirmed that our method maintained the bacterial diversity of the environmental samples, enabling the isolation of novel Acidobacteriaceae and expanding the list of cultivated species to include two strains that may define new species within the genera Terracidiphilus and Acidobacterium.

Total Synthesis and Structure Revision of (-)-Avicennone C.

All four possible stereoisomers of the natural product (-)-avicennone C were synthesized using two different methods for ring closure. The absolute stereochemistry was elucidated unambiguously by comparison of the analytical data with those of the reported natural product and by single X-ray crystal diffraction of synthetic intermediates. The proposed structure needed to be revised with regard to the absolute configuration of the stereogenic center bearing the secondary hydroxyl group. The reported synthesis offers a flexible, selective, and efficient access to all possible stereoisomers and may be of value for the stereoselective synthesis of other epoxyquinone natural products.

Molecular Networking-Guided Discovery and Characterization of Stechlisins, a Group of Cyclic Lipopeptides from a Pseudomonas sp.

Increasingly sensitive analytical instruments and robust downstream data processing tools have revolutionized natural product research over the past decade. A molecular networking-guided survey led to the identification of 33 new cyclic lipopeptides (CLPs) from the culture broth of the proteobacterium Pseudomonas sp. FhG100052. The compound family resembles members of the amphisin group of CLPs that possess a 3-hydroxy fatty acid linked to the N-terminus of an undecapeptide core. Culture optimization led to the isolation and subsequent structure elucidation of one known and five new derivatives by extensive MS/MS and NMR experiments in combination with Marfey's analysis. The data were in agreement with in silico analysis of the corresponding biosynthetic gene cluster. Most strikingly, the length of the incorporated fatty acid defined the growth inhibitory effects against Moraxella catarrhalis FH6810, as observed by MIC values ranging from no inhibition (>128 µg/mL) to 4 µg/mL.

Antimicrobial Peptides from Rat-Tailed Maggots of the Drone Fly Eristalis tenax Show Potent Activity against Multidrug-Resistant Gram-Negative Bacteria.

The spread of multidrug-resistant Gram-negative bacteria is an increasing threat to human health, because novel compound classes for the development of antibiotics have not been discovered for decades. Antimicrobial peptides (AMPs) may provide a much-needed breakthrough because these immunity-related defense molecules protect many eukaryotes against Gram-negative pathogens. Recent concepts in evolutionary immunology predict the presence of potent AMPs in insects that have adapted to survive in habitats with extreme microbial contamination. For example, the saprophagous and coprophagous maggots of the drone fly Eristalis tenax (Diptera) can flourish in polluted aquatic habitats, such as sewage tanks and farmyard liquid manure storage pits. We used next-generation sequencing to screen the E. tenax immunity-related transcriptome for AMPs that are synthesized in response to the injection of bacterial lipopolysaccharide. We identified 22 AMPs and selected nine for larger-scale synthesis to test their activity against a broad spectrum of pathogens, including multidrug-resistant Gram-negative bacteria. Two cecropin-like peptides (EtCec1-a and EtCec2-a) and a diptericin-like peptide (EtDip) displayed strong activity against the pathogens, even under simulated physiological conditions, and also achieved a good therapeutic window. Therefore, these AMPs could be used as leads for the development of novel antibiotics.

Profiling antimicrobial peptides from the medical maggot Lucilia sericata as potential antibiotics for MDR Gram-negative bacteria.

Background: The ability of MDR Gram-negative bacteria to evade even antibiotics of last resort is a severe global challenge. The development pipeline for conventional antibiotics cannot address this issue, but antimicrobial peptides (AMPs) offer an alternative solution. Objectives: Two insect-derived AMPs (LS-sarcotoxin and LS-stomoxyn) were profiled to assess their suitability for systemic application in humans. Methods: The peptides were tested against an extended panel of 114 clinical MDR Gram-negative bacterial isolates followed by time-kill analysis, interaction studies and assays to determine the likelihood of emerging resistance. In further in vitro studies we addressed cytotoxicity, cardiotoxicity and off-target interactions. In addition, an in vivo tolerability and pharmacokinetic study in mice was performed. Results: LS-sarcotoxin and LS-stomoxyn showed potent and selective activity against Gram-negative bacteria and no cross-resistance with carbapenems, fluoroquinolones or aminoglycosides. Peptide concentrations of 4 or 8 mg/L inhibited 90% of the clinical MDR isolates of Escherichia coli, Enterobacter cloacae, Acinetobacter baumannii and Salmonella enterica isolates tested. The 'all-d' homologues of the peptides displayed markedly reduced activity, indicating a chiral target. Pharmacological profiling revealed a good in vitro therapeutic index, no cytotoxicity or cardiotoxicity, an inconspicuous broad-panel off-target profile, and no acute toxicity in mice at 10 mg/kg. In mouse pharmacokinetic experiments LS-sarcotoxin and LS-stomoxyn plasma levels above the lower limit of quantification (1 and 0.25 mg/mL, respectively) were detected after 5 and 15 min, respectively. Conclusions: LS-sarcotoxin and LS-stomoxyn are suitable as lead candidates for the development of novel antibiotics; however, their pharmacokinetic properties need to be improved for systemic administration.

Biological Profiling of Coleoptericins and Coleoptericin-Like Antimicrobial Peptides from the Invasive Harlequin Ladybird Harmonia axyridis.

The spread of antibiotic-resistant human pathogens and the declining number of novel antibiotics in the development pipeline is a global challenge that has fueled the demand for alternative options. The search for novel drug candidates has expanded to include not only antibiotics but also adjuvants capable of restoring antibiotic susceptibility in multidrug-resistant (MDR) pathogens. Insect-derived antimicrobial peptides (AMPs) can potentially fulfil both of these functions. We tested two coleoptericins and one coleoptericin-like peptides from the invasive harlequin ladybird Harmonia axyridis against a panel of human pathogens. The AMPs displayed little or no activity when tested alone but were active even against clinical MDR isolates of the Gram-negative ESKAPE strains when tested in combination with polymyxin derivatives, such as the reserve antibiotic colistin, at levels below the minimal inhibitory concentration. Assuming intracellular targets of the AMPs, our data indicate that colistin potentiates the activity of the AMPs. All three AMPs achieved good in vitro therapeutic indices and high intrahepatic stability but low plasma stability, suggesting they could be developed as adjuvants for topical delivery or administration by inhalation for anti-infective therapy to reduce the necessary dose of colistin (and thus its side effects) or to prevent development of colistin resistance in MDR pathogens.

Total Synthesis and Structural Revision of the Antibiotic Tetrapeptide GE81112A.

The total synthesis of the naturally occurring antibiotic GE81112A, a densely functionalized tetrapeptide, is reported. Comparison of spectral data with those of the natural product and the lack of biological activity of the synthesized compound led us to revise the published configuration of the 3-hydroxypipecolic acid moiety. This hypothesis was fully validated by the synthesis of the corresponding epimer.