Sophisticated natural products as antibiotics.

In this Review, we explore natural product antibiotics that do more than simply inhibit an active site of an essential enzyme. We review these compounds to provide inspiration for the design of much-needed new antibacterial agents, and examine the complex mechanisms that have evolved to effectively target bacteria, including covalent binders, inhibitors of resistance, compounds that utilize self-promoted entry, those that evade resistance, prodrugs, target corrupters, inhibitors of 'undruggable' targets, compounds that form supramolecular complexes, and selective membrane-acting agents. These are exemplified by ß-lactams that bind covalently to inhibit transpeptidases and ß-lactamases, siderophore chimeras that hijack import mechanisms to smuggle antibiotics into the cell, compounds that are activated by bacterial enzymes to produce reactive molecules, and antibiotics such as aminoglycosides that corrupt, rather than merely inhibit, their targets. Some of these mechanisms are highly sophisticated, such as the preformed ß-strands of darobactins that target the undruggable ß-barrel chaperone BamA, or teixobactin, which binds to a precursor of peptidoglycan and then forms a supramolecular structure that damages the membrane, impeding the emergence of resistance. Many of the compounds exhibit more than one notable feature, such as resistance evasion and target corruption. Understanding the surprising complexity of the best antimicrobial compounds provides a roadmap for developing novel compounds to address the antimicrobial resistance crisis by mining for new natural products and inspiring us to design similarly sophisticated antibiotics.

Structural insights into ClpP protease side exit pore-opening by a pH drop coupled with substrate hydrolysis.

The ClpP serine peptidase is a tetradecameric degradation molecular machine involved in many physiological processes. It becomes a competent ATP-dependent protease when coupled with Clp-ATPases. Small chemical compounds, acyldepsipeptides (ADEPs), are known to cause the dysregulation and activation of ClpP without ATPases and have potential as novel antibiotics. Previously, structural studies of ClpP from various species revealed its structural details, conformational changes, and activation mechanism. Although product release through side exit pores has been proposed, the detailed driving force for product release remains elusive. Herein, we report crystal structures of ClpP from Bacillus subtilis (BsClpP) in unforeseen ADEP-bound states. Cryo-electron microscopy structures of BsClpP revealed various conformational states under different pH conditions. To understand the conformational change required for product release, we investigated the relationship between substrate hydrolysis and the pH-lowering process. The production of hydrolyzed peptides from acidic and basic substrates by proteinase K and BsClpP lowered the pH values. Our data, together with those of previous findings, provide insight into the molecular mechanism of product release by the ClpP self-compartmentalizing protease.

Chemical Predictive Modelling and Natural Product-based Divergent Synthesis - Design of Type B PPAPs with Nanomolar Activities against MRSA.

In response to the pressing global challenge of antibiotic resistance, time efficient design and synthesis of novel antibiotics are of immense need. Polycyclic polyprenylated acylphloroglucinols (PPAP) were previously reported to effectively combat a range of gram-positive bacteria. Although the exact mode of action is still not clear, we conceptualized a late-stage divergent synthesis approach to expand our natural product-based PPAP library by 30 additional entities to perform SAR studies against methicillin-resistant Staphylococcus aureus (MRSA). Although at this point only data from cellular assays are available and understanding of molecular drug-target interactions are lacking, the experimental data were used to generate 3D-QSAR models via an artificial intelligence training and to identify a common pharmacophore model. The experimentally validated QSAR model enabled the estimation of anti-MRSA activities of a virtual compound library consisting of more than 100,000 in-silico generated B PPAPs, out of which the 20 most promising candidates were synthesized. These novel PPAPs revealed significantly improved cellular activities against MRSA with growth inhibition down to concentrations less than 1 µm.

Structure of Staphylococcus aureus ClpP Bound to the Covalent Active-Site Inhibitor Cystargolide A.

The caseinolytic protease is a highly conserved serine protease, crucial to prokaryotic and eukaryotic protein homeostasis, and a promising antibacterial and anticancer drug target. Herein, we describe the potent cystargolides as the first natural ß-lactone inhibitors of the proteolytic core ClpP. Based on the discovery of two clpP genes next to the cystargolide biosynthetic gene cluster in Kitasatospora cystarginea, we explored ClpP as a potential cystargolide target. We show the inhibition of Staphylococcus aureus ClpP by cystargolide A and B by different biochemical methods in vitro. Synthesis of semisynthetic derivatives and probes with improved cell penetration allowed us to confirm ClpP as a specific target in S. aureus cells and to demonstrate the anti-virulence activity of this natural product class. Crystal structures show cystargolide A covalently bound to all 14 active sites of ClpP from S. aureus, Aquifex aeolicus, and Photorhabdus laumondii, and reveal the molecular mechanism of ClpP inhibition by ß-lactones, the predominant class of ClpP inhibitors.

The Cyanobacterial "Nutraceutical" Phycocyanobilin Inhibits Cysteine Protease Legumain.

The blue biliprotein phycocyanin, produced by photo-autotrophic cyanobacteria including spirulina (Arthrospira) and marketed as a natural food supplement or "nutraceutical," is reported to have anti-inflammatory, antioxidant, immunomodulatory, and anticancer activity. These diverse biological activities have been specifically attributed to the phycocyanin chromophore, phycocyanobilin (PCB). However, the mechanism of action of PCB and the molecular targets responsible for the beneficial properties of PCB are not well understood. We have developed a procedure to rapidly cleave the PCB pigment from phycocyanin by ethanolysis and then characterized it as an electrophilic natural product that interacts covalently with thiol nucleophiles but lacks any appreciable cytotoxicity or antibacterial activity against common pathogens and gut microbes. We then designed alkyne-bearing PCB probes for use in chemical proteomics target deconvolution studies. Target identification and validation revealed the cysteine protease legumain (also known as asparaginyl endopeptidase, AEP) to be a target of PCB. Inhibition of this target may account for PCB's diverse reported biological activities.

High Plasticity of the Amicetin Biosynthetic Pathway in Streptomyces sp. SHP 22-7 Led to the Discovery of Streptcytosine P and Cytosaminomycins F and G and Facilitated the Production of 12F-Plicacetin.

A chemical reinvestigation of the Indonesian strain Streptomyces sp. SHP 22-7 led to the isolation of three new pyrimidine nucleosides, along with six known analogues and zincphyrin. The structures of the new compounds (6, 7, 10) were elucidated by employing spectroscopic techniques (NMR, MS, CD, and IR) as well as enantioselective analyses of methyl branched side chain configurations. Application of the precursor-directed feeding approach led to the production and partial isolation of nine further pyrimidine analogues. The new compounds 6, 7, and 11 and three of the known compounds (2-4) were found to possess antimycobacterial and cytotoxic properties.

Human commensals producing a novel antibiotic impair pathogen colonization.

The vast majority of systemic bacterial infections are caused by facultative, often antibiotic-resistant, pathogens colonizing human body surfaces. Nasal carriage of Staphylococcus aureus predisposes to invasive infection, but the mechanisms that permit or interfere with pathogen colonization are largely unknown. Whereas soil microbes are known to compete by production of antibiotics, such processes have rarely been reported for human microbiota. We show that nasal Staphylococcus lugdunensis strains produce lugdunin, a novel thiazolidine-containing cyclic peptide antibiotic that prohibits colonization by S. aureus, and a rare example of a non-ribosomally synthesized bioactive compound from human-associated bacteria. Lugdunin is bactericidal against major pathogens, effective in animal models, and not prone to causing development of resistance in S. aureus. Notably, human nasal colonization by S. lugdunensis was associated with a significantly reduced S. aureus carriage rate, suggesting that lugdunin or lugdunin-producing commensal bacteria could be valuable for preventing staphylococcal infections. Moreover, human microbiota should be considered as a source for new antibiotics.

Discovery of Ircinianin Lactones B and C-Two New Cyclic Sesterterpenes from the Marine Sponge Ircinia wistarii.

Two new ircinianin-type sesterterpenoids, ircinianin lactone B and ircinianin lactone C (7 and 8), together with five known entities from the ircinianin compound family (1, 3-6) were isolated from the marine sponge Ircinia wistarii. Ircinianin lactones B and C (7 and 8) represent new ircinianin terpenoids with a modified oxidation pattern. Despite their labile nature, the structures could be established using a combination of spectroscopic data, including HRESIMS and 1D/2D NMR techniques, as well as computational chemistry and quantum-mechanical calculations. In a broad screening approach for biological activity, the class-defining compound ircinianin (1) showed moderate antiprotozoal activity against Plasmodium falciparum (IC50 25.4 µM) and Leishmania donovani (IC50 16.6 µM).

Synthesis of tryptophan-dehydrobutyrine diketopiperazine and biological activity of hangtaimycin and its co-metabolites.

An improved synthesis for tryptophan-dehydrobutyrine diketopiperazine (TDD), a co-metabolite of the hybrid polyketide/non-ribosomal peptide hangtaimycin, starting from ʟ-tryptophan is presented. Comparison to TDD isolated from the hangtaimycin producer Streptomyces spectabilis confirmed its S configuration. The X-ray structure of the racemate shows an interesting dimerisation through hydrogen bridges. The results from bioactivity testings of hangtaimycin, TDD and the hangtaimycin degradation product HTM222 are given.

The Antibiotic Negamycin Crosses the Bacterial Cytoplasmic Membrane by Multiple Routes.

Negamycin is a natural pseudodipeptide antibiotic with promising activity against Gram-negative and Gram-positive bacteria, including Enterobacteriaceae, Pseudomonas aeruginosa, and Staphylococcus aureus, and good efficacy in infection models. It binds to ribosomes with a novel binding mode, stimulating miscoding and inhibiting ribosome translocation. We were particularly interested in studying how the small, positively charged natural product reaches its cytoplasmic target in Escherichia coli Negamycin crosses the cytoplasmic membrane by multiple routes depending on environmental conditions. In a peptide-free medium, negamycin uses endogenous peptide transporters for active translocation, preferentially the dipeptide permease Dpp. However, in the absence of functional Dpp or in the presence of outcompeting nutrient peptides, negamycin can still enter the cytoplasm. We observed a contribution of the DppA homologs SapA and OppA, as well as of the proton-dependent oligopeptide transporter DtpD. Calcium strongly improves the activity of negamycin against both Gram-negative and Gram-positive bacteria, especially at concentrations around 2.5 mM, reflecting human blood levels. Calcium forms a complex with negamycin and facilitates its interaction with negatively charged phospholipids in bacterial membranes. Moreover, decreased activity at acidic pH and under anaerobic conditions points to a role of the membrane potential in negamycin uptake. Accordingly, improved activity at alkaline pH could be linked to increased uptake of [3H]negamycin. The diversity of options for membrane translocation is reflected by low resistance rates. The example of negamycin demonstrates that membrane passage of antibiotics can be multifaceted and that for cytoplasmic anti-Gram-negative drugs, understanding of permeation and target interaction are equally important.

Generation of Lasso Peptide-Based ClpP Binders.

The Clp protease system fulfills a plethora of important functions in bacteria. It consists of a tetradecameric ClpP barrel holding the proteolytic centers and two hexameric Clp-ATPase rings, which recognize, unfold, and then feed substrate proteins into the ClpP barrel for proteolytic degradation. Flexible loops carrying conserved tripeptide motifs protrude from the Clp-ATPases and bind into hydrophobic pockets (H-pockets) on ClpP. Here, we set out to engineer microcin J25 (MccJ25), a ribosomally synthesized and post-translationally modified peptide (RiPP) of the lasso peptide subfamily, by introducing the conserved tripeptide motifs into the lasso peptide loop region to mimic the Clp-ATPase loops. We studied the capacity of the resulting lasso peptide variants to bind to ClpP and affect its activity. From the nine variants generated, one in particular (12IGF) was able to activate ClpP from Staphylococcus aureus and Bacillus subtilis. While 12IGF conferred stability to ClpP tetradecamers and stimulated peptide degradation, it did not trigger unregulated protein degradation, in contrast to the H-pocket-binding acyldepsipeptide antibiotics (ADEPs). Interestingly, synergistic interactions between 12IGF and ADEP were observed.

Nocathioamides, Uncovered by a Tunable Metabologenomic Approach, Define a Novel Class of Chimeric Lanthipeptides.

The increasing number of available genomes, in combination with advanced genome mining techniques, unveiled a plethora of biosynthetic gene clusters (BGCs) coding for ribosomally synthesized and post-translationally modified peptides (RiPPs). The products of these BGCs often represent an enormous resource for new and bioactive compounds, but frequently, they cannot be readily isolated and remain cryptic. Here, we describe a tunable metabologenomic approach that recruits a synergism of bioinformatics in tandem with isotope- and NMR-guided platform to identify the product of an orphan RiPP gene cluster in the genomes of Nocardia terpenica IFM 0406 and 0706T . The application of this tactic resulted in the discovery of nocathioamides family as a founder of a new class of chimeric lanthipeptides I.

Comparison of Proteomic Responses as Global Approach to Antibiotic Mechanism of Action Elucidation.

New antibiotics are urgently needed to address the mounting resistance challenge. In early drug discovery, one of the bottlenecks is the elucidation of targets and mechanisms. To accelerate antibiotic research, we provide a proteomic approach for the rapid classification of compounds into those with precedented and unprecedented modes of action. We established a proteomic response library of Bacillus subtilis covering 91 antibiotics and comparator compounds, and a mathematical approach was developed to aid data analysis. Comparison of proteomic responses (CoPR) allows the rapid identification of antibiotics with dual mechanisms of action as shown for atypical tetracyclines. It also aids in generating hypotheses on mechanisms of action as presented for salvarsan (arsphenamine) and the antirheumatic agent auranofin, which is under consideration for repurposing. Proteomic profiling also provides insights into the impact of antibiotics on bacterial physiology through analysis of marker proteins indicative of the impairment of cellular processes and structures. As demonstrated for trans-translation, a promising target not yet exploited clinically, proteomic profiling supports chemical biology approaches to investigating bacterial physiology.

Acyldepsipeptide Probes Facilitate Specific Detection of Caseinolytic Protease†P Independent of Its Oligomeric and Activity State.

Caseinolytic protease P (ClpP) is a tetradecameric peptidase that assembles with chaperones such as ClpX to gain proteolytic activity. Acyldepsipeptides (ADEPs) are small-molecule mimics of ClpX that bind into hydrophobic pockets on the apical site of the complex, thereby activating ClpP. Detection of ClpP has so far been facilitated with active-site-directed probes which depend on the activity and oligomeric state of the complex. To expand the scope of ClpP labeling, we took a stepwise synthetic approach toward customized ADEP photoprobes. Structure-activity relationship studies with small fragments and ADEP derivatives paired with modeling studies revealed the design principles for suitable probe molecules. The derivatives were tested for activation of ClpP and subsequently applied in labeling studies of the wild-type peptidase as well as enzymes bearing mutations at the active site and an oligomerization sensor. Satisfyingly, the ADEP photoprobes provided a labeling readout of ClpP independent of its activity and oligomeric state.

New Nocobactin Derivatives with Antimuscarinic Activity, Terpenibactins A-C, Revealed by Genome Mining of Nocardia terpenica IFM 0406.

We report a genomics-guided exploration of the metabolic potential of the brasilicardin producer strain Nocardia terpenica IFM 0406. Bioinformatics analysis of the whole genome sequence revealed the presence of a biosynthetic gene cluster presumably responsible for the generation of formerly unknown nocobactin derivatives. Mass spectrometry-assisted isolation led to the identification of three new siderophores, terpenibactins A (1), B (2) and C (3), which belong to the class of nocobactins. Their structures were elucidated by employing spectroscopic techniques. Compounds 1-3 demonstrated inhibitory activity towards the muscarinic M3 receptor, while exhibiting only a low cytotoxicity.

Functional Characterisation of ClpP Mutations Conferring Resistance to Acyldepsipeptide Antibiotics in Firmicutes.

Acyldepsipeptide (ADEP) is an exploratory antibiotic with a novel mechanism of action. ClpP, the proteolytic core of the caseinolytic protease, is deregulated towards unrestrained proteolysis. Here, we report on the mechanism of ADEP resistance in Firmicutes. This bacterial phylum contains important pathogens that are relevant for potential ADEP therapy. For Staphylococcus aureus, Bacillus subtilis, enterococci and streptococci, spontaneous ADEP-resistant mutants were selected in vitro at a rate of 10-6 . All isolates carried mutations in clpP. All mutated S. aureus ClpP proteins characterised in this study were functionally impaired; this increased our understanding of the mode of operation of ClpP. For molecular insights, crystal structures of S. aureus ClpP bound to ADEP4 were determined. Well-resolved N-terminal domains in the apo structure allow the pore-gating mechanism to be followed. The compilation of mutations presented here indicates residues relevant for ClpP function and suggests that ADEP resistance will occur at a lower rate during the infection process.

Nostotrebin 6 Related Cyclopentenediones and δ-Lactones with Broad Activity Spectrum Isolated from the Cultivation Medium of the Cyanobacterium Nostoc sp. CBT1153.

Cyanobacteria are an interesting source of biologically active natural products, especially chemically diverse and potent protease inhibitors. On our search for inhibitors of the trypanosomal cysteine protease rhodesain, we identified the homodimeric cyclopentenedione (CPD) nostotrebin 6 (1) and new related monomeric, dimeric, and higher oligomeric compounds as the active substances in the medium extract of Nostoc sp. CBT1153. The oligomeric compounds are composed of two core monomeric structures, a trisubstituted CPD or a trisubstituted unsaturated δ-lactone. Nostotrebin 6 thus far has been the only known cyanobacterial CPD. It has been found to be active in a broad variety of assays, indicating that it might be a pan-assay interference compound (PAIN). Thus, we compared the antibacterial and cytotoxic activities as well as the rhodesain inhibition of selected compounds. Because a compound with a δ-lactone instead of a CPD core structure was equally active as nostotrebin 6, the bioactivities of these compounds seem to be based on the phenolic substructures rather than the CPD moiety. While the dimers were roughly equally potent, the monomer displayed slightly weaker activity, suggesting that the compounds show unspecific activity depending upon the number of free phenolic hydroxy groups per molecule.

Ubiquitously expressed Human Beta Defensin 1 (hBD1) forms bacteria-entrapping nets in a redox dependent mode of action.

Ever since the discovery of endogenous host defense antimicrobial peptides it has been discussed how these evolutionary conserved molecules avoid to induce resistance and to remain effective. Human ß-defensin 1 (hBD1) is an ubiquitously expressed endogenous antimicrobial peptide that exhibits qualitatively distinct activities between its oxidized and reduced forms. Here, we explore these antimicrobial mechanisms. Surprisingly, using electron microscopy we detected a so far unknown net-like structure surrounding bacteria, which were treated with the reduced but not the oxidized form of hBD1. A transmigration assay demonstrated that hBD1-derived nets capture bacteria and inhibit bacterial transmigration independent of bacterial killing. The presence of nets could completely prevent migration of hBD1 resistant pathogens and are stable in the presence of human duodenal secretion with a high amount of proteases. In contrast to HD6, cysteins are necessary for net formation. This redox-dependent function serves as an additional mechanism of action for hBD1 and differs from net formation by other defensins such as Paneth cell-derived human α-defensin 6 (HD6). While hBD1red and hBD1ox have distinct antimicrobial profiles and functions, only the reduced form provides additional host protection by entrapping bacteria in extracellular net structures preventing bacterial invasion. Better understanding of the modes of action of endogenous host peptides will help to find new antimicrobial strategies.

Consequences of dosing and timing on the antibacterial effects of ADEP antibiotics.

Antibiotic acyldepsipeptides (ADEPs) exert potent antibacterial activity in rodent models of bacterial infection and exceptional efficacy against persister cells of methicillin-resistant Staphylococcus aureus (MRSA). The mechanism of ADEP action is unusual in that the antibiotic releases the destructive capacity of over-activated ClpP, the proteolytic core of the bacterial Clp protease. The essential bacterial cell division protein FtsZ had emerged in a previous study as a preferred protein substrate of ADEP-activated ClpP but it is definitely not the only cellular substrate. In the current study, we set out to follow the morphological changes that lead to ADEP-mediated bacterial death in S. aureus and Bacillus subtilis, differentiating between antibacterial effects at low and high ADEP concentrations. Here, fluorescence and time-lapse microscopy data show that cells adopt a characteristic phenotype of cell division inhibition at ADEP levels close to the MIC, but retain the capacity to form viable daughter cells for a substantial period of time when transferred to ADEP-free growth medium. After extended exposure to low ADEP concentrations, nucleoids of B. subtilis started to disorganize and upon compound removal many cells failed to re-organize nucleoids, re-initiate cytokinesis and consequently died. Survival versus cell death of filamentous cells attempting recovery depended on the timing of completion of new septa in relation to the loss of cell envelope integrity. We show that the potential to recover after ADEP removal depends on the antibiotic concentration as well as the treatment duration. When exposed to ADEP at concentrations well above the MIC, biomass production ceased rapidly as did the potential to recover. In time-kill studies both long-time exposure to low ADEP levels as well as short-time exposure to high concentrations proved highly effective, while intermittent concentrations and time frames were not. We here provide new insights into the antimicrobial activity of ADEP antibiotics and the consequences of dosing and timing for bacterial physiology which should be considered in view of a potential therapeutic application of ADEPs.

Environmental and cellular factors affecting the localization of T6SS proteins in Burkholderia thailandensis.

The type VI secretion system (T6SS) injects effector proteins into neighboring bacteria and host cells. Effector translocation is driven by contraction of a tubular sheath in the cytoplasm that expels an inner needle across the cell envelope. The AAA + ATPase ClpV disassembles and recycles the contracted sheath. While ClpV-1-GFP of the Burkholderia T6SS-1, which targets prokaryotic cells, assembles into randomly localized foci, ClpV-5-GFP of the virulence-associated T6SS-5 displays a polar distribution. The mechanisms underlying the localization of T6SSs to a particular site in the bacterial cell are currently unknown. We recently showed that ClpV-5-GFP retains its polar localization in the absence of all T6SS-5 components during infection of host cells. Herein, we set out to identify factors involved in the distribution of ClpV-5 and ClpV-1 in Burkholderia thailandensis. We show that focal assembly and polar localization of ClpV-5-GFP is not dependent on the intracellular host cell environment, known to contain the signal to induce T6SS-5 gene expression. In contrast to ClpV-5-GFP, localization of ClpV-1-GFP was dependent on the cognate T6SS. Foci formation of both ClpV5-GFP and ClpV-1-GFP was decreased by D cycloserine-mediated inhibition of peptidoglycan synthesis while treatment of B. thailandensis with A22 blocking the cytoskeletal protein MreB did not affect assembly of ClpV-5 and ClpV-1 into single discrete foci. Furthermore, we found that surface contact promotes but is not essential for localization of ClpV-5-GFP to the pole whereas expression of clpV-1-gfp appears to be induced by surface contact. In summary, the study provides novel insights into the localization of ClpV ATPases of T6SSs targeting prokaryotic and eukaryotic cells.