Heike Brötz-Oesterhelt

Dysregulation of bacterial proteolytic machinery by a new class of antibiotics.

Here we show that a new class of antibiotics—acyldepsipeptides—has antibacterial activity against Gram-positive bacteria in vitro and in several rodent models of bacterial infection. The acyldepsipeptides are active against isolates that are resistant to antibiotics in clinical application, implying a new target, which we identify as ClpP, the core unit of a major bacterial protease complex. ClpP is usually tightly regulated and strictly requires a member of the family of Clp-ATPases and often further accessory proteins for proteolytic activation. Binding of acyldepsipeptides to ClpP eliminates these safeguards. The acyldepsipeptide-activated ClpP core is capable of proteolytic degradation in the absence of the regulatory Clp-ATPases. Such uncontrolled proteolysis leads to inhibition of bacterial cell division and eventually cell death.

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.

Structures of ClpP in complex with acyldepsipeptide antibiotics reveal its activation mechanism

Clp-family proteins are prototypes for studying the mechanism of ATP-dependent proteases because the proteolytic activity of the ClpP core is tightly regulated by activating Clp-ATPases. Nonetheless, the proteolytic activation mechanism has remained elusive because of the lack of a complex structure. Acyldepsipeptides (ADEPs), a recently discovered class of antibiotics, activate and disregulate ClpP. Here we have elucidated the structural changes underlying the ClpP activation process by ADEPs. We present the structures of Bacillus subtilis ClpP alone and in complex with ADEP1 and ADEP2. The structures show the closed-to-open-gate transition of the ClpP N-terminal segments upon activation as well as conformational changes restricted to the upper portion of ClpP. The direction of the conformational movement and the hydrophobic clustering that stabilizes the closed structure are markedly different from those of other ATP-dependent proteases, providing unprecedented insights into the activation of ClpP.

Specific and Potent Inhibition of NAD+-dependent DNA Ligase by Pyridochromanones

Pyridochromanones were identified by high throughput screening as potent inhibitors of NAD+-dependent DNA ligase from Escherichia coli. Further characterization revealed that eubacterial DNA ligases from Gramnegative and Gram-positive sources were inhibited at nanomolar concentrations. In contrast, purified human DNA ligase I was not affected (IC50 > 75 μm), demonstrating remarkable specificity for the prokaryotic target. The binding mode is competitive with the eubacteria-specific cofactor NAD+, and no intercalation into DNA was detected. Accordingly, the compounds were bactericidal for the prominent human pathogen Staphylococcus aureus in the low μg/ml range, whereas eukaryotic cells were not affected up to 60 μg/ml. The hypothesis that inhibition of DNA ligase is the antibacterial principle was proven in studies with a temperature-sensitive ligase-deficient E. coli strain. This mutant was highly susceptible for pyridochromanones at elevated temperatures but was rescued by heterologous expression of human DNA ligase I. A physiological consequence of ligase inhibition in bacteria was massive DNA degradation, as visualized by fluorescence microscopy of labeled DNA. In summary, the pyridochromanones demonstrate that diverse eubacterial DNA ligases can be addressed by a single inhibitor without affecting eukaryotic ligases or other DNA-binding enzymes, which proves the value of DNA ligase as a novel target in antibacterial therapy.

Antibiotic acyldepsipeptides activate ClpP peptidase to degrade the cell division protein FtsZ

The worldwide spread of antibiotic-resistant bacteria has lent urgency to the search for antibiotics with new modes of action that are devoid of preexisting cross-resistances. We previously described a unique class of acyldepsipeptides (ADEPs) that exerts prominent antibacterial activity against Gram-positive pathogens including streptococci, enterococci, as well as multidrug-resistant Staphylococcus aureus. Here, we report that ADEP prevents cell division in Gram-positive bacteria and induces strong filamentation of rod-shaped Bacillus subtilis and swelling of coccoid S. aureus and Streptococcus pneumoniae. It emerged that ADEP treatment inhibits septum formation at the stage of Z-ring assembly, and that central cell division proteins delocalize from midcell positions. Using in vivo and in vitro studies, we show that the inhibition of Z-ring formation is a consequence of the proteolytic degradation of the essential cell division protein FtsZ. ADEP switches the bacterial ClpP peptidase from a regulated to an uncontrolled protease, and it turned out that FtsZ is particularly prone to degradation by the ADEP–ClpP complex. By preventing cell division, ADEP inhibits a vital cellular process of bacteria that is not targeted by any therapeutically applied antibiotic so far. Their unique multifaceted mechanism of action and antibacterial potency makes them promising lead structures for future antibiotic development.

Inducing Secondary Metabolite Production by the Endophytic Fungus Fusarium tricinctum through Coculture with Bacillus subtilis

Coculturing the fungal endophyte Fusarium tricinctum with the bacterium Bacillus subtilis 168 trpC2 on solid rice medium resulted in an up to 78-fold increase in the accumulation in constitutively present secondary metabolites that included lateropyrone (5), cyclic depsipeptides of the enniatin type (6-8), and the lipopeptide fusaristatin A (9). In addition, four compounds (1-4) including (-)-citreoisocoumarin (2) as well as three new natural products (1, 3, and 4) were not present in discrete fungal and bacterial controls and only detected in the cocultures. The new compounds were identified as macrocarpon C (1), 2-(carboxymethylamino)benzoic acid (3), and (-)-citreoisocoumarinol (4) by analysis of the 1D and 2D NMR and HRMS data. Enniatins B1 (7) and A1 (8), whose production was particularly enhanced, inhibited the growth of the cocultivated B. subtilis strain with minimal inhibitory concentrations (MICs) of 16 and 8 μg/mL, respectively, and were also active against Staphylococcus aureus, Streptococcus pneumoniae, and Enterococcus faecalis with MIC values in the range 2-8 μg/mL. In addition, lateropyrone (5), which was constitutively present in F. tricinctum, displayed good antibacterial activity against B. subtilis, S. aureus, S. pneumoniae, and E. faecalis, with MIC values ranging from 2 to 8 μg/mL. All active compounds were equally effective against a multiresistant clinical isolate of S. aureus and a susceptible reference strain of the same species.

How many modes of action should an antibiotic have

All antibiotics that have been successfully employed for decades as monotherapeutics in the treatment of bacterial infections rely on mechanisms of bacterial growth inhibition which are by far more complex than inhibition of a single enzyme. Such successful antibiotics have in common that they address several targets in parallel and/or that their targets are encoded by multiple genes. Such multiplicity of targets and of target genes has the advantage that the emergence of spontaneous target-related resistance is a comparatively slow process. Recently registered antibiotics and novel antibiotics in development are discussed in the light of this promising concept of antibacterial polypharmacology.

Medicinal chemistry optimization of acyldepsipeptides of the enopeptin class antibiotics.

The therapy of life-threatening infections is significantly weakened by the global spread of antibiotic resistance. Among Gram-positive bacteria, the development of resistance of staphylococci, streptococci, and enterococci is of particular concern. Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of complicated nosocomial infections, and its prevalence in hospitals has increased during the last decade. Furthermore, the frequent reliance on vancomycin as a last line of defense has led to a high level of vancomycin-resistant nosocomial isolates. Streptococcus pneumoniae is an important community-acquired respiratory tract pathogen, and a leading cause of morbidity and mortality. More than 30% of US isolates are penicillin-resistant (penicillin-resistant S. pneumoniae, PRSP) and many of these strains are also resistant towards a large number of other antibiotics. Novel antibacterial agents with unprecedented mechanisms of action, which are devoid of pre-existing cross-resistances, are therefore very necessary. Most of the currently marketed antibiotic classes originate from the secondary metabolism of bacteria or fungi, which emphasizes that natural products are a valuable source of novel antibacterial agents. Thus, depsipeptides of the enopeptin family are of interest. In 1982, the isolation of depsipeptide antibiotics A54556A and B (Scheme 1, 1 and 2, respectively), from a mixture of eight individual depsipeptidic factors (A– H), produced by aerobic fermentation of Streptomyces hawaiiensis (NRRL 15010) was described. Enopeptin A (3) and B (4) (Scheme 1) were isolated in 1991 from a culture broth of Streptomyces sp. RK-1051, found in a soil sample from Tsuruoka City, Japan. The enopeptin structure consists of a lactone core composed of five S-configured amino acids and a lipophilic acylated phenylalanine side chain attached to a serine nitrogen. This macrocyclic, peptidic structure made these acyldepsipeptides interesting candidates for total synthesis. The level of interest was further increased by mode of action studies with B. subtilis, demonstrating impaired bacterial cell division and induction of filamentation as the underlying causes of antibacterial activity (Figure 1). Applying reversed genomics technologies, Brçtz-Oesterhelt and co-workers were able to demonstrate that the lead structure 1 acts by binding to caseine lytic protease (ClpP), the core unit of a major bacterial–protease complex. In order to protect the bacterial cell from the destructive power of this universal protease, ClpP is tightly regulated and requires a ClpATPase, and often other accessory proteins for activation. Binding of 1 to ClpP eliminates the requirement of Clp-ATPases and other regulatory factors for proteolytic degradation. Therefore, uncontrolled proteolysis leads to inhibition of bacterial cell division and eventually cell death. With respect to drug discovery, natural acyldepsipeptides 1 and 2 displayed only limited in vitro activity against the Grampositive pathogen S. aureus (Table 1) and Gram-negative bacteria were not susceptible. Furthermore, 1 and 2 were not effective in standard mouse models of lethal bacterial infection, and their physicochemical and pharmacokinetic profile was dominated by poor aqueous solubility and high clearance. Thus, 1 and 2 were not favorable candidates for drug development. Finally, natural enopeptin acyldepsipeptide antibiotics 1 and 2 are challenging lead structures from a chemical and synthetic viewpoint. Several functional groups limited their stability: (a) the lactone core was readily hydrolyzed in basic and acidic aqueous media; (b) the acylated serine hydroxy group eliminated readily under non-aqueous basic conditions; (c) the conjugated triene was sensitive to temperature and light (cyclization and aromatization reactions) ; and (d) solubility was not sufficient for parenteral application. However, the novel target and the absence of cross-resistance to established antibiotics strongly encouraged the initiation of a medicinal chemistry program, the objective of which was to improve these deficiencies using a thorough understanding of the lead conformation, based on x-ray structure analysis of the synthetic congener 5 (Figure 2). Crystallization of 5 from toluene gave a solvate with two toluene molecules, whereas solvent free crystals were obtained from aqueous acetonitrile. In the case of the solvate crystal, a Scheme 1. Natural enopeptin depsipeptide antibiotics.

New Class of Bacterial Phenylalanyl-tRNA Synthetase Inhibitors with High Potency and Broad-Spectrum Activity

ABSTRACT Phenylalanyl (Phe)-tRNA synthetase (Phe-RS) is an essential enzyme which catalyzes the transfer of phenylalanine to the Phe-specific transfer RNA (tRNAPhe), a key step in protein biosynthesis. Phenyl-thiazolylurea-sulfonamides were identified as a novel class of potent inhibitors of bacterial Phe-RS by high-throughput screening and chemical variation of the screening hit. The compounds inhibit Phe-RS of Escherichia coli, Haemophilus influenzae, Streptococcus pneumoniae, and Staphylococcus aureus, with 50% inhibitory concentrations in the nanomolar range. Enzyme kinetic measurements demonstrated that the compounds bind competitively with respect to the natural substrate Phe. All derivatives are highly selective for the bacterial Phe-RS versus the corresponding mammalian cytoplasmic and human mitochondrial enzymes. Phenyl-thiazolylurea-sulfonamides displayed good in vitro activity against Staphylococcus, Streptococcus, Haemophilus, and Moraxella strains, reaching MICs below 1 μg/ml. The antibacterial activity was partly antagonized by increasing concentrations of Phe in the culture broth in accordance with the competitive binding mode. Further evidence that inhibition of tRNAPhe charging is the antibacterial principle of this compound class was obtained by proteome analysis of Bacillus subtilis. Here, the phenyl-thiazolylurea-sulfonamides induced a protein pattern indicative of the stringent response. In addition, an E. coli strain carrying a relA mutation and defective in stringent response was more susceptible than its isogenic relA+ parent strain. In vivo efficacy was investigated in a murine S. aureus sepsis model and a S. pneumoniae sepsis model in rats. Treatment with the phenyl-thiazolylurea-sulfonamides reduced the bacterial titer in various organs by up to 3 log units, supporting the potential value of Phe-RS as a target in antibacterial therapy.

Functional genomics in antibacterial drug discovery.

Antibacterial drug discovery has experienced a paradigm shift from phenotypic screening for antibacterial activity to rational inhibition of preselected targets. Functional genomics techniques are implemented at various stages of the early drug discovery process and play a central role in target validation and mode of action determination. The spectrum of methods ranges from genetic manipulations (e.g. knockout studies, mutation analyses and the construction of conditional mutants) to transcriptome and proteome expression profiling. Functional genomics supports antibacterial drug discovery by improving knowledge on gene function, bacterial physiology and virulence and the effects of antibiotics on bacterial metabolism.