ARTICLES Dysregulation of bacterial proteolytic machinery by a new class of antibiotics Heike Bro¨tz-Oesterhelt1, Dieter Beyer1, Hein-Peter Kroll1, Rainer Endermann1, Christoph Ladel1, Werner Schroeder2, Berthold Hinzen3, Siegfried Raddatz3, Holger Paulsen3, Kerstin Henninger4, Julia E Bandow5, Hans-Georg Sahl6 & Harald Labischinski1 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 http://www.nature.com/naturemedicine 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. Worldwide spread of antibiotic resistance greatly impairs the treat- (Table 1). Gram-negative bacteria were only affected when efflux ment of life-threatening infections and antibacterial agents with new pumps were deleted or permeabilizing agents were added to the mechanisms of action are urgently needed1,2. Among Gram-positive culture broth, indicating that penetration across the outer membrane bacteria, multidrug-resistant isolates of Staphylococcus aureus, Strepto- is hampered, as expected for molecules of that size. coccus pneumoniae and Enterococcus cause special concern because of their rising prevalence in hospitals and community settings2–5. In vivo pharmacology, pharmacokinetics and toxicology A group of eight closely related acyldepsipeptides (ADEPs) was ADEP 2 and ADEP 4 proved active in the treatment of bacterial previously isolated from the fermentation broth of Streptococcus infections in rodents and surpassed in all models the activity of 2005 Nature Publishing Group Group 2005 Nature Publishing © hawaiiensis NRRL 15010 and briefly described as the ‘A54556 complex’ the marketed competitor linezolid. When we challenged mice with a in a patent6. The report suggested notable in vitro activity against lethal systemic infection of E. faecalis, 1 mg/kg ADEP 2 or 0.5 mg/kg staphylococci and streptococci but provided no information about ADEP 4 were sufficient for 100% survival (Fig. 2a). In lethal sepsis in vivo potency or mechanism of action. To assess the antibacterial caused by S. aureus, 12.5 mg/kg ADEP 4 rescued 80% of the mice activity of this class of compounds and to exploit their full potential, (Fig. 2b) and reduced the bacterial loads in liver, spleen and lung by we determined the correct structure of ‘factor A’, the main component 2–3 log units compared to an untreated control (Fig. 2c). Moreover, of the A54556 complex, which we designate here ADEP 1, established in a S. pneumoniae bacteremia in rats, ADEP 4 was again superior to a route for its de novo synthesis and synthesized largely improved linezolid (Fig. 2d), thus showing promising efficacy in two rodent congeners in a derivatization program. Here, we describe the anti- species and against three major problem pathogens among Gram- bacterial activity of these optimized ADEPs as well as the mechanism positive bacteria. of action of this new class of antibiotics. Exploratory pharmacokinetic studies in mice and dogs showed moderate-to-high clearances, a moderate-to-high volume of distribu- RESULTS tion and half-lives of 1–2 h (Supplementary Note online). In addi- ADEPs are potent against multidrug-resistant bacteria tion, initial toxicological studies yielded promising results Our improved congeners, exemplified here by ADEP 2 and ADEP 4 (Supplementary Note and Supplementary Table 1 online). (Fig. 1), showed potent antibacterial activity against a broad range of Gram-positive bacteria, including multidrug-resistant clinical isolates ADEPs act through a novel antibacterial mechanism (minimal inhibitory concentrations (MIC), 0.01–0.05 mg/ml) and Here we focus on our discovery of the molecular mechanism by which largely surpassed the activity of the natural product ADEP 1 ADEPs cause bacterial death. The best-characterized Gram-positive 1Departments of Anti-infectives, 2Enabling Technologies, 3Chemistry and 4Pharmacokinetics, Bayer HealthCare AG, Pharma Research, Aprather Weg 18a, D-42096 Wuppertal, Germany. 5Institute for Microbiology, University of Greifswald, F.-L.-Jahn Strasse 15, D-17487 Greifswald, Germany. 6Institute for Medical Microbiology, University of Bonn, Sigmund-Freud-Strasse 25, D-53105 Bonn, Germany. Present addresses: RBM Serono, Via Ribes1, 10010 Colleretto Giacosa, Italy (C.L.). Pfizer Inc., Global Research and Development, Ann Arbor, Michigan 48105, USA (J.E.B.). Combinature Biopharm AG, Robert-Roessle-Str. 10, Berlin, Germany (H.L.). Correspondence should be addressed to H.B.-O. ([email protected]). Received 16 March; accepted 6 September; published online 2 October 2005; doi:10.1038/nm1306 1082 VOLUME 11 [ NUMBER 10 [ OCTOBER 2005 NATURE MEDICINE ARTICLES Table 1 Antibacterial activity of selected ADEPs F O F O N N O O IC50 (mg/ml) O O O O O NH O N O NH O N O (S) O H HN H HN N * N N Resistance ADEP ADEP ADEP ADEP N O I O Strain phenotype 1 2 3 4 , , ADEP 1 ( factor A ) ADEP 2 Bacillus subtilis 168 0.2 0.05 4100 0.01 F O F F O F Streptococcus pneumoniae 665 PRSP 1.6 0.05 4100 0.02 N N O O Streptococcus pyogenes Wacker 0.4 0.01 4100 0.02 O O O O O O O NH N Enterococcus faecalis ICB 27159 0.4 r0.01 4100 r0.01 O NH N (R) O H O H N HN N HN* N Enterococcus faecium L 4001 VRE 0.4 0.02 4100 r0.01 N O O Staphylococcus aureus NRS 119 MRSA 6.3 0.4 4100 0.05 ADEP 3 ADEP 4 MIC values for representative multidrug-resistant clinical isolates as a measure of antibacterial in vitro potency. PRSP, penicillin-resistant S. pneumoniae;VRE, F F F F O O vancomycin-resistant E. faecium; MRSA, methicillin-resistant S. aureus. N N O O O O O O O 3 O NH O N N – O NH N H O H N+ O H with stable resistance to ADEPs (MIC Z 100 mg/ml versus 3 mg/ml for N HN N N HN NH2 N N O I O 3 the susceptible wild-type). Transformation of the susceptible HN818 O I O H wild-type strain with a genomic library of the resistant isolate (on a O O ADEP 5 ADEP 6 plasmid vector) generated daughter clones with plasmid-encoded Figure 1 Structures of ADEPs. Natural product ADEP 1 (‘factor A’) high-level resistance to ADEPs. Sequencing of the respective plasmids compared to its optimized congeners ADEP 2 and ADEP 4. The R-epimer identified ClpP, the catalytic core unit of a major bacterial protease 9,10 http://www.nature.com/naturemedicine ADEP 3, which differs from ADEP 2 (S configuration) only by the (caseinolytic protease) , as the resistance determinant (Fig. 3b). All conformation of the difluorophenylalanine side chain (stereocenter indicated daughter clones carried a point mutation leading to an amino-acid by an asterisk), is antibacterially inactive and was included as a negative exchange (Thr182Ala) close to the active center. Sequencing of the control in the mode-of-action studies described here. ADEP 5 and ADEP 6 gene encoding ClpP of the resistant parent clone confirmed the are further tools for mode-of-action studies. The NH2 functionality introduced in the southwestern region of ADEP 5 allows coupling to NHS- mutation, suggesting that the activity of ClpP is impaired and that activated Sepharose and ADEP 6 carries a tritium label and an arylazide intact ClpP is indispensable for ADEP-mediated bacterial death. moiety for crosslinking studies. To confirm the requirement of functional ClpP for ADEP activity in a Gram-positive background, we next investigated a ClpP-deletion mutant of B. subtilis.StrainB. subtilis trpC2 QB4916 (DclpP::specti- species, Bacillus subtilis, served as a model bacterium for our studies. nomycinR)11 was highly resistant to ADEP 1 (MIC Z 100 mg/ml Treatment of B. subtilis with 1.6 mg/ml of ADEP 1 (eightfold the MIC) versus 0.2 mg/ml for the isogenic wild-type). Similarly, a ClpP-deletion reduced the number of viable cells by 2 log units over a period of 6 h. strain of S. aureus was ADEP resistant (MIC 450 mg/ml versus In incorporation assays with radiolabeled precursors, the biosynthesis 0.6 mg/ml for the wild-type) and analysis of resistant colonies of 2005 Nature Publishing Group Group 2005 Nature Publishing of DNA, RNA, protein, cell wall and fatty acid proceeded unhindered E. faecalis and S. pneumoniae, which occurred on ADEP-containing © for 1 h at 2 mg/ml ADEP 1, whereas classical antibiotics were clearly agar plates with frequencies in the range of 10À6, showed point distinguished by preferential inhibition of their target pathway in this mutations in ClpP. These results indicate that the presence of func- type of assay. Microscopic examination showed that immediately after tional ClpP is a requirement for ADEP activity in all species tested and addition of ADEP 1 at concentrations as low as 0.4 mg/ml, B. subtilis that its absence or inactivity is sufficient for ADEP resistance. started to form filaments that reached a length of 200 mM after 5 h ClpP forms the proteolytic core of major protein degradation (Fig. 3a). These results indicate a mechanism of action that does not machinery in eubacteria and is highly conserved and broadly fall into one of the classical target areas, but involves direct or indirect inhibition of cell division.