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BIOLOGICAL SCIENCES / MICROBIOLOGY
Discovery of platencin, a dual FabF and FabH inhibitor with in vivo antibiotic properties
*Merck Research Laboratories, Rahway, NJ 07065; and Centro de Investigación Básica, Merck Sharp & Dohme de España, S.A. Josefa Valcárcel 38, Madrid 28027, Spain
Edited by Arnold L. Demain, Drew University, Madison, NJ, and approved March 9, 2007 (received for review January 26, 2007)
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-ketoacyl-[acyl carrier protein (ACP)] synthase II (FabF) and III (FabH) with IC50 values of 1.95 and 3.91 µg/ml, respectively, whereas platensimycin targets only FabF (IC50 = 0.13 µg/ml) in S. aureus, emphasizing the fact that more antibiotics with novel structures and new modes of action can be discovered by using this antisense differential sensitivity whole-cell screening paradigm.
platensimycin | natural product | thiolactomycin | antisense | fatty acid synthesis
Type II fatty acid synthesis (FASII) is essential to bacterial cell viability and therefore is a potential target for new antibiotics. Significant differences between bacterial and human fatty acid synthesis systems exist that include the organization and structure of enzymes and the specific roles played by fatty acids making this system an attractive target for antibacterial drug discovery (6–11). One of the essential enzymes, FabI, in the fatty acid biosynthetic pathway is a proven drug target with two marketed antibacterial agents, triclosan (antiseptic) and isoniazid (an anti-Mycobacterium tuberculosis agent) (12, 13). The initiation condensing enzyme, FabH, and elongation condensing enzymes, FabF/B, are essential components of this biosynthetic pathway (14–17) and consequently are highly conserved among key pathogens. Although no drugs targeting condensing enzymes are used in the clinic, two inhibitors, cerulenin (18) and thiolactomycin (19), with poor antibacterial activities were discovered more than two decades ago. Cerulenin selectively targets FabF/B forming a covalent bond with the catalytic cysteine in the active site with its tail occupying the long hydrophobic cavity that normally contains the growing acyl chain of the natural substrate (20, 21). Thiolactomycin and its analogs (22, 23) target the malonyl binding site of both FabH and FabF/B. Several other inhibitors targeting the bacterial condensing enzymes have been reported, but none were suitable drug candidates because of poor penetration and/or lack of target selectivity in whole cells and/or in vivo toxicity. In previous studies we have described the discovery and full characterization of platensimycin (24), a novel class highly selective FabF inhibitor with potent antibiotic properties, by screening >250,000 natural product extracts using a target-based whole-cell strategy with antisense silencing technology (25) in concert with biochemical validation (26). In the present study we report the discovery of platencin, a natural product from a strain of Streptomyces platensis MA7339 recovered from a soil sample collected in Spain. The chemical structure of platencin contains a unique ketolide portion that is different from platensimycin. Platencin showed broad-spectrum Gram-positive antibacterial activity and in vivo efficacy in a mouse model. Characterization of platencin in biochemical assays showed that platencin targets both FabH and FabF enzymes.
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90 µg/h, which is consistent with the relative in vitro antibacterial activities of the two drugs in the presence of serum [MIC = 8 µg/ml for platencin (Table 1) and 2 µg/ml for platensimycin (24)], suggesting that the MIC in the presence of serum mimics the animal test condition.
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In S. aureus, fabH is upstream of fabF and both genes are members of the same operon. Therefore, overexpression of fabF antisense RNA, targeting fabF mRNA, causes a complete degradation of the 5' portion of the transcript, which in turn causes down-regulation of both FabF and FabH expression (25). Inhibition of phospholipid biosynthesis by platencin demonstrated that it targets fatty acid synthesis pathway. The activity and selectivity of platencin in the two-plate antisense assay indicated that platencin targeted FabF, FabH, or both enzymes. To determine the target of platencin, first we performed a direct binding assay (24) with purified FabF. In this assay platencin showed direct binding to acyl-enzyme intermediate; however, its binding affinity was 5.9-fold lower (IC50 = 0.113 ± 0.089 µM) than platensimycin. We have already reported that platensimycin poorly inhibited the FabH enzyme (IC50 = 67 µM) in a purified FabH single-enzyme assay (24). In this assay, platencin exhibited 4.1-fold higher inhibitory activity (IC50 = 16.2 ± 4.2 µM) compared with platensimycin. These results suggested that platencin tends to inhibit both condensing enzymes whereas platensimycin selectively targets FabF. The differences between the IC50 values of the single-enzyme assay (IC50 = 0.048 µM) and whole-cell labeling [IC50 = 0.1 µg/ml (0.23 µM)] remained unexplained (24). This could be because of poor cell uptake including membrane penetration (29) and active efflux. Another possibility is that the in vitro single-enzyme assay does not represent the measurement of the precise reaction inside the cell.
As shown in Fig. 3A, fatty acid biosynthesis enzymes in bacteria work sequentially as a biofactory. A product from one individual enzyme becomes a substrate to the next enzyme, which requires that all enzymes work closely, possibly forming a complex through protein–protein interactions. To address this question in as precise a way as possible, we developed a FabH/FabF PAGE elongation assay, which allowed us not only to test but also to quantitatively differentiate inhibitors against FabF and/or FabH simultaneously. This assay directly uses the crude S. aureus cytosolic proteins, which should provide an enzyme complex similar to what exists in the cell in the natural state. The schematic description of the assay is detailed in Fig. 3A. The basic idea is that, when the assay is performed with all natural enzymes, but without addition of ATP and NADPH2/NADH2, both the AccABCD reaction and the elongation cycle cannot proceed. As a result, addition of radiolabeled malonyl-CoA, lauryl-CoA, and the natural substrate acetyl-CoA produced only three radiolabeled protein [acyl carrier protein (ACP)] products (shown in yellow boxes in Fig. 3A). These are identified as malonyl-ACP (FabD product), acetoacetyl-ACP (FabH products), and long-chain -ketoacyl-ACP (C14, FabF products) and can be separated on 0.5 M urea PAGE. In a control experiment, when a known FabH inhibitor (Fig. 3D) (30) was tested in this assay it blocked FabH activity, leading to loss of accumulation of acetoacetyl-ACP [IC50 = 2 µg/ml (3.96 µM) for FabH] (Fig. 3B and data not shown). Platensimycin, a selective FabF inhibitor, led to the significant reduction of accumulation of long-chain -ketoacyl-ACP [IC50 = 0.13 µg/ml (0.29 µM) for FabF] and loss of accumulation of acetoacetyl-ACP [IC50 = 109 µg/ml (247 µM) for FabH] (Fig. 3 B and C). In a similar experiment platencin blocked both FabF and FabH and reduced the level of accumulated acetoacetyl ACP and -ketoacyl-ACP with IC50 values of 1.95 µg/ml (4.58 µM) and 3.91 µg/ml (9.17 µM), respectively (Fig. 3B), suggesting that platencin is a dual inhibitor with similar inhibition efficiency for both condensing enzymes. Platencin and platensimycin did not inhibit FabD, as its product (malonyl-ACP) did not decrease, but increased slightly because of inhibition of the downstream FabH and/or FabF enzymes that consume malonyl-ACP. This further demonstrates that the compounds specifically targeted the condensing enzymes. Interestingly, the FabF IC50 value (0.13 µg/ml) of platensimycin in the FabH/FabF PAGE elongation assay is similar to the IC50 value (0.1 µg/ml) (Table 1) obtained from the whole-cell labeling assay, emphasizing the fact that (i) the FabF enzyme in S. aureus is the target that platensimycin selectively inhibits and (ii) the FabH/FabF PAGE elongation assay with the crude S. aureus cytosolic proteins indeed closely mimics what takes place inside living cells. Compared with platensimycin, both IC50 values of platencin (FabF IC50 = 1.95 µg/ml and FabH IC50 = 3.91 µg/ml) are >10-fold higher than that obtained from the whole-cell labeling assay (0.19 µg/ml) (Fig. 2B), suggesting that platencin inhibits fatty acid synthesis in S. aureus through a synergistic effect by targeting both condensing enzymes.
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The discovery of a second structure from natural products with dual mode of action, similar to thiolactomycin but with >200-fold higher antibacterial activity against S. aureus, emphasizes the applicability of the target-based whole-cell antisense screening approach on FabF/FabH targets. This universal approach could be applied to other targets in bacteria, particularly the 90% of essential proteins that remain unexplored. The antisense strategy provides a great opportunity for the discovery of novel antibiotic chemical structures from natural products. In addition, the structure of platencin provides more insight for the structure–function relationship of FASII inhibitors and for the possible development of critically needed antibacterial agents.
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Materials and Methods |
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Preparation of Total FASII Enzymes. The details of the preparation of FASII enzymes from S. aureus were described previously (26). Briefly, cells were grown to stationary phase and centrifuged at 8,000 rpm for 10 min with a Beckman JA-10 rotor. The pellets were washed twice with ice-cold buffer A (0.1 M sodium phosphate, pH 7/1 mM EDTA/5 mM 2-mercaptoethanol) and resuspended in the same buffer. The cells were lysed in a cold microfluidizer (M-110EH; Microfluidics) at 18,000 lbs/in2 and centrifuged at 20,000 rpm for 15 min at 4°C. The supernatant was collected, and ammonium sulfate was added into the supernatant with low-speed stirring at 4°C to reach 45% of ammonium sulfate concentration. The mixture was centrifuged at 10,000 rpm for 5 min, the supernatant was collected, and ammonium sulfate was added again to reach 80% of ammonium sulfate. The mixture was centrifuged again, and the pellet, containing all necessary fatty acid synthesis (FASII) enzymes, was dissolved in buffer A, dialyzed at 4°C against four changes of buffer A using 10-kDa molecular mass cutoff dialysis tubing (15961-022; Invitrogen), and then concentrated. The protein concentration was determined by using the standard Bio-Rad protocol. The protein was aliquoted, flash-frozen with liquid nitrogen, and stored at –80°C.
FabH/FabF PAGE Elongation Assay. The assay was performed by preincubating 0.5 µg of the S. aureus FASII enzymes with a serial dilution of the inhibitors at room temperature for 20 min in 50 µl of buffer containing 100 mM sodium phosphate (pH 7.0), 10 µM lauroyl-CoA, 10 µM acetyl-CoA, 1% Me2SO, and 5 µM S. aureus ACP pretreated with 3 mM DTT. The reaction was initiated by addition of 5 µM [14C]malonyl-CoA (60 mCi/mmol, NEC612, labeled at C-2 of the malonyl group). The reaction was incubated at 30°C for 10 min, terminated by adding 10 µl of native sample buffer, and kept on ice or kept at –20°C. The samples were applied to and resolved by a 20% polyacrylamide gel containing 0.5 M urea. The gel was blotted to a polyvinylidene difluoride membrane and visualized by using a PhosphorImager.
RBC Assays. Human erythrocytes were collected (at least 5 ml of whole blood) in a vacutainer tube with EDTA from a single human donor on the day of the experiment. Two milliliters of the freshly drawn whole blood was added to 6 ml of sterile saline and gently mixed, then centrifuged at 5°C for 5 min at 2,000 rpm. The supernatant was discarded, and the human RBC were resuspended with 6 ml of sterile saline and centrifuged again at 5°C for 5 min at 2,000 rpm. This procedure was repeated two times. Three percent suspension of the washed RBCs was prepared by adding 0.3 ml of the washed RBCs to 9.7 ml of sterile saline. The final concentration of test compounds ranging from 64 µg/ml to 0.06 µg/ml in 100 µl of sterile saline containing 3.2% Me2SO was prepared in 96-well Microtest U-bottom plates (BD-353227; Becton Dickinson). The RBC lysis assay (32) was initiated by addition of 5 µl of 3% washed human RBC suspension to the each well in the plates. The plates were then immediately shaken for 1–2 min at 550 rpm to disperse the RBCs and incubated at room temperature for 60 min. The plates were centrifuged at 2,000 rpm for 5 min and read immediately. Hemolysis of RBCs is indicated by complete or partial clearing (lysis) of the supernatants with either no or a rough RBC pellet in the bottom of the wells. No hemolysis is judged by clear supernatant with a smooth RBC pellet in the bottom of the well. The MLC is defined as the lowest concentration of a test compound to visibly produce complete or partial lysis of RBCs. Amphotericin B was used as a positive control (MLC = 2–4 µg/ml), and chloramphenicol was used as a negative control (MLC > 64 µg/ml).
Other Assays. Screening of natural products (25), in vivo studies (24), whole-cell labeling assay (26), agar-diffusion two-plate differential sensitivity assay (two-plate assay) (25), and determination of antibacterial activity (MIC) (28) were described previously.
Isolation of Platencin.
Platencin was isolated from a fermentation broth of S. platensis (MA7339) by extraction with acetone followed by successive reversed-phase chromatographies on amberchrome followed by HPLC using aqueous methanol and aqueous acetonitrile gradient as elution solvents, respectively. The production yield of platencin was 1 mg/liter (33).
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Abbreviations: MRSA, methicillin-resistant S. aureus; ACP, acyl carrier protein; MIC, minimum inhibitory concentration; MDC, minimum detection concentration; FASII, type II fatty acid synthesis; MLC, minimum lytic concentration.
To whom correspondence concerning biological matters should be addressed at: Merck & Co., P.O. Box 2000, R80W-250, Rahway, NJ 07065. E-mail: jun_wang2{at}merck.com
To whom correspondence concerning chemical matters should be addressed at: Merck & Co., P.O. Box 2000, R80Y-350, Rahway, NJ 07065. E-mail: sheo_singh{at}merck.com
Author contributions: J.W., S.K., S.H.L., A.G., R.P., K.D., F.R., M.M., L.H., E.T., S.L.C., K.H., R.C., O.S., I.G., A.B., F.V., O.G., F.P., H.J., K.Y., D.F.C., and S.B.S. performed research; and J.W. and S.B.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0700746104/DC1.
© 2007 by The National Academy of Sciences of the USA
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) and a significant inhibition of phospholipid synthesis (
) with an IC50 value of 0.19 µg/ml. Error bars show the standard deviation of two individual experiments.