Báo cáo khoa học: Mode of action of the microbial metabolite GE23077, a novel potent and selective inhibitor of bacterial RNA polymerase

doi:10.1111/j.1432-1033.2004.04244.x

Eur. J. Biochem. 271, 3146–3154 (2004) (cid:1) FEBS 2004

Mode of action of the microbial metabolite GE23077, a novel potent and selective inhibitor of bacterial RNA polymerase

Edoardo Sarubbi(cid:1), Federica Monti*, Emiliana Corti, Anna Miele(cid:2) and Enrico Selva

Vicuron Pharmaceuticals, Gerenzano, Varese, Italy

for effective antimicrobial activity of the antibiotic. Bio- chemical studies were also conducted with purified enzymes to obtain further insights into the mode of action of GE23077. Interestingly, the compound displays a behaviour similar to that of rifampicin, an antibiotic structurally unrelated to GE23077: both compounds act at the level of transcription initiation, but not on the r subunit and not on the formation of the promoter DNA–RNAP complex. Tests on different rifampicin-resistant E. coli RNAPs did not show any cross-resistance between the two compounds, indicating distinct binding sites on the target enzyme. In conclusion, GE23077 is an interesting new molecule for future mechanistic studies on bacterial RNAP and for its potential in anti-infective drug discovery.

Keywords: antibiotic; cell permeabilization; natural product; rifampicin; transcription initiation.

GE23077, a novel microbial metabolite recently isolated from Actinomadura sp. culture media, is a potent and selective inhibitor of bacterial RNA polymerase (RNAP). It inhibits Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) RNAPs with IC50 values (i.e. the concen- tration at which the enzyme activity is inhibited by 50%) in the 10)8 M range, whereas it is not active on E. coli DNA polymerase or on eukaryotic (wheat germ) RNAP II (IC50 values > 10)4 M in both cases). In spite of its potent activity on purified bacterial RNAPs, GE23077 shows a narrow spectrum of antimicrobial activity on Gram-positive and Gram-negative bacteria. To investigate the molecular basis of this behaviour, the effects of GE23077 on macromolecular biosynthesis were tested in E. coli cells permeabilized under different conditions. The addition of GE23077 to plasmo- lyzed cells resulted in an immediate and specific inhibition of intracellular RNA biosynthesis, in a dose–response manner, strongly suggesting that cell penetration is the main obstacle

DNA-directed RNA polymerase (EC 2.7.7.6; RNAP) is the central enzyme of bacterial gene expression, responsible for all cellular RNA synthesis [1]. The catalytically competent (cid:3)core(cid:4) RNAP consists of five subunits (a2bb¢x, with a combined molecular mass of (cid:1) 400 kDa) and is capable of elongation and termination. The initiation-competent (cid:3)holo(cid:4) RNAP is composed of the core enzyme and of an additional subunit, r, which confers on RNAP the ability to initiate transcription at specific promoter sites [2,3]. After over four decades of intensive research, RNAP is currently the subject of renewed interest and excitement, owing to recent publication of the crystal structures of the core [4] and holo [5,6] enzymes, and of an RNAP–DNA complex [7].

The transcription process consists of three main stages: initiation, elongation and termination. Transcription initiation is a multistep process [8] in which holo RNAP specifically binds to promoter DNA at positions )35 and )10 to form an RNAP–promoter closed complex, melts the DNA duplex around the )10 region to yield an RNAP– promoter open complex, and then initiates transcription in the presence of nucleoside triphosphates. After the synthesis of an RNA chain of about 9–12 nucleotides, the transcription complex enters the elongation stage. This transition is marked by a significant conformational change, which leads to r dissociation and the formation of a highly processive RNAP–DNA elongation complex, with changes in the positions of all structural domains of the enzyme by 2 A˚ to 12 A˚

[1].

Owing to its central role in DNA transcription, RNAP is an essential enzyme in bacterial cells and the target of different natural antibiotics. Rifampicin, a potent and broad-spectrum anti-infective agent [9], is undoubtedly the best-known RNAP inhibitor. As a result of its property to freely diffuse into tissues, living cells and bacteria, rifampicin is particularly effective against intracellular pathogens, such as Mycobacterium tuberculosis, for which it is one of the most widely used chemotherapeutic agents [10]. However, because bacteria develop resistance to rifampicin with high frequency, the discovery of novel RNAP inhibitors remains of great interest for the biomedical community. Several different series of compounds (isolated from natural sources [11–14] or, more recently, from chemical libraries [15]),

Correspondence to E. Sarubbi, Lead Discovery Technologies, Aventis Pharma, 13 quai Jules Guesde, 94403 Vitry-sur-Seine, France. Fax: + 33 1 58933087, E-mail: Edoardo.Sarubbi@aventis.com Abbreviations: c.p.m., counts per minute; DNAP, DNA polymerase; IC50, the concentration of compound at which the enzyme activity is inhibited by 50%; RNAP, RNA polymerase; rifR, rifampicin resistant. Enzyme: DNA-directed RNA polymerase (EC 2.7.7.6). Present address: (cid:1)Lead Discovery Technologies, Aventis Pharma, France. *Arpida Ltd, Munchenstein, Switzerland. (cid:2)Aventis Pharma, Anagni (Frosinone), Italy. (Received 2 April 2004, revised 29 May 2004, accepted 3 June 2004)

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which act on RNAP, have been reported in the literature, but none has thus far been marked for clinical use.

50%) ¼ 20 nM], the antimicrobial activity of GE23077, tested on a variety of Gram-positive and Gram-negative strains, shows a narrow species range. Its spectrum of activity is essentially restricted to Moraxella catarrhalis isolates and, to a lesser extent, Neisseria gonorrhoeae and Mycobacterium smegmatis, where relatively high antibiotic concentrations (10)4 M) must be used [19]. Such restricted cellular activity might be a result of the inability of the antibiotic to penetrate most bacterial cell membranes or, alternatively, GE23077 might be blocked, inactivated or pumped out by unknown enzymatic activities.

Besides their potential interest as therapeutic agents, these compounds are also valuable tools for using to characterize the complex activity of their target enzyme. RNAP inhib- itors have been discovered which act at different stages of the transcription process, for example (a) lipiarmycin inhibits the formation of the first dinucleotide of the nascent RNA chain [11], (b) rifampicin blocks the synthesis of RNA molecules longer than two or three nucleotides, preventing the transition from initiation to elongation, but it does not inhibit the elongation complex itself [16,17], (c) strepto- lydigin prevents RNA chain elongation by inhibiting the translocation step [12,18], and (d) the recently reported CRB703 series of compounds specifically inhibit the nuc- leotide addition reaction in the elongation complex [15]. The availability of RNAP inhibitors, acting at different steps of the transcription process, has been very helpful for charac- terizing the various conformational changes that RNAP undergoes during DNA transcription, a process that, however, still remains incompletely understood.

In this study, we determined the following. First, the in vitro potency and selectivity of GE23077, assessing its activity on different purified polymerases. Second, its mode of action on whole bacteria, using permeabilized cells to confirm the specificity of RNA synthesis inhibition. Third, its mechanism of inhibition of purified E. coli RNAP, determining at which stage of the transcription process it exerts its action. Finally, its activity on different rifampicin- resistant (rifR) RNAPs, assessing its propensity for cross- resistance with rifampicin to obtain information on its binding site on the RNAP molecule.

Materials and methods

Enzymes and antibiotics

GE23077 is a novel microbial metabolite, recently discovered in the fermentation broth of an Actinomadura sp. during the screening of natural products for specific inhibitors of bacterial RNAP [19]. It is structurally unrelated to any other known compound and is composed of two, almost identical, components (GE23077-A and GE23077-B) which only differ slightly in a side-chain of otherwise identical cyclic peptides (Fig. 1). When isolated, the two components show similar biochemical activity [19], suggest- ing that the small variations in the side-chain result in only minor effects on GE23077 activity.

In spite of its potent inhibitory activity on purified Escherichia coli RNAP [i.e. the IC50 (concentration of compound at which the enzyme activity is inhibited by

Purified E. coli holo and core RNAP, E. coli DNA polymerase (DNAP) and wheat germ RNAP II were from Epicentre Technologies (Madison, WI, USA). The RNAP holo and core enzymes, isolated from E. coli strain MRE- 600 (ATCC 29417; ATCC), were checked for the presence and absence of the r subunit by SDS/PAGE. Bacillus subtilis RNAP was a kind gift of A. Galizzi (Institute of Genetics, University of Pavia, Italy) [20]. Rifampicin- resistant (rifR) E. coli RNAP (rpoB3) was from Promega (Madison, WI, USA); rifR RNAP (rpoB7) and rifR RNAP (rpoB3595) were purified, respectively, from E. coli strains CAG3516 and CAG3595 [21], following the purification procedure described previously [22]. The antibiotics rif- ampicin, streptolydigin, ciprofloxacin and chloramphenicol were obtained from Sigma; lipiarmycin was prepared in our laboratories, as previously described [23]; GE23077 was isolated and its physico-chemical properties characterized as described previously [19].

All other chemicals were purchased from standard

commercial sources as analytical grade reagents.

RNAP assays

The inhibition of RNAP activity was determined in an in vitro transcription system, following the incorporation of tritium-labelled uracil in trichloroacetic acid-precipitable material. The reaction mixtures (50 lL total volume in 96- well microplates) contained different dilutions of inhibitors in 50 mM Tris/HCl (pH 8.0), 50 mM KCl, 10 mM MgCl2, 0.1 mM EDTA, 5 mM dithiothreitol, 10 lgÆmL)1 BSA (Sigma), 20 lgÆmL)1 E. coli DNA or sonicated calf thymus DNA (from Boehringer Mannheim), 1 mM ATP, 1 mM GTP, 1 mM CTP, 2 lM UTP and 0.5 lCi 3H-labelled UTP (from Amersham Biosciences). The reactions were started by the addition of enzyme (0.5–1.0 U). Samples were

Fig. 1. Chemical structure of GE23077-A and GE23077-B.

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phenylalanine

3H-labelled

pH 8.0, containing 40 mM KCl, 10 mM magnesium acetate, 0.2 mM MnCl2, 0.5 mM each of ATP, CTP, GTP and UTP, 5 mM phosphoenolpyruvate, 50 lgÆmL)1 pyruvate kinase, (59 CiÆmmol)1, 0.135 lM 1 mCiÆmL)1), 0.5 mM of each of the 19 remaining amino acids, and different concentrations of antibiotics.

incubated at 37 (cid:2)C for 15 min (1 h for wheat germ RNAP II) and quenched with 150 lL of ice-cold 10% trichloro- acetic acid. After 30 min on ice, samples were passed through glass fibre filters using a Cell Harvester device (Wallac, Turku, Finland). Radioactivity not incorporated in the precipitate was washed away with water (25 s) and ethanol (15 s). Finally, filters were counted using a Beta- Plate System (Amersham Biosciences). The RNAP inhibi- tion observed in the presence of different concentrations of in counts per inhibitors was calculated and expressed, minute (c.p.m.), as follows:

RNAP inhibition ¼ ½1 (cid:2) ðsample c:p:m: (cid:2) background c:p:m:Þ=ðno inhibitor c:p:m: (cid:2) background c:p:m:Þ(cid:3) (cid:4)100:

In all cases, reactions were carried out at 30 (cid:2)C for 30 min, then 110 lL of a 10% solution of trichloroacetic acid in water was added and the mixtures were incubated at 4 (cid:2)C for 30 min (for protein biosynthesis assays, after the addition of trichloroacetic acid, samples were preincubated for 10 min at 80 (cid:2)C and then at 4 (cid:2)C for 30 min). All samples were passed through glass fibre filters, using the Wallac Cell Harvester, and washed with water (25 s) and ethanol (15 s). Finally, radioactivity on the filters was counted using a BetaPlate System (Amersham Biosciences).

DNAP assays

Results

Activity of GE23077 on purified RNAPs

Inhibition of DNAP activity was also tested in 96-well microplates using a procedure similar to the RNAP assay. Reactions (50 lL total volume) were performed in 50 mM Tris/HCl, pH 8.0, 5 mM MgCl2, 0.2 mM dithiothreitol, 10 lgÆmL)1 BSA, 20 lgÆmL)1 calf thymus DNA, 20 lM dATP, 20 lM dCTP, 20 lM dGTP, 0.3 lCi 3H-labelled dTTP (0.1 lM, from Amersham Biosciences) and 1 U of E. coli DNAP. Incubation (15 min at 37 (cid:2)C), trichloroace- tic acid precipitation, filtration and radioactivity counting were performed as described above for the RNAP assay.

Table 1 shows the in vitro inhibitory activity of GE23077 on different polymerases, as compared with other known inhibitors of bacterial RNAP. The new antibiotic behaves as a highly selective inhibitor of bacterial RNAPs, active on enzymes from both Gram-negative (E. coli) and Gram- positive (B. subtilis) species, but not active against eukary- otic (wheat germ) RNAP II or E. coli DNAP. Its inhibition potency and selectivity for bacterial RNAPs are comparable with those of rifampicin, and higher than those of strepto- lydigin and lipiarmycin.

Cell plasmolyzation

Effect of GE23077 on intracellular macromolecular biosynthesis

E. coli K12 G210 cells were grown to log phase in 50 mL of Antibiotic Medium 3 (Difco). At an absorbance (A) of 0.75 at 550 nm, cells were harvested, washed with 1 mL of buffer A (20 mM Hepes, pH 8.0) and resuspended in 0.5 mL of 20 mM Hepes, pH 8.0, containing 5 mM EGTA and 2 M sucrose. After 5 min at 25 (cid:2)C, the cell suspension was diluted with 1 mL of buffer A and centrifuged. The cell pellet was washed with 1 mL of the same buffer to remove any residual sucrose and EGTA, and then frozen at )80 (cid:2)C. Each cell pellet was resuspended in ice-cold buffer A (1.5 mL) immediately before use.

Despite its potent inhibitory activity on bacterial RNAP, GE23077 shows a narrow range of antimicrobial activity [19]. To test whether this is a result a potential inability to penetrate bacterial membranes and, at the same time, to confirm in whole cells the specificity of action observed in biochemical assays, it was decided to study the effects of GE23077 on macromolecular biosynthesis in permeabilized E. coli cells.

Macromolecular biosynthesis in permeabilized cells

As a first, mild approach, bacterial cells were treated with Mg2+-chelating agents, compounds that have been reported to weaken bacterial membranes [24], increasing the penetration of antibiotics such as actinomycin [25],

DNA biosynthesis was assayed by incubating 10 lL of plasmolyzed cells (containing (cid:1) 5 · 109 cells per mL) in a total volume of 50 lL of 20 mM Hepes, pH 8.0, containing 100 mM KCl, 10 mM magnesium acetate, 1 mM dithiothre- itol, 2 mM ATP, 0.1 mM NAD, 0.5 mM each of dATP, dGTP and dCTP, 0.05 lM methyl[3H]thymidine (0.2 lL of 79 CiÆmmol)1, 1 mCiÆmL)1), and different concentrations of antibiotics.

Table 1. Activity of GE23077 and other RNA polymerase (RNAP) inhibitors on purified polymerases. Results are expressed as IC50 values (i.e. the lM concentration of the compound at which the enzyme activity is inhibited by 50%). ND, not determined.

RNA biosynthesis was assayed by incubating 10 lL of plasmolyzed cells in a total volume of 50 lL of 20 mM Hepes, pH 8.0, containing 10 mM KCl, 10 mM magnesium acetate, 0.2 mM MnCl2, 0.5 mM each of ATP, GTP and CTP, 10 lM 3H-labelled UTP (0.5 lL of 50 CiÆmmol)1, UTP, 0.2 lM 1 mCiÆmL)1) and different concentrations of antibiotics.

B. subtilis RNAPa Wheatgerm RNAP II E. coli DNAP E. coli RNAPa

a Holoenzyme. b Described previously [23].

Protein biosynthesis was assayed by incubating 10 lL of plasmolyzed cells in a total volume of 50 lL of 2 mM Hepes,

GE23077 Rifampicin Streptolydigin Lipiarmycinb 0.020 0.030 7.5 5.0 0.025 0.028 ND 0.60 > 100 > 100 > 100 ND > 100 > 100 > 100 65

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kirromycin [26] and pulvomycin [27], normally poorly active on Gram-negative bacteria. However, whereas both EDTA and EGTA increased the activity of rifampicin, respectively, by a factor of 30 and 16 – i.e. from a minimum inhibitory concentration (MIC) of 4 lM (control) to an MIC of 0.13 lM (1 mM EDTA) and an MIC of 0.25 lM (5 mM EGTA) – no significant improvement in antimicrobial activity was observed with GE23077 (MIC > 200 lM in all cases). An alternative approach, based on the use of polymyxin B to increase the permeability of E. coli cells under different conditions, also failed to significantly improve the antimicrobial activity of GE23077.

It was then decided to test cell plasmolyzation, i.e. the incubation of bacterial cells in hypertonic medium (2 M sucrose). This treatment, more drastic than the previous ones, makes the outer membrane adhere tightly to the cell wall and the inner membrane contract away from it, producing a small amount of damage to both membranes and thereby increasing their permeability [28]. Although cells do not replicate in these conditions, and consequently MIC values cannot be determined, such a method allows assessment of the effect of added compounds on macro- molecular biosynthesis [29,30]. As shown in Fig. 2, when 30 lM GE23077 is added to plasmolyzed cells, RNA synthesis is totally inhibited within few minutes, in the same manner as the rifampicin control, while no effect is observed on DNA or protein synthesis. Thus, the specificity of action observed with purified enzymes (Table 1) is confirmed in bacterial cells.

As shown in Fig. 3, the inhibition of RNA synthesis by GE23077 is also dose-dependent, like that of rifampicin, although higher concentrations of the former are required to achieve comparable inhibition levels: in our experimental conditions, the IC50 values were 2 lM for GE23077 and 0.12 lM for rifampicin.

In summary, these data confirm the specificity of action of GE23077 on cellular RNA synthesis and strongly suggest that its restricted antimicrobial activity is a result of its inability to cross bacterial membranes.

Mechanism of action of GE23077 on E.coliRNAP

In order to obtain some basic information on the mechan- ism of action of GE23077 on its target enzyme, different biochemical assays were performed using purified enzymes and known RNAP inhibitors as reference compounds.

GE23077 to the elongating complex did not result into an immediate stop, as observed with streptolydigin, but rather in a slowing down of the process, a behaviour similar to that shown by rifampicin, thereby indicating that GE23077 acts at the level of transcription initiation.

r-dependent vs. r-independent transcription initiation. The r subunit of RNAP plays a central role in promoter recognition and transcription initiation in bacterial cells

Transcription initiation vs. chain elongation. As a first step in the elucidation of the mechanism of action of GE23077, it is crucial to assess whether it exerts its action at the level of transcription initiation, like lipiarmycin [11] and rifampicin [16], or chain elongation, like streptolydigin [12]. To obtain such information, the time course of RNAP inhibition was measured comparing the effect of adding GE23077 to the reaction solution either before the start of transcription or during RNA synthesis (Fig. 4). Rifampicin and streptolydigin were used as reference inhibitors of, respectively, transcription initiation and chain elongation. As expected, all three compounds behaved similarly when added to the reaction mixture before the start of transcrip- tion (induced by DNA addition), resulting in complete inhibition of RNA synthesis. Conversely, the addition of

Fig. 2. Effect of GE23077 and other agents on macromolecular bio- synthesis in permeabilized Escherichia coli cells. Bacteria were perme- abilized by preincubation in hypertonic medium, as described in the Materials and methods. The concentration of compounds used in this experiment were as follows: GE23077, 30 lM (in all three cases); ciprofloxacin, 2 lM (a positive control for DNA biosynthesis); rifampicin, 3 lM (a positive control for RNA biosynthesis); and chloramphenicol, 20 lM (a positive control for protein biosynthesis).

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the compound’s effects on RNAP were compared under conditions of either r-dependent or r-independent tran- scription initiation, using streptolydigin as reference inhib- itor, which, by acting on chain elongation, is known to inhibit RNAP regardless of the transcription initiation conditions used [12]. As shown in Fig. 5, it was found that GE23077 is able to inhibit RNA synthesis in both cases, although with different potency (IC50 values of 20 nM for r-dependent and 100 nM for r-independent initiation). Even though this finding clearly indicates that the molecular target of GE23077 is not the r subunit itself, the fivefold lower activity and the different shape of the inhibition curve observed in the absence of r indicate that the presence of this factor potentiates the inhibitory activity of GE23077. As expected, such differential behaviour in the presence or absence of r is not shown by streptolydigin, which, by acting at a stage when the r factor has already dissociated from the transcription complex [12], displays similar inhi- bition curves and IC50 values in both cases.

Hence, besides adding new information on the mechan- ism of action of GE23077, the results shown in Fig. 5 also provide direct confirmation of the findings, reported in the previous paragraph, that it acts at the level of transcription initiation.

[2,3]. However, it is known that core (i.e. r-free) bacterial RNAP is able to perform in vitro transcription using fragmented or nicked DNA molecules as templates, in a promoter-independent manner. Although less efficient than the physiologically relevant r-dependent process ((cid:3)holo(cid:4) RNAP and E. coli genomic DNA as template), such r-independent transcription activity ((cid:3)core(cid:4) RNAP and fragmented eukaryotic DNA as template) is nevertheless sufficiently high to be exploited for studies on the mechan- ism of action of RNAP inhibitors. As a specific inhibitor of

transcription initiation, GE23077 might exert its action by directly binding and inhibiting the RNAP r subunit, or by acting exclusively on holo (and not core) RNAP. To investigate this hypothesis,

RNAP–DNA complex formation. To further elucidate the mechanism of action of GE23077 on E. coli RNAP, the possibility was investigated that the compound might inhibit RNA synthesis by preventing RNAP from binding to DNA. Binding of RNAP to DNA is indeed one of the earliest steps of the transcription process and a possible molecular target of a transcription initiation inhibitor. In such cases, a preformed RNAP–DNA complex would be less sensitive to the action of the inhibitor than an isolated, unbound RNAP molecule. To test such a possibility, the E. coli holoenzyme was preincubated with DNA to allow complex formation before the addition of the inhibitor, and then the effect of GE23077 on RNA synthesis was assessed. Two antibiotics known to show different behaviour, in that respect, were used as controls: lipiarmycin, whose inhibitory activity is known to be largely reduced when it is added after the formation of the RNAP–DNA complex [11]; and rifampicin, which, conversely, binds and inhibits RNAP equally well if added when the enzyme is already bound to DNA [16]. As shown in Fig. 6, all three compounds totally inhibited RNA synthesis when added before DNA, whereas

Fig. 3. Dose–response analysis of RNA biosynthesis inhibition by rifampicin and GE23077 in permeabilized Escherichia coli cells.

Fig. 4. Effect of GE23077 and other RNA polymerase (RNAP) inhibitors on in vitro RNA synthesis: comparison of the effects of com- pound addition before vs. after reaction start. The concentration of compounds used in this experiment were as follows: GE23077, 10 lM; rifampicin, 1 lM; streptolydigin, 100 lM. j, No inhibitor controls; d, compounds were added before the reaction start, marked by the addition of DNA to mixtures containing all the other components and the indicated inhibitor; m, compounds were added 5 min after reaction start, as indicated by the arrows.

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molecule and, consequently, the possibility of cross-resist- ance between them. To test such a hypothesis, we studied the effect of GE23077 on different rifR RNAPs, purified from E. coli strains containing known rpoB mutations [21,31]. As shown in Table 2, GE23077 behaved very differently from rifampicin in these tests, inhibiting RNA synthesis with similar potency in all cases. These data show that cross-resistance between the two compounds is not a common event and suggest that they have distinct binding sites on their target enzyme.

Discussion

This report describes the biochemical activity of GE23077, a novel microbial metabolite identified in the course of a screening program aimed at the discovery of selective inhibitors of bacterial RNAP [19]. Its high potency and selectivity, comparable to those of rifampicin (Table 1), together with its novel chemical structure, render this compound very interesting from a scientific perspective and for its therapeutic potential. The narrow range of anti- microbial activity of GE23077 might explain why this potent RNAP inhibitor had previously been undetected, an observation which supports and validates the notion of using target-oriented biochemical assays (rather than more traditional microbiological assays) to find novel, unex- ploited chemical leads for drug development.

lipiarmycin was significantly less active than rifampicin and GE23077 when added after preincubation of the enzyme with DNA. The observation that, in these experiments, GE23077 behaves like rifampicin, strongly suggests that its mode of action is not based on the prevention of RNAP binding to DNA.

Activity of GE23077 on purified rifR RNAPs

The molecular basis for the low activity of GE23077 in microbiological assays was investigated in this study. In experiments with permeabilized E. coli cells, it was found that the antibiotic is able to exert its action, i.e. to block RNA synthesis, when cell membranes are damaged. Its activity on macromolecular biosynthesis is dose-dependent and selective, not showing any effect on either DNA or protein synthesis, thereby confirming on whole cells the specificity of action observed with purified enzymes.

Although GE23077 is structurally very different from rifampicin, the data shown in the previous paragraphs indicate that the two compounds share a number of common features. Both are potent and selective inhibitors of bacterial RNAPs (Table 1) and cellular RNA biosyn- thesis (Figs 2 and 3), both act at the level of transcription initiation (Fig. 4), and both show similar activity on their target enzyme when added before or after RNAP–DNA complex formation (Fig. 6). This might suggest overlapping binding sites for the two compounds on the RNAP

It is tempting to conclude from these findings that GE23077 is poorly active on whole bacterial cells, simply because it is not able to cross bacterial membranes, which would act like physical barriers to the action of the antibiotic. This idea is also supported by its hydrophilic molecular structure, which includes the presence of a

Fig. 5. Effect of GE23077 and streptolydigin on in vitro RNA synthesis: comparison of the effects of compounds under conditions of r-dependent vs. r-independent transcription initiation. The inhibition of (cid:3)holo(cid:4) RNA polymerase (RNAP) with Escherichia coli genomic DNA as template (r-dep.) is compared with the inhibition of (cid:3)core(cid:4) RNAP with sonicated calf thymus DNA (r-ind.), at different concentrations of GE23077 and streptolydigin (strept.). The data shown are the mean of triplicate readings ± SD.

Fig. 6. Effect of GE23077 and other RNA polymerase (RNAP) inhibitors on in vitro RNA synthesis: comparison of the effects of compound addition either before or after RNAP–DNA complex formation. The concentration of compounds used in this experiment are as follows: GE23077, 1 lM; rifampicin, 1 lM; lipiarmycin, 100 lM. j, No inhibitor controls; d, compounds were added before the reaction start, marked by the addition of DNA to mixtures containing all the other components and the indicated inhibitor; m, nucleotides and the indicated inhibitor were added to mixtures that contained all the other components, and that had been preincubated for 5 min at 37 (cid:2)C to allow RNAP–DNA complex formation.

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Table 2. Activity of GE23077 and rifampicin on purified Escherichia coli rifampicin resistant (rifR) RNA polymerases (RNAPs). Results are expressed as IC50.

rpoB allele (mutation) Wild-type rpoB3595 (Ser522 fi Phe)b rpoB7 (Ile572 fi Phe)b rpoB3 (Ser531 fi Phe)a

a Described previously [31]. b Described previously [21].

mapped thus far are located in the rpoB gene [21,31]. When the activity of GE23077 was compared with that of rifampicin on three independent rifR RNAP mutants, the the two compounds was very different behaviour of (Table 2), indicating that cross-resistance is not a common event and hence that the two compounds possess distinct binding sites on RNAP.

negative charge around neutral pH (Fig. 1). However, it is important to note that even minor damage to the cell membrane may have far-reaching consequences on cellular in particular, on membrane-associated activities, and, transport systems. Although our data suggest that impair- ment in cell penetration should be the main reason for the observed low antimicrobial activity of GE23077, the possibility exists that other mechanisms, such as efflux pumps, might contribute to the in vivo inactivation of the antibiotic.

In this respect, it is interesting to note that GE23077 is about one order of magnitude less potent than rifampicin in permeabilized cells (Fig. 3), which contrasts with the similar potency displayed by the two antibiotics on purified enzymes (Table 1). Such a difference might simply reflect a still-incomplete penetration of GE23077 in plasmolyzed bacteria, but alternative explanations, such as only partial inactivation of efflux pumps, are possible. In addition, the observation that the E. coli strain used for the cell-perme- abilization studies (i.e. K12 G210) is different from that used for purified RNAP production (i.e. MRE-600), also suggests the possibility that the lower activity in permeabi- lized cells might be the result of a pre-existing partial resistance to GE23077 in that particular strain.

Rifampicin resistance is known to arise spontaneously with a relatively high frequency, e.g. (cid:1) 10)8 in E. coli [21]. The similarity in the mode of action of the two antibiotics, together with the observation that GE23077 is active on rifR RNAP mutants, raises the question of what is the resistance mutation frequency of the new antibiotic. In view of the low antimicrobial activity of GE23077 on E. coli and other bacteria, such a question might be addressed using the M. catarrhalis clinical isolates on which GE23077 shows significant activity [19]. However, the cell penetration issue discussed above suggests that a considerable fraction of GE23077-resistant colonies might contain alterations in cell permeability, rather than genuine RNAP mutations. The isolation and sequencing of a statistically significant number of mutants could assess the extent of such phenomenon. (see below) of obtaining Considering the prospects GE23077 derivatives with enhanced cell-penetration capa- bilities (and consequently higher antimicrobial activity and wider spectrum), such improved molecules should also allow a more straightforward and accurate determination of a bona fide resistant RNAP mutation frequency.

In general, it is important to consider that different mechanisms might operate in different bacteria to confer resistance to GE23077. The variety of bacterial species showing very low or no sensitivity to the antibiotic [19] raises the question of whether some might carry an intrinsically resistant RNAP target. Further studies will help to elucidate this issue.

The data reported in the present report indicate that GE23077 is an interesting RNAP inhibitor, worthy of further investigation for the wealth of structural informa- tion that it can provide on the functioning of a crucial enzyme like RNAP. It would be interesting to establish whether the resemblance in the inhibitory action of GE23077 and rifampicin is also observed at a more detailed level, i.e. the specific step inhibited during the initiation process. Further mechanistic studies, e.g. experiments based on the abortive initiation reaction [16], or on fluorescence resonance energy transfer (FRET) analyses [32], might elucidate whether GE23077, like rifampicin, blocks the translocation step that would ordinarily follow the forma- tion of the first phosphodiester bond, or whether it acts at a different step, as might be suggested by the lack of cross- resistance. Also, further information might be obtained through structural elucidation of the RNAP–GE23077 complex, in a study similar to the one recently performed on the Thermus aquaticus RNAP–rifampicin complex [17]. A high-resolution structure determination of the RNAP– GE23077 complex should provide insights into GE23077 binding and its mechanism of inhibition, together with new information on the transcription process itself.

In this work, information was also obtained on the mechanism of action of GE23077 on its target enzyme. It was found that the compound acts at the level of transcrip- tion initiation and that even though the presence of the RNAP r subunit potentiates its activity, its molecular target is not the r subunit itself, or the interaction of RNAP with promoter DNA to form the transcription complex (i.e. GE23077 inhibits the enzyme equally well even when this is already engaged in the RNAP–DNA complex). Strikingly, this behaviour is similar to that shown by rifampicin [16] and hence the two compounds, although structurally unrelated, show analogies that go beyond potency and specificity, an observation that might suggest similar binding sites for the two molecules on the target enzyme. This hypothesis prompted us to investigate whether such similarities would also entail cross-resistance between the two compounds. The rifampicin-binding site has been well characterized and is located in a pocket between two structural domains of the RNAP b subunit [17]. Accord- ingly, the large majority of rifR mutations identified and

GE23077 Rifampicin 0.020 0.030 0.050 > 100 0.062 > 100 0.031 15

GE23077, a novel bacterial RNA polymerase inhibitor (Eur. J. Biochem. 271) 3153

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7. Murakami, K.S., Masuda, S., Campbell, E.A., Muzzin, O. & Darst, S.A. (2002) Structural basis of transcription initiation: an RNA polymerase holoenzyme–DNA complex. Science 296, 1285– 1290.

8. Record, M.T.J., Reznikoff, W., Craig, M., McQuade, K. & Schlax, P. (1996) Escherichia coli RNA polymerase (Er70), pro- moters and the kinetics of the steps of transcription initiation. In: Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. (Neidhart, F.C., ed.), pp. 792–820. ASM Press, Washington, DC. 9. Sensi, P. (1983) History of the development of rifampin. Rev. Infect. Dis. 3, 402–406.

In addition to the scientific interest of GE23077 as novel RNAP inhibitor, it is also interesting to speculate on its potential as a chemical lead for novel anti-infective chemo- therapeutic agents. Considering the emergence of bacterial resistance to drug therapy and the observation that, with the exception of oxazolidinones, no new scaffolds of antibac- terial agents for human use have been developed in the past 30 years [33], the novel structure of GE23077 becomes particularly attractive. Its activity on clinical isolates of M. catarrhalis [19] is interesting, as such a bacterium is considered to be the third commonest pathogen of the respiratory tract in humans after Streptococcus pneumoniae and Haemophilus influenzae, responsible for otitis media in children and lower respiratory tract infections in the elderly [34]. In addition, the widespread production of b-lactamase renders M. catarrhalis resistant to penicillins [35], as also observed in GE23077-sensitive M. catarrhalis strains (E. Selva, unpublished data).

10. Parenti, F. & Lancini, G. (1997) Rifamycins. In Antibiotics and Chemotherapy (O’Grady, F., Lambert, H.P., Finch, R.G. & Greenwood, D., eds), 7th edn, pp. 453–459. Churchill Livingstone, New York, NY.

11. Sonenshein, A.L. & Alexander, H.B. (1979) Initiation of tran- scription in vitro inhibited by lipiarmycin. J. Mol. Biol. 127, 55–72. 12. McClure, W.R. (1980) On the mechanism of streptolydigin inhi- bition of Escherichia coli RNA polymerase. J. Biol. Chem. 255, 1610–1616.

the

cell-penetration issue

13. Reichenbach, H. & Hofle, G. (1999) Myxobacteria as producers of secondary metabolites. In Drug Discovery from Nature (Grabley, S. & Thiericke, R., eds), pp. 149–178. Springer-Verlag, Berlin, Germany.

14. O’Neill, A., Oliva, B., Storey, C., Hoyle, A., Fishwick. C. & Chopra, I. (2000) RNA polymerase inhibitors with activity against rifampicin-resistant mutants of Staphylococcus aureus. Antimicrob. Agents Chemother. 44, 3163–3166.

15. Artsimovitch, I., Chu, C., Lynch, A.S. & Landick, R. (2003) A new class of bacterial RNA polymerase inhibitors affects nucleo- tide addition. Science 302, 650–654.

The activity found against clinical isolates of M. catar- rhalis suggests that GE23077 can be considered as a natural template for chemical modifications to extend its anti- microbial spectrum to include other pathogens. Given its potent and selective activity on its biochemical target, appropriate chemical derivation programmes might over- come and yield potent molecules with a wider range of antimicrobial activity. In this respect, it is interesting to note that rifampicin, the widely used antibiotic that has become an important component of today’s anti-infective chemotherapy arsenal, is indeed a semisynthetic derivative of the naturally occurring microbial metabolite, rifamycin SV [10]. In a comparable scenario, GE23077 derivatives possessing sim- ilar activity on RNAP and, at the same time, improved cell-membrane permeability, might be promising leads for the development of antibacterial drugs.

16. McClure, W.R. & Cech, C.L. (1978) On the mechanism of rifampicin inhibition of RNA synthesis. J. Biol. Chem. 253, 8949– 8956.

17. Campbell, E.A., Korzheva, N., Mustaev, A., Murakami, K., Nair, S., Goldfarb, A. & Darst, S.A. (2001) Structural mechanism for rifampicin inhibition of bacterial RNA polymerase. Cell 104, 901–912.

Acknowledgements

18. Yang. X. & Price, C.W. (1995) Streptolydigin resistance can be conferred by alterations to either the beta or beta¢ subunits of Bacillus subtilis RNA polymerase. J. Biol. Chem. 270, 23930– 23933. We are grateful to P. Landini, B. Goldstein, G. Lancini and M. Denaro for suggestions and helpful discussions. We also thank F. Parenti for critical reading of the manuscript.

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