Mode of action of the microbial metabolite GE23077, a novel potent and selective inhibitor of bacterial RNA polymerase Edoardo Sarubbià, Federica Monti*, Emiliana Corti, Anna Miele and Enrico Selva Vicuron Pharmaceuticals, Gerenzano, Varese, Italy GE23077, a novel microbial metabolite r ecently isolated from Actinomadura sp. culture media, is a potent and selective inhibitor of bacterial RNA polymerase (RNAP). It inhibits Gram-positive (Bacillus s ubtilis) a nd Gram- negative (Escherichia coli)RNAPswithIC 50 values (i.e. the concen- tration a t which the enzyme a ctivity i s inhibited by 50%) i n the 10 )8 M range, whereas it is not active on E. coli DNA polymerase or on eukaryotic (wheat germ) RNAP I I (IC 50 values > 10 )4 M in both cases). In spite of its potent activity on pur ified b acterial 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 for effective antimicrobial activity of the antibiotic. Bio- chemical studies were also conducted w ith purified enzymes to obtain further insights into the mode of action of GE23077. Interestingly, the compound d isplays a behaviour similar to that of rifampicin, an antibiotic structurally unrelated to GE23077: both c ompounds act at t he level of transcription initiation, but not on the r subunit a nd not on the f ormation of the promoter DNA–RNAP complex. T ests on different rifampicin-resistant E. coli RNAPs did not show any cross-resistance between the two compoun ds, indicating distinct binding sites on the target enzyme. In conclusion, GE23077 is an interesting new molecule for future mechanistic s tudies on bacterial R NAP and for its potential in anti-infective drug discovery. Keywords: antibiotic; cell permeabilization; natural p roduct; rifampicin; t ranscription initiation. DNA-directed RNA polymeras e (EC 2.7.7.6; RNAP) i s the central enzyme of bacterial gene expression, responsible for all cellular RNA synthesis [ 1]. T he catalytic ally competent ÔcoreÕ RNAP consists of five subunits (a 2 bb¢x,witha combined molecular m ass of % 400 kDa) and is capable of elongation and termination. The initiation-competent Ôholo Õ RNAP is compo sed of the core enzyme and of an additional subunit, r, which confers o n RNAP t he 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 o f the crystal structures o f t he 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 c losed complex, melts the DNA du plex around th e )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 about9–12nucleotides,the transcription complex enters the elongation stage. This transition is marked by a s ignificant 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 b y 2 A ˚ to 12 A ˚ [1]. Owing to its central role i n DNA transcription, RNAP is an essential enzyme in bacterial cells and t he 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 p athogens, such as Mycobacterium tuberculosis, for which i t 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 remain s of great i nterest for the biomedical c ommunity. Several different series of compounds (isolated f rom 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: E doardo.Sarubbi@aventis.com Abbreviations: c.p.m., counts per minute; DNAP, DNA polymerase; IC 50 , the concentration of compound at which the enzyme activity is inhibited by 50%; RNAP, RNA p olymerase; rif R , rifampicin resistant. Enzyme: D NA-directed RNA polymerase (EC 2.7.7.6). Present add ress: àLead Disc overy Technologies, Ave ntis Pharma, France. * Arpida Lt d, Munchenstein, S witzerland. Aventis Pharma, Anagni (Frosinone), Italy. (Received 2 April 2004, r evised 29 May 2004, accepted 3 June 2004) Eur. J. Biochem. 271, 3146–3154 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04244.x which act on RNAP, h ave b een reported in the literature, but none has thus far been marked for c linical use. Besides their potential interest as therapeutic a gents, these compounds ar e also valuable tools for using to c haracterize the complex activity of their target enzyme. RNAP inhib- itors have been discovered which a ct at different stages of the t ranscription process, for example (a) lipiarmycin inhibits the formation of the fi rst 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 e longation by i nhibiting the translocation step [ 12,18], and (d) the r ecently r eported CRB703 series of compounds specifically inhibit the nuc- leotide addition reaction in the elongation c omplex [15]. T he availability of R NAP i nhibitors, acting at different steps of the transcription process, has been very helpful for charac- terizing the various conformational changes that RNAP undergoes during DNA transcripti on, a process that, however, still remains incompletely understood. GE23077 is a novel microbial metabolite, recently discovered in the f ermentation broth of an Actinomadura sp. during the screening of natural products for specific inhibitors of bacterial R NAP [ 19]. I t is structurally unrelated to any other known c ompound and i s 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 s how similar b iochemical activity [19], suggest- ing that the small v ariations in the side-chain result in only minor effects on G E23077 activity. In spite of its potent inhibitory activity on purified Escherichia coli RNAP [i.e. the IC 50 (concentration of compound at which the enzym e activity is inhibited by 50%) ¼ 20 n M ], the antimicrobial activity of GE23077, tested on a variety of Gram-positive and Gram-negative strains, shows a narrow s pecies range. Its s pectrum of activity is essentially restricted to Moraxella catarrhalis isolates and, to a lesser extent, N eisseria gonorrhoeae and Mycobacterium smegmatis , w here relatively high antibiotic concentrations (10 )4 M ) must be used [19]. Such r estricted cellular a ctivity m ight 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. 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 p ermeabilized cells to confirm the specificity of RNA synthesis inhibition. Third, its mechanism of inhibition of purified E. coli RNAP, determining a t which stage of the transcription process it exerts its action. Finally, its activity on different r ifampicin- resistant ( rif R ) R NAPs, assessing its propensity f or cross- resistance with rifampicin to obtain information on its binding site on the RNAP molecule. Materials and methods Enzymes and antibiotics Purified E. coli holo and core RNAP, E. coli DNA polymerase (DNAP) and wheat germ RNAP II w ere from Epicentre T echnologies (Madison, WI, USA). The RNAP holo a nd core enzymes, isolated from E. coli strain MRE- 600 (ATCC 29417; ATCC), were checked for t he presence and absence of the r subunit b y SDS/PAGE. Bacillus subtilis RNAP was a kind gift of A. Galizzi (Institute of Genetics, University of Pavia, Italy) [20]. Rifampicin- resistant ( rif R ) E. coli RNAP (rpoB3) was f rom Promega (Madison, WI, U SA); r if R RNAP (rpoB7) and r if R 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 c hloramphenicol were obtained from Sigma; lipiarmycin was prepared in our laboratories, as previously described [23]; G E23077 was isolated and i ts physico-chemical properties c haracterized as described previously [19]. All o ther 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 s ystem, following the i ncorporation 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 m M Tris/HCl (pH 8.0), 50 m M KCl, 10 m M MgCl 2 , 0.1 m M EDTA, 5 m M dithiothreitol, 10 lgÆmL )1 BSA (Sigma), 20 lgÆmL )1 E. coli DNA o r sonicated calf thymus DNA (from Boehringer Mannheim), 1 m M ATP, 1 m M GTP, 1 m M CTP, 2 l M UTP a nd 0.5 lCi 3 H-labelled U TP (fromAmershamBiosciences).Th e reactions were started by the addition of enzyme (0.5–1.0 U). Samples were Fig. 1. Che mical structure of GE23077-A and GE23 077-B. Ó FEBS 2004 GE23077, a novel bacterial RNA polymerase inhibitor (Eur. J. Biochem. 271) 3147 incubated at 37 °C for 15 min (1 h for wheat germ RNAP II) and quenched w ith 150 lL of ice-cold 10% t richloro- acetic acid. After 30 min on ice, samples were passed through glass fi bre filters using a Cell Harvester d evice (Wallac, Turku, Finland). Radioactivity no t i ncorporated in the precipitate was washed away w ith water (25 s) and ethanol (15 s). Finally, filters were c ounted using a Beta- Plate System ( Amersham Bioscien ces). The R NAP i nhibi- tion observed i n the presence of different concentrations of inhibitors was calculated and expressed, in counts per minute (c.p.m.), as follows: RNAP inhibition ¼½1 Àðsample c:p:m: À background c:p:m:Þ=ðno inhibitor c:p:m: À background c:p:m:Þ Â100: DNAP assays Inhibition of DNAP activity was also tested in 96-well microplates using a procedure similar t o the RNAP assay. Reactions (50 lL t otal vo lume) were performed in 50 m M Tris/HCl, pH 8.0, 5 m M MgCl 2 ,0.2m M dithiothreitol, 10 lgÆmL )1 BSA, 20 lgÆmL )1 calf thymus DNA, 20 l M dATP, 2 0 l M dCTP, 2 0 l M dGTP, 0 .3 lCi 3 H-labelled dTTP (0.1 l M , from Amersham B iosciences) and 1 U of E. coli DNAP. I ncubation (15 min at 37 °C), trichloroace- tic acid p recipitation, filtration a nd radioactivity counting were performed as described above for the RNAP a ssay. Cell plasmolyzation E. coli K12 G210 cells were grown to log phase in 50 mL of Antibiotic Medium 3 (Difco). A t an absorbance (A)of0.75 at 550 nm, cells were harvested, washed with 1 mL of buffer A(20m M Hepes, pH 8.0) and resu spended i n 0.5 mL of 20 m M Hepes, pH 8.0, containing 5 m M EGTA and 2 M sucrose. After 5 min at 25 °C, the cell suspension was diluted with 1 mL of buffer A and centrifuged. The cell pellet was washed with 1 mL o f the same buffer to remove any residual sucrose and E GTA, and then f rozen at )80 °C. Each cell pellet was resuspended in ice-cold buffer A (1.5 mL) i mmediately before use. Macromolecular biosynthesis in permeabilized cells DNA biosynthesis was assayed by incubating 10 lLof plasmolyzed cells (containing % 5 · 10 9 cells per mL) in a total v olume of 50 lLof20m M Hepes, pH 8.0, containing 100 m M KCl, 10 m M magnesium acetate , 1 m M dithiothre- itol, 2 m M ATP, 0.1 m M NAD, 0.5 m M each of dATP, dGTP and d CTP, 0.05 l M methyl[ 3 H]thymidine (0.2 lLof 79 Ci Æmmol )1 ,1mCiÆmL )1 ), and different concentrations of antibiotics. RNA biosynthesis was as sayed by incubating 10 lLof plasmolyzed cells in a t otal volume of 50 lLof20 m M Hepes, pH 8.0, containing 10 m M KCl, 10 m M magnesium acetate, 0.2 m M MnCl 2 ,0.5 m M each of ATP, GTP and C TP, 10 l M UTP, 0.2 l M 3 H-labelled UTP (0.5 lLof50CiÆmmol )1 , 1mCiÆmL )1 ) and different concentrations of antibiotics. Protein biosynthesis w as as sayed b y i ncubating 10 lLof plasmolyzed cells in a total volume of 50 lLof2 m M Hepes, pH 8.0, containing 40 m M KCl, 10 m M magnesium acetate, 0.2 m M MnCl 2 ,0.5m M each of ATP, CTP, GTP and UTP, 5m M phosphoenolpyruvate, 50 lgÆmL )1 pyruvate k inase, 0.135 l M 3 H-labelled phenylalanine (59 C iÆmmol )1 , 1mCiÆmL )1 ), 0.5 m M of each of the 19 r emaining amino acids, and different concentrations of antibiotics. In all c ases, r eactions were c arried out at 30 °Cfor 30 min, then 110 lL of a 10% s olution of trichloroacetic acid in water w as added and the m ixtures were incubated at 4 °C for 30 min (for protein biosynthesis assays, after the addition of trichloroacetic a cid, samples were p reincubated for 10 min at 80 °Candthenat4°C for 30 min). All samples were passed through g lass fibre filters, using t he Wallac Cell Harvester, a nd washed with water ( 25 s) and ethanol (15 s). Finally, radioactivity on the filters was counted using a BetaPlate System (Amersham Biosciences). Results Activity of GE23077 on purified RNAPs Table 1 shows the in vitro inhibitory activity of GE2 3077 on different polymerases, as compared with other known inhibitors of bacterial RNAP. The new antibiotic behaves as a highly s elective inhibitor of bacterial RNAPs, active on enzymes f rom both Gram-negative (E. coli)andGram- positive ( B. subtilis) species, but not a ctive against euk ary- otic (wheat germ) RNAP I I or E. coli DNAP. Its i nhibition potency and s electivity for bacterial RNAPs a re comparable with those of rifampicin, and higher than t hose of strepto- lydigin and lipiarmycin. Effect of GE23077 on intracellular macromolecular biosynthesis Despite i ts potent inhibitory activity on bacterial RNAP, GE23077 shows a narrow r ange of antimicrobial activity [19]. To test whether this is a r esult a potential inability to penetrate bacterial membranes a nd, at the s ame time, to confirm in whole cells the s pecificity o f action observed in biochemical a ssays, i t was decided to s tudy the effects o f GE23077 on macromolecular biosynthesis i n permeabilized E. coli cells. As a first, mild approach, bacterial cells were treated with Mg 2+ -chelating agents, compounds that have been reported to weaken bacterial membranes [24], increasing the penetration of antibiotics such a s a ctinomycin [25], Table 1. A ctivity of GE23077 and o ther R NA p olymerase (RNAP) inhibitors on purified polymerases. Results are expressed as IC 50 values (i.e. t he l M concentration of the compound a t which the enzym e activity is inhib ited by 50%). ND, not determined. E. coli RNAP a B. subtilis RNAP a Wheatgerm RNAP II E. coli DNAP GE23077 0.020 0.025 > 100 > 100 Rifampicin 0.030 0.028 > 100 > 100 Streptolydigin 7.5 ND > 100 > 100 Lipiarmycin b 5.0 0.60 ND 65 a Holoenzyme. b Described previously [23]. 3148 E. Sarubbi et al. (Eur. J. Biochem. 271) Ó FEBS 2004 kirromycin [26] and pulvomycin [ 27], normally poorly active on Gram-negative b acteria. However, whereas both EDTA and EGTA increased the a ctivity o f rifampicin, respectively, by a f actor of 3 0 and 16 – i.e. from a minimum inhibitory concentration (MIC) of 4 l M (control) to an MIC of 0.13 l M (1 m M EDTA) a nd an MIC o f 0.25 l M (5 m M EGTA) – no significant improvement in antimicrobial activity was observed with GE23077 (MIC > 200 l M in all cases). An alternative a pproach, based on the use of polymyxin B to increase the permeability of E. coli cells under different condition s, also failed t o significantly improve the antimicrobial activity of GE23077. It was then d ecided to test c ell 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 m embranes and thereby increasing th eir permeability [28]. A lthough cells do not replicate in these conditions, a nd consequently MIC values c annot be determined, such a meth od allows assessment of the effect of added compounds on macro- molecular biosynthesis [29,30]. As shown in Fig. 2, when 30 l M GE23077 is added to plasmolyzed cells, RNA synthesis i s t otally inhibited within f ew minutes, i n the same manner as the rifampicin control, while no effect is observed on DNA or protein s ynthesis. 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 c oncentrations of the former a re required to achieve c omparable inhibition levels: i n our experimental conditions, the IC 50 values were 2 l M for GE23077 and 0.12 l M for rifampicin. In summary, these data confirm t he specificity of action of GE23077 on cellular RNA synthesis a nd strongly suggest that its restricte d antimicrobial activity is a result of i ts inability to cross bacterial membranes. Mechanism of action of GE23077 on E. coli RNAP In order to obtain some basic information on the mechan- ism o f action o f G E23077 on its target e nzyme, diff erent biochemical assays were performed using purified enzymes and known RNAP inhibitors as reference compounds. Transcription initiation vs. chain elongation. As a first step in the elucidation of the mechanism of action of GE23077, it is crucial t o assess whether it e xerts its action at the l eve l o f transcription i nitiation, like lipiarmycin [11] and rifampicin [16], or c hain e longation, lik e streptolydigin [12]. To obtain such information, the time course of RNAP inhibition was measured comparing the effect of adding GE23077 to the reaction solution e ither before the start of transcription o r during RNA synthesis ( Fig. 4). Rifampicin and stre ptolydigin w ere used as reference inhibitors of, respectively, transcriptio n initiation and ch ain elongation. As expected, a ll three c ompounds 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, t he addition of GE23077 to the elongating complex did not result into an immediate stop, as observed with streptolydigin, but rather in a s lowing down o f the proces s, a b ehaviour similar t o that shown by rifampicin, thereby i ndicating 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 i n bacterial cells Fig. 2. Effect of GE23077 and other ag ents on macromolecular bio- synthesis in permeabilized Escherichia coli cells. Bacteria were perme- abilized by preincubation in hype rtonic me dium, as described in the Materials and methods. The concentration of compounds used in this experiment were as follows: GE23077, 30 l M (in all three cases); ciprofloxacin, 2 l M (a positive control for DNA biosynthesis); rifampicin, 3 l M (a positive c ontrol for RNA biosynthesis); and chloramphenicol, 20 l M (a positive co ntrol for protein biosynthe sis). Ó FEBS 2004 GE23077, a novel bacterial RNA polymerase inhibitor (Eur. J. Biochem. 271) 3149 [2,3]. However, it is known that core ( i.e. r-free) bacterial RNAP is able to perform in vitro transcription using fragmented or n icked DNA molecules as templates, in a promoter-independent manner . Although less efficient t han the physiologically relevant r-dependent process (ÔholoÕ RNAP and E. coli genomic DNA as template), such r-independent transcription activity (ÔcoreÕ RNAP and fragmented eukaryotic DNA as template) is nevertheless sufficiently high to be ex ploited for studie s 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 t his hypothesis, the compound’s effects on RNAP were compared under conditions o f either r-dependent or r-independent tran- scription initiation, using streptolydigin as reference inhib- itor, w hich, by acting on chain elongation, is known to inhibit RNAP regardless of the transcription initiation conditions used [12]. As shown in F ig. 5, it was found that GE23077 is able to inhibit RNA synthesis in both c ases, although with different potency (IC 50 values of 20 n M for r-dependent and 100 n M for r-independent initiation). Even though this finding clearly indicates that the molecular target of GE23077 is not the r subunit itself, the fi vefold lower activity and the different shape o f the inhibition curve observed in t he absence o f r indicate tha t the presence of this factor potentia tes the inhibitory activity of GE23077. As expected, such differential behaviour in the presence o r absence of r is not shown by streptolydigin, which, by acting at a stage when the r factor has a lready dissociated from the t ranscription complex [12], displays similar inhi- bition curves and IC 50 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. RNAP–DNA complex formation. To further elucidate the mechanism of action of GE23077 on E. coli RNAP, the possibility was investigate d that the compound might inhibit RNA synthesis by p reventing 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 t arget o f a transcription i nitiation inhibitor. In such cases, a p reformed RNAP–DNA complex would b e less sensitive to t he action of the inhibitor than an i solated, unbound RNAP molecule. To test such a possibility, the E. coli holoenzyme was preincubated with DNA to allow complex formation befor e the addition o f the inhibitor, and then the e ffect of GE23077 on RNA synthesis was a ssessed. Two a ntibiotics known t o show different behaviour, i n that respect, were used as controls: lipiarmycin, whose inhibitory activity is known t o b e largely reduced when it is added a fter the formation of the RNAP–DNA complex [11]; and rifampicin, which, conversely, binds and inhibits RNAP equally well if added when t he enzyme is already bound to DNA [16]. As s hown i n F ig. 6, all three compounds totally inhibited RNA synthesis when a dded before DNA, w hereas Fig. 3. Dose –response analysis of RNA biosynthesis inhibition by rifampicin and GE23077 in p ermeabilized Escherichia coli cells. Fig. 4. Effect of G E23077 and oth er RNA polymerase (RNAP) inhibitors on in vitro RNA synthesis: c omparison of the effects of com- pound a ddition before v s. after reaction start. The concentration of c ompo unds used in t his experiment were as follows: GE23077, 10 l M ; rifampicin, 1 l M ; streptolydigin, 100 l M . j, No in hibitor controls; d, compounds were added b efore t he reaction start, m arked by the addition of DNA t o mixtures c ontaining all the other compon ents and the in dica ted inhibitor; m, compo unds were added 5 min after r eaction s tart, as indicated by the arrows. 3150 E. Sarubbi et al. (Eur. J. Biochem. 271) Ó FEBS 2004 lipiarmycin was significantly l ess active than rifampicin and GE23077 when a dded a fter preincubation of the enzyme with DNA. T he observation that, in t hese experiments, GE23077 behaves like rifampic in, strongly suggests that its mode of action is not based on t he prevention of RN AP binding to DNA. Activity of GE23077 on purified rif R RNAPs 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 (Fig s 2 and 3 ), both act at the level of transcription initiation (Fig. 4), and both show similar activity on their target enzyme when added b efore or a fter RNAP–DNA complex formation (Fig. 6). This might suggest overlapping binding sites for th e two compounds on the RNAP molecule and, consequently, the possibility of c ross-resist- ance between them. T o test such a hypothesis, we studied the effect of GE23077 on different rif R RNAPs, purified from E. co li strains containing known rpoB mutations [21,31]. As shown in Table 2, GE23077 behaved very differently f rom rifampicin i n these tests, inhibiting RNA synthesis w ith s imilar 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 enz yme. Discussion This report d escribes the b iochemical a ctivity of G E23077, a novel microbial metabolite i dentified in the course of a screening program aimed at the discovery of selective inhibitors of bacterial R NAP [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 a nd for its therapeutic potential. The narrow range of anti- microbial activity of GE23077 might explain why this potent R NAP 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. The molecular basis f or the l ow activity of GE23077 in microbiological assays was investigated in this s tudy. In experiments with permeabilized E. coli cells, i t was found that the antibiotic is ab le t o exert its action, i.e. to block RNA synthesis, when cell membranes are damaged. It s activity on macromolecular biosynthesis is dose-depen dent and selective, no t showing any effect o n either DNA or protein synthesis, thereby confirming on whole cells the specificity of action observed with purified enz ymes. It is tempting to conclude from these findings that GE23077 is poorly active on whole bacterial cells, s imply because i t i s 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. Effec t o f G E23077 and streptolydigin on in vitro RNA s ynthesis: comparison of the effects of compounds under conditions of r-dependent vs. r-independent transcription initiation. The inhibition of ÔholoÕ RNA polymerase (RNAP) with Escherichia coli genomic DNA as template (r-dep.) is compared with the inhibition of ÔcoreÕ RNAP with sonicated calf thymus DNA (r-ind.), a t different concentrations of GE23077 and streptolydigin (strept.). The data s hown are t he 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 befo re or after RNAP–DNA co mplex formation. The concentration of co mpo unds used in t his experiment are as follows : GE23077, 1 l M ; rifampicin, 1 l M ; lipiarmy c in , 100 l M . j, No inhibitor controls; d, compounds were ad ded before t he reaction start, marked by the addition of DNA to m ixtures containing all the other components a nd the indicated inh ibitor; m, nucleotides and the indicated inhibitor were added to mixtures that con tained a ll the o ther co mp onents, a nd that had been preincubated for 5 min at 37 °C t o a llow RN AP–D NA c omplex f ormation. Ó FEBS 2004 GE23077, a novel bacterial RNA polymerase inhibitor (Eur. J. Biochem. 271) 3151 negative charge around neutra l pH (Fig. 1). However, i t is important to note that e ven minor damage to the cell membrane may have far -reaching consequences on cellular activities, and, in particular, on membrane-associated transport s ystems. Although our data suggest that impair- ment in cell penetration should b e the main reason fo r 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 t han 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 s imply r eflect a still-incomplete pe netration of G E23077 in plasmolyzed bacteria, but alternative e xplanations, such as only partial inactivation o f efflux pumps, a re possible. In addition, the observation that the E. coli strain used for t he 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 t hat the lower activity in p ermeabi- lized cells might be the result of a pre-existing partial resistance to GE23077 in that particular strain. In general, it is important to consider that different mechanisms might operate in different b acteria to confer resistance to GE23077. The variety of bacterial species showing v ery low or no sensitivity to the antibiotic [19] raises the question of w hether some might carry an intrinsically resistant RNAP t arget. F urther studies will help to elucidate this issue. In this work, information was also obtained o n the mechanism of action of GE23077 on its target enzyme. It was f ound that the compound acts at the level of transcrip- tion initiation and that even though the presence of the RNAP r subunit potentiates i ts a ctivity, its molecular target is not the r subunit itself, or the i nteraction of RNAP with promoter DNA to form the t ranscription complex (i.e. GE23077 inhibit s the enzyme equally well even when this is already engaged in the RNAP–DNA complex). Strikingly, this behaviour is similar to that s hown by rifampicin [16] and hence the two compounds, although structurally unrelated, s how analogies that go b eyond potency and specificity, an observation that might suggest similar binding sites for the two molecules on the target enzyme. This hypothesis p rompted 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 rif R mutations identified and mapped thus far are located in the rpoB gene [21,31]. When the activity of G E23077 was compared with t hat of rifampicin on three in dependent rif R RNAP mutants, the behaviour of the two compounds was very different (Table 2), indicating that cross-resistance is not a common event and hence that the two c ompounds possess distinct binding sites on RNAP. Rifampicin resistance is known to arise spontaneously with a relatively h igh frequency, e.g. % 10 )8 in E. coli [21]. The similarity in the mode of action of the t wo antibiotics, together with the observation that GE23077 is a ctive on rif R RNAP mutants, raises the question o f what is the resistance mutation frequency of the new antibiotic. In view of the l ow antimicrobial activity of GE23077 on E. coli and other bacteria, such a question might be a ddressed using the M. catarrhalis clinical isolates on which GE23077 shows significant activity [19]. However, the cell penetration issue discussed above suggests that a con siderable fraction of GE23077-resistant colonies might contain alterations in cell permeability, rather than genuine RNAP mutations. The isolation and sequencing o f a statistically significant number of mutants c ould a ssess the extent of such phenomenon. Considering the prospects (see below) of obtaining 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 o f a bona fide resistant RNAP mutation frequency. 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 r ifampicin is also observed at a more deta iled 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 s tep that would ordinarily follow the forma- tion of the first phosphodiester b ond, or whether i t acts a t a different step, as might be suggested by the lack of cross- resistance. Also, f urther information might be obtained through structural elucidation of the RNAP–GE23077 complex, in a s tudy similar t o the one recently performed on the Thermus aquaticus RNAP–rifampicin complex [17]. A high-resolution structure determination of the RNAP– GE23077 complex s hould p rovide insights into GE23077 binding and its me chanism o f i nhibition, together with new information on the transcription process itself. Table 2. A ctivity of G E23077 and r ifampicin on purified Escherichia c oli rifampicin resistant ( rif R ) RNA polymerases (RNAPs). R esults are expressed as IC 50 . rpoB allele (mutation) Wild-type rpoB3 (Ser531 fi Phe) a rpoB3595 (Ser522 fi Phe) b rpoB7 (Ile572 fi Phe) b GE23077 0.020 0.050 0.062 0.031 Rifampicin 0.030 > 100 > 100 15 a Described previously [31]. b Described previously [21]. 3152 E. Sarubbi et al. (Eur. J. Biochem. 271) Ó FEBS 2004 In addition t o the s cientific interest of GE23077 as novel RNAP inhibitor, it is also interesting to speculate on its potential as a c hemical lead f or novel an ti-infective chemo- therapeutic agents. Considering the emergence of bacterial resistance to drug therapy and the observation that, with t he exception of oxazo lidinones, no new scaffolds of antibac- terial agents f or human use have been developed i n the past 30 y ears [33], the novel structure of GE23077 b ecomes particularly attractive. Its activity on clinical isolates of M. catarrhalis [19] is inte resting, as such a bac terium is considered to be the third commo nest pathogen o f the respiratory tract in humans after Streptococcus pneumoniae and Haemophilus influenzae, responsible for otitis media in children a nd lower respiratory tract i nfections in the elderly [34]. I n addition, the widespread production of b-lact amase renders M. catarrhalis resistant to penicillins [ 35], as also observed in GE23077-sensitive M. catarrhalis strains (E. Selva, unpublished data). The activity found again st clinical isolates of M. catar- rhalis suggests t hat GE23077 can be considered as a natural template for chemical modifications to extend its anti- microbial spectrum to includ e other pathogen s. Given its potent and selective activity on its biochemical target, appropriate chemical d erivation programmes m ight over- come the cell-penetration issue and yield potent molecules with a wider r ange of antimicrobial activit y. 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 poss essing s im- ilar activity o n RNAP and, at the same time, improved cell-membrane permeability, might be promising leads for the development of antibacterial drugs. 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(1997) Moraxella (Branhamella) catarrhalis: clinical and molecular aspects of a rediscovered pathogen. J. Me d. Microbiol. 46, 360–371. 35. McGregor, K., Chang, B.J., Mee, B.J. & Riley, T.V. (1998) Moraxel la catarrhalis: clinical significance, antimicrobial suscept- ibility and BRO b-lactamases. Eur. J. Microbiol. I nfect. Dis. 17, 219–234. 3154 E. Sarubbi et al. (Eur. J. Biochem. 271) Ó FEBS 2004 . Mode of action of the microbial metabolite GE23077, a novel potent and selective inhibitor of bacterial RNA polymerase Edoardo Sarubbià, Federica Monti*, Emiliana Corti, Anna Miele and Enrico. 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. Selva Vicuron Pharmaceuticals, Gerenzano, Varese, Italy GE23077, a novel microbial metabolite r ecently isolated from Actinomadura sp. culture media, is a potent and selective inhibitor of bacterial