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 initiati
Trang 1Mode of action of the microbial metabolite GE23077, a novel potent and selective inhibitor of bacterial RNA polymerase
Edoardo Sarubbi, Federica Monti*, Emiliana Corti, Anna Miele and Enrico Selva
Vicuron Pharmaceuticals, Gerenzano, Varese, Italy
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 IC50values (i.e the
concen-tration at which the enzyme activity is inhibited by 50%) in
the 10)8Mrange, whereas it is not active on E coli DNA
polymerase or on eukaryotic (wheat germ) RNAP II (IC50
values > 10)4Min 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
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
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
core RNAP consists of five subunits (a2bb¢x, with a
combined molecular mass of 400 kDa) and is capable of
elongation and termination The initiation-competent holo
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)35and )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;
IC 50 , the concentration of compound at which the enzyme
activity is inhibited by 50%; RNAP, RNA polymerase;
rif R , rifampicin resistant.
Enzyme: DNA-directed RNA polymerase (EC 2.7.7.6).
Present address: Lead Discovery Technologies, Aventis Pharma,
France *Arpida Ltd, Munchenstein, Switzerland Aventis Pharma,
Anagni (Frosinone), Italy.
(Received 2 April 2004, revised 29 May 2004,
accepted 3 June 2004)
Trang 2which act on RNAP, have been reported in the literature,
but none has thus far been marked for clinical use
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
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
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)4M) 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
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 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); rifRRNAP (rpoB7) and rifRRNAP (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 vitrotranscription 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 mMTris/HCl (pH 8.0), 50 mMKCl, 10 mMMgCl2, 0.1 mM EDTA, 5 mM dithiothreitol, 10 lgÆmL)1 BSA (Sigma), 20 lgÆmL)1E coliDNA or sonicated calf thymus DNA (from Boehringer Mannheim), 1 mM ATP, 1 mM GTP, 1 mMCTP, 2 lMUTP and 0.5 lCi3H-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.
Trang 3incubated at 37C 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 concentrainhibi-tions of
inhibitors was calculated and expressed, in counts per
minute (c.p.m.), as follows:
RNAP inhibition ¼ ½1 ðsample c:p:m: background
c:p:m:Þ=ðno inhibitor c:p:m: background c:p:m:Þ
100:
DNAP assays
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 coliDNAP Incubation (15 min at 37C),
trichloroace-tic acid precipitation, filtration and radioactivity counting
were performed as described above for the RNAP assay
Cell plasmolyzation
E coliK12 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 2M
sucrose After 5 min at 25C, 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 C
Each cell pellet was resuspended in ice-cold buffer A
(1.5 mL) immediately before use
Macromolecular biosynthesis in permeabilized cells
DNA biosynthesis was assayed by incubating 10 lL of
plasmolyzed cells (containing 5 · 109cells per mL) in a
total volume of 50 lL of 20 mMHepes, pH 8.0, containing
100 mMKCl, 10 mMmagnesium acetate, 1 mM
dithiothre-itol, 2 mM ATP, 0.1 mM NAD, 0.5 mM each of dATP,
dGTP and dCTP, 0.05 lMmethyl[3H]thymidine (0.2 lL of
79 CiÆmmol)1, 1 mCiÆmL)1), and different concentrations
of antibiotics
RNA biosynthesis was assayed by incubating 10 lL of
plasmolyzed cells in a total volume of 50 lL of 20 mMHepes,
pH 8.0, containing 10 mMKCl, 10 mMmagnesium acetate,
0.2 mMMnCl2, 0.5 mMeach of ATP, GTP and CTP, 10 lM
UTP, 0.2 lM 3H-labelled UTP (0.5 lL of 50 CiÆmmol)1,
1 mCiÆmL)1) and different concentrations of antibiotics
Protein biosynthesis was assayed by incubating 10 lL of
plasmolyzed cells in a total volume of 50 lL of 2 m Hepes,
pH 8.0, containing 40 mMKCl, 10 mMmagnesium acetate, 0.2 mMMnCl2, 0.5 mMeach of ATP, CTP, GTP and UTP,
5 mM phosphoenolpyruvate, 50 lgÆmL)1pyruvate kinase, 0.135 lM 3H-labelled phenylalanine (59 CiÆmmol)1,
1 mCiÆmL)1), 0.5 mMof each of the 19 remaining amino acids, and different concentrations of antibiotics
In all cases, reactions were carried out at 30C for
30 min, then 110 lL of a 10% solution of trichloroacetic acid in water was added and the mixtures were incubated at
4C for 30 min (for protein biosynthesis assays, after the addition of trichloroacetic acid, samples were preincubated for 10 min at 80C and then at 4 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)
Results
Activity of GE23077 on purified RNAPs 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 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
Effect of GE23077 on intracellular macromolecular biosynthesis
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 colicells
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],
Table 1 Activity of GE23077 and other RNA polymerase (RNAP) inhibitors on purified polymerases Results are expressed as IC 50 values (i.e the l M concentration of the compound at which the enzyme activity is inhibited by 50%) ND, not determined.
E coli RNAPa
B subtilis RNAPa
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.bDescribed previously [23].
Trang 4kirromycin [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 (2M
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 lMfor 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 onE coli RNAP
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
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 additranscrip-tion), resulting in complete
inhibition of RNA synthesis Conversely, the addition of
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
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 l M (in all three cases); ciprofloxacin, 2 l M (a positive control for DNA biosynthesis); rifampicin, 3 l M (a positive control for RNA biosynthesis); and chloramphenicol, 20 l M (a positive control for protein biosynthesis).
Trang 5[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 (holo
RNAP and E coli genomic DNA as template), such
r-independent transcription activity (core 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,
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 (IC50values of 20 nMfor 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 IC50values 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 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 coliholoenzyme 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 l M ; rifampicin, 1 l M ; streptolydigin, 100 l M 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.
Trang 6lipiarmycin 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 rifRRNAPs
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
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
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
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 holo RNA
polymerase (RNAP) with Escherichia coli genomic DNA as template
(r-dep.) is compared with the inhibition of core 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 l M ; rifampicin, 1 l M ; lipiarmycin, 100 l M 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 C to allow RNAP–DNA complex formation.
Trang 7negative 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
activities, and, in particular, on membrane-associated
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
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
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 initiatranscrip-tion 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
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 rifRRNAP 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 compounds possess distinct binding sites on RNAP
Rifampicin resistance is known to arise spontaneously with a relatively high frequency, e.g. 10)8in 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 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 of
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 funcinforma-tioning 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
Table 2 Activity of GE23077 and rifampicin on purified Escherichia coli rifampicin resistant (rifR) RNA polymerases (RNAPs) Results 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
a
Described previously [31].bDescribed previously [21].
Trang 8In 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)
The activity found against clinical isolates of M
catar-rhalissuggests 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 the cell-penetration issue 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
Acknowledgements
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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