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

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
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.
Acknowledgements
WearegratefultoP.Landini,B.Goldstein,G.LanciniandM.Denaro
for suggestions and h elpful discus sions. W e a lso t hank F. Parenti for
critical reading of the manuscript.
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