Download Resistance to anti-peptide deformylase drugs

Key Paper Evaluation
Resistance to anti-peptide
deformylase drugs
1. Introduction
2. Results from the paper by Apfel
et al.
3. Significance of the results
4. Expert opinion
Bibliography
Evaluation of: APFEL CM, LOCHER H, EVERS S et al.: Peptide deformylase as an
antibacterial drug target: target validation and resistance development. Antimicrob.
Agents. Chemother. (2001) 45(4):1058-1064.
Carmela Giglione & Thierry Meinnel
Institut des Sciences du Végétal, UPR2355, Centre National de la Recherche Scientifique, Bâtiment 23,
1 Avenue de la Terrasse, F-91198 Gif-sur-Yvette cedex, France
Recent work has assessed the potential of peptide deformylase (PDF) as a target for broad spectrum antibacterial agents. By using a number of
approaches, including proteomics, researchers at Roche have shown that the
molecules they had selected in vitro were able to target PDF in vivo. However,
the authors, having observed resistance occurring at a rather high frequency
and on the basis of the recent discovery of a deformylase homologue in
humans, suggest that PDF ‘may not be an optimal target for broad spectrum
antibacterial agents’. We link these data to results published by other laboratories and conclude that PDF deserves to still be considered a valuable target
for new antibiotics.
Keywords: antibiotics, deformylase, human homologue, resistance, side effects, toxicity
Expert Opin. Ther. Targets (2001) 5(3):
1.
Ashley Publications
www.ashley-pub.com
Introduction
Infectious diseases caused by bacteria, fungi and other parasitic organisms affect
hundreds of millions of people worldwide and cause millions of deaths each year.
Renewed interest in the discovery of new antibiotics has been driven by the development by these organisms of resistance to the drugs commonly used against them.
Although the number of antibiotics is very large (>160), the diversity of chemical
classes that they represent is, however, rather limited and resistance mechanisms are
most often connected to each antibiotic class. Up-to-date approaches towards the
search for new antibiotics therefore take advantage of new targets involved in novel
mechanisms yet to be explored in drug discovery, for which resistance mechanisms
have not evolved widely amongst bacterial populations. The search for a broad spectrum drug is clearly preferable for several reasons, including the high cost development of each individual drug and the possibility for physicians to take immediate
action in case of obvious infection. Recently, the study of microbial genomics has
had a huge impact on the field of drug target discovery and validation. Comparative
genomics data, together with systematic transposon mutagenesis, have revealed that
a common set of only 250-350 genes is essential in bacteria and therefore useful for
target validation experiments. Among this plethora of potential new targets are several that seem better suited to the drug discovery process, in that they closely match
defined criteria, such as the ability to assay activity in vitro. Today, pharmaceutical
research for new antibiotics involves much preliminary work with the purified target
in vitro, the isolation of low affinity inhibitors (‘hits’) and the further improvement
of the potency of the first lead compounds to obtain orally bioavailable drugs.
A recent paper by Apfel et al. (F. Hoffmann-La Roche, Ltd., Switzerland) [1]
attempts to validate one such promising target, PDF (for a complete review on PDF
see [2] ). PDF (encoded by the def gene) is the enzyme responsible for the cleavage of
the N-formyl group occurring at the N-termini of nascent polypeptides synthesised
2001 © Ashley Publications Ltd ISSN 1472-8222
1
Resistance to anti-peptide deformylase drugs
in eubacteria. The presence of this group comes from the
activity of the methionyl-t-RNAfMet transformylase (encoded
in bacteria by fmt) which adds a formyl group to the free
amino group of methionine, which is already bound to the
initiator tRNAfMet (for a review see [3]). PDF activity is essential for bacterial survival and is believed to be absent from
mammals, although a PDF homologue has been recently
described [4]. A number of specific anti-PDF drugs, which all
have more or less the same structure (as proposed in [2]) have
recently been described and shown to display broad spectrum
antibacterial activities [5-8]. The Apfel paper assesses whether
the inhibitors of PDF that they previously selected in vitro:
• do indeed interfere with PDF activity in vivo
• lead to bacterial cell growth arrest or death
• cause resistance development.
2.
Results from the paper by Apfel et al.
In their report, Apfel et al. [1] have shown first that an overexpression of PDF activity in Escherichia coli, Haemophilus influenzae or Streptococcus pneumoniae leads to a decrease of
susceptibility to the inhibitor Ro 66-0376. Next, a S. pneumoniae strain with a tetracyline-inducible def gene was constructed. In the absence of tetracycline i.e., upon significant
decrease of PDF concentration compared to the wild type,
this strain:
• did not display any growth rate difference
• displayed more than a 20-fold increase in sensitivity to
inhibitor Ro 66-0376
Upon treatment of H. influenzae and S. pneumoniae with
sub-inhibitory concentrations of actinonin (a potent antiPDF agent), the authors analysed pulse-labelled proteins by
2D gel electrophoresis and compared the protein patterns to
that of a control, an untreated culture. A systematic shift of
most of the low-molecular-weight proteins was observed
towards a more acidic pI. The authors assigned the acidic shift
of the actinonin-treated protein sample to the presence of
N-formyl groups instead of the free amino-groups of
deformylated proteins. Indeed, deformylation removes one
positive charge, making the pI of proteins more acidic and it
is expected that this effect is stronger on small proteins.
The authors also have studied drug resistance in E. coli
strains grown in the presence of trimethoprim and thymidine.
Under such conditions, mimicking a fmt inactivation (see
references quoted in [9]) i.e., a translation initiation with
methionine instead of N-formyl-methionine, the anti-PDF
drugs were ineffective. Taken together, these data indicate that
PDF is indeed the molecular target of the drug used and a
bacteriostatic effect was observed. Finally, Apfel et al. have
searched for resistance development in an XL2-blue strain
(Stratagene) and obtained a frequency of 10-7 at 64 µg/ml of
Ro 66-6976. Resistance was caused by inactivation of the fmt
gene through Tn10 transposition.
2
3. Significance
of the results
The paper by Apfel et al. [1] is part of a series of data recently
published by various pharmaceutical companies in an effort to
validate PDF as a target for novel antibiotics. This paper provides, firstly, a very convincing demonstration that PDF is the
molecular target of such drugs. In fact, the use of bacterial
strains with reduced expression of PDF and possible induction by an external component has been already described by
Versicor. Chen et al. [6] constructed an E. coli strain with PDF
expression induced by arabinose. Roche has used a similar
strategy and reached the same conclusions with the pathogen
S. pneumoniae. The most interesting data of this paper is
clearly the analysis of proteins by 2D electrophoresis and the
demonstration of the acidic shift of many small proteins upon
treatment with PDF inhibitors. To our knowledge, this is the
first large-scale analysis showing the general effect caused by
PDF inhibition on the proteome of any bacterium. The
authors should be commended for bringing these new data to
the field. Furthermore, as the authors did not manage to get a
fully inactivated strain, they conclude that the def gene is
essential in S. pneumonia. Up until now, definitive demonstration of the essential character of the def gene has only been
made in E. coli [9,10] and Staphylococcus aureus [11].
Apfel et al. have also investigated the resistance mechanism
triggered upon anti-PDF treatment. Unfortunately, the
authors have only reported results obtained with E. coli. The
fact that they obtain inactivation of the fmt gene is not surprising and could have been predicted from earlier work [9,12]
as discussed previously [2]. In contrast to def, fmt is not, in
fact, strictly required for cell growth. E. coli fmt knockout
mutants grow only at temperatures below 37°C, although
their growth rate is impaired more than 10-fold [9,12]. Nevertheless, faster growing mutants were observed and one locus
responsible for the phenotype was shown to be metZ, a gene
encoding the initiator tRNAfMet, which was overexpressed
[13]. The effect of fmt inactivation appeared to be weaker in
Pseudomonas aeruginosa than in E. coli and a growth rate difference of only 3-fold was reported [14]. The non-essential
character of fmt does not appears to be restricted to Gramnegative bacteria, since the Gram-positive Streptococcus faecalis
can grow in the absence of formylation [13]. In addition to the
data published by Roche, several recent studies, mainly by
Versicor [6,11], have described the mechanism of resistance to
anti-PDF drugs in pathogenic bacteria. As expected, fmt was
by far the most frequent target leading to resistance to such
drugs (Table 1). Two interesting results were recently provided
by British-Biotech (Table 1 [7]). First, E. coli fmt mutants had
shorter doubling times to those previously reported. In our
opinion, this may be due to the fast growing mechanisms
already described (see above). Second, mutations at a lower
rate were mapped outside of the def-fmt region. It will be very
interesting to characterise these loci or further examine the
region around def-fmt, since an increased expression of the def
product could cause such resistance. The most recent reports
Expert Opin. Ther. Targets (2001) 5(3)
Giglione & Meinnel
Table 1: Resistance mechanisms to anti-PDF drugs.
Bacterium
Staphylococcus aureus
ATCC25923
Staphylococcus aureus
ATCC29213
Drug used Frequency of
(Effect)
drug
resistance
Actinonin
(Bacteriostatic)
10-6.3
BB-3497
(Bacteriostatic)
2x10-7
-7
BB-3497
(Bacteriostatic)
10
BB-3497
(Bacteriostatic)
10-7
Ro 66.6976
(Bacteriostatic)
-7
10
Haemophilus influenzae
Actinonin
(Bacteriostatic)
10-8
Streptococcus pneumoniae
Actinonin
(Bacteriostatic)
10-8
Escherichia coli
ATCC25922
Escherichia coli
ATCC25922
Escherichia coli
XL2-blue
Resistance yield
(µg/ml)
(wild type ⇒
resistant)
Growth rate
(min.)
(wild type ⇒
resistant)
Resistance
mechanism
Company
16 ⇒ 128
37 ⇒ 60
fmt (frameshift; stop,
missense A108E,
G117V)
Versicor
32 ⇒ >256
27 ⇒ 37
fmt
(frameshift; stop)
B-Biotech
fmt
frameshift; stop;
missense A10P)
B-Biotech
8 ⇒ >256
30 ⇒ 60
8 ⇒ 32
30 ⇒ 32
8 ⇒ >128
16 ⇒ 128
dealing with anti-PDF drugs (Table 1) suggest that mutations
within the def gene itself can lead also to resistance mechanisms, although less efficiently than with fmt inactivation. In
this context, we have recently made the hypothesis that some
bacteria - such as the actinomycetes, which produce actinonin, the most powerful anti-PDF drug known so far [6] could express drug-resistant PDFs [15]. Finally, it should be
noted that Gram-negative bacteria like E. coli or H. influenzae
have efficient efflux systems which counteract anti-PDF drugs
like actinonin [6]. One cannot exclude therefore that such
efflux pumps could also participate in low frequency resistance mechanisms, acquired for instance by horizontal transfer.
4.
Expert opinion
PDF is now recognised as a promising target for new-generation, broad spectrum antibiotics since it fulfils a list of many
decisive advantages for this purpose (see introduction in [2]).
However, several major issues including in vivo drug targeting, possible resistance mechanisms and their frequency of
occurrence and anticipation of toxicity to humans, have not
yet been fully assessed. The work by Apfel and co-workers
attempts to answer some of these questions. Although the
demonstration of drug targeting is convincing, the conclusion
that PDF ‘may not be an optimal target’ because of excessive
resistance frequency is, to us, questionable. In the Roche
study, anti-PDF drugs were used at bacteriostatic concentrations, which favours the development of drug resistance. As
pointed out in the paper, it is also important to stress that
anti-PDF drugs with bactericidal activity were previously
described and shown to not lead to resistance with a frequency higher than 10 -9 [8]. Moreover, the fact that resistance
24 ⇒120
37 ⇒ 60
[Ref.]
[11]
[7]
[7]
? neither def nor fmt B-Biotech
[7]
fmt
(Tn10 insertion)
Roche
fmt
(frameshift; stop)
Versicor
def
Versicor
[1]
is observed at a rather high frequency does not necessarily lead
to a ‘bad mark’ for the selected target, especially when the
resistance mechanism is a new one. This is the case of fmt
mutations arising from the use of anti-PDF drugs. Finally, the
fact that fmt mutants have a systematically reduced growth
rate compared to the wild type (Table 1) suggests to us that
PDF is still an extremely attractive target.
We agree with the conclusion that the most serious reservation for the use of anti-PDF drugs could come from the
recent identification of a PDF orthologue in humans [2,4]. The
existance of this human protein raises the idea that treatment
with such compounds could result in toxicity. In this context,
it should be remembered that a key challenge in the search for
new antibiotics is that innovation is necessary, not only due to
resistance but also to avoid side effects. Before entering Phase
I clinical trials, it is, however, difficult to predict ab initio the
side effects associated with the new drugs. However, in the
case of PDF, its structural resemblance to the metalloproteases
has led to the systematic testing of the effects of anti-PDF
drugs on, e.g., matricins (see conclusion in [2]). Recent results
showing the high selectivity of actinonin and BB-3497 for
PDF over mammalian metalloproteases have been made available [7]. This selectivity suggests strongly that anti-PDF drugs
should not be toxic due to inhibition of matricin activity in
humans.
It now appears that the anticipation of side effect may benefit from genomic studies. Analyses involving the exclusion of
genes with homologues in the sequenced genome of eukaryotes, such as yeast, may be helpful to restrict the number of target candidates still further [16]. Similar analyses can be
performed now that the human genome is complete. However, the recent identification of the approximately 32,000
Expert Opin. Ther. Targets (2001) 5(3)
3
Resistance to anti-peptide deformylase drugs
human genes [17], together with the functional annotation of a
collection of more than 13,000 unique mouse cDNAs [18],
have revealed that many of them have orthologues in bacteria.
These genes may have come either from horizontal gene transfer or from transfer from the mitochondrion, which itself is
believed to have a bacterial origin, according to the endosymbiont hypothesis [19,20]. This is likely to be the case in the origin of the human PDF homologue. Clearly, as our knowledge
increases, we believe that it will probably become more and
more difficult to select attractive bacterial targets that lack any
orthologue in the human genome. We think therefore that the
discovery of the orthologue of a given target in humans should
not lead us to discard the target or slow down the search for
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Affiliation
Carmela Giglione & Thierry Meinnel†
†Author for correspondence
Institut des Sciences du Végétal, UPR2355, Centre National
de la Recherche Scientifique, Bâtiment 23, 1 avenue de la
Terrasse, F-91198 Gif-sur-Yvette cedex, France
Tel.: 33 1 69 82 36 12; Fax: 33 1 69 82 36 07;
E-mail: [email protected]