U.S. patent application number 09/845335 was filed with the patent office on 2002-05-16 for ef-tu protein encoded on the plastid dna of the malaria parasite and protein synthesis inhibitors effective as anti-malarial compounds.
This patent application is currently assigned to MEDICAL RESEARCH COUNCIL. Invention is credited to Clough, Barbara, Preiser, Peter, Wilson, Robert John Macleod.
Application Number | 20020058266 09/845335 |
Document ID | / |
Family ID | 26735152 |
Filed Date | 2002-05-16 |
United States Patent
Application |
20020058266 |
Kind Code |
A1 |
Clough, Barbara ; et
al. |
May 16, 2002 |
EF-Tu protein encoded on the plastid DNA of the malaria parasite
and protein synthesis inhibitors effective as anti-malarial
compounds
Abstract
The plastid DNA of the malaria parasite Plasmodium falciparum
has been sequenced and found to contain a gene encoding an EF-Tu
protein. Inhibitors of the protein are effective as anti-malarial
compounds and the protein can be used to screen for such
inhibitors. Furthermore, the 23S ribosomal RNA encoded on the
malaria parasite plastid DNA is a target for anti-malarial
compounds and the antibiotic thiostrepton acts as an anti-malarial
by binding to the RNA.
Inventors: |
Clough, Barbara; (London,
GB) ; Preiser, Peter; (London, GB) ; Wilson,
Robert John Macleod; (London, GB) |
Correspondence
Address: |
Nixon & Vanderhye P.C.
1100 N. Glebe Road, 8th Floor
Arlington
VA
22201
US
|
Assignee: |
MEDICAL RESEARCH COUNCIL
|
Family ID: |
26735152 |
Appl. No.: |
09/845335 |
Filed: |
May 1, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09845335 |
May 1, 2001 |
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09140466 |
Aug 26, 1998 |
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6268160 |
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60056246 |
Aug 28, 1997 |
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Current U.S.
Class: |
435/6.15 |
Current CPC
Class: |
G01N 2333/445 20130101;
Y10S 530/822 20130101; C12Q 1/18 20130101; Y02A 50/30 20180101;
Y10S 435/947 20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 001/68 |
Claims
We claim:
1. The EF-Tu protein encoded on the plastid DNA of the malaria
parasite Plasmodium falciparum.
2. The protein according to claim 1 which has the sequence labelled
"eftu_pf" in FIG. 2A (SEQ ID NO:2).
3. The protein according to claim 1 or 2 in purified form.
4. A DNA molecule encoding the protein of claim 1 or 2.
5. The DNA molecule according to claim 4 which comprises the
sequence shown in FIG. 2B (SEQ ID NO:1).
6. The DNA molecule according to claim 4 in purified form.
7. The DNA molecule according to claim 4 which is a cloning or
expression vector.
8. A host cell transformed with the vector of claim 7.
9. A method of producing the EF-Tu protein encoded on the plastid
DNA of the malaria parasite Plasmodium falciparum, which method
comprises (i) culturing a host cell containing a DNA molecule
encoding the protein under conditions such that the protein is
expressed; and (ii) recovering the protein from the culture.
10. A method of identifying an anti-malarial compound, which method
comprises (i) contacting a compound with the EF-Tu protein encoded
on the plastid DNA of the malaria parasite Plasmodium falciparum;
and (ii) determining whether the compound binds to or inhibits the
protein, any such binding or inhibition being indicative that the
compound is an anti-malarial.
11. A compound identified by the method of claim 10.
12. A method of preventing or treating infection of a patient with
the malaria parasite Plasmodium falciparum, which method comprises
administering to the patient a compound which inhibits the EF-Tu
protein encoded on the plastid DNA of said malaria parasite.
13. The method according to claim 12 wherein the compound is an
antibiotic.
14. The method according to claim 13 wherein the compound is a
member of the kirromycin series of antibiotics.
15. The method according to claim 14 wherein the compound is
selected from the group consisting of kirromycin (mocimycin),
aurodox (1-methylmocimycin), efrotomycin, enacyloxin IIa and
GE2270.
16. An antibody specific for the EF-Tu protein encoded on the
plastid DNA of the malaria parasite Plasmodium falciparum.
17. A method of identifying an anti-malarial compound, which method
comprises (i) contacting a test compound with the 23S ribosomal RNA
encoded on the plastid DNA of the malaria parasite Plasmodium
falciparum (pf 23S rRNA.sub.pl) or with a fragment of said RNA
containing the GTPase domain; and (ii) determining whether the
compound binds to said RNA or said fragment, any such binding being
indicative that the compound is an anti-malarial.
18. A method according to claim 17 which comprises (i) incubating
the Pf 23S rRNA.sub.pl or the fragment thereof with the test
compound and a reference compound known to bind to the rRNA or the
fragment; (iia) determining the amount of reference compound that
is bound to the rRNA or the fragment; and (iib) comparing the
amount of reference compound bound to the rRNA or the fragment with
the amount that is bound in the absence of the test compound;
wherein any reduction in the binding of the reference compound in
the presence of the test compound compared to the binding in the
absence of the test compound is indicative that the test compound
is competing for binding to the rRNA and that the test compound
could be an anti-malarial.
19. The method according to claim 18 wherein the reference compound
is thiostrepton.
20. The method according to claim 17 wherein said RNA or said
fragment contains an A residue at the position corresponding to
position 1067 in the 23S rRNA of Escherichia coli.
21. The method according to claim 20 wherein said fragment
comprises the pf 23S rRNA.sub.pl sequence corresponding to the
sequence from about position 1051 to about position 1108 of the 23S
rRNA of Escherichia coli.
22. A compound identified by the method of claim 17.
Description
FIELD OF THE INVENTION
[0001] This invention relates to a new protein encoded in the
plastid DNA of the malaria parasite Plasmodium falciparum, to DNA
encoding the protein, to methods of producing the protein, to
methods of screening for anti-malarial compounds, to compounds
identified by such screening methods and to methods of preventing
or treating growth of the malaria parasite.
BACKGROUND TO THE INVENTION
[0002] The malarial 35 kb circular DNA molecule central to this
invention corresponds to a minor species of DNA distinct from
nuclear DNA discovered in the 1960s (Gutteridge et al. 1971). In
the mid-80s the first study on its purification and molecular
analysis was published (Williamson et al. 1985). Its similarity was
noted to a circular DNA in the related organism Toxoplasma
gondii--a well known opportunistic pathogen in AIDS cases.
[0003] It is important to stress that the malaria parasite and
related apicomplexans are unusual amongst non-photosynthetic
organisms in that they possess two forms of organellar DNA,
typically a property of plants. One form of organellar DNA has been
identified as mitochondrial DNA (mtDNA), whereas the other, the 35
kb circle, we have proposed is the remnant of a plastid DNA
(plDNA), a provenance hitherto unsuspected for these organisms
(Wilson et al. 1991, 94). This plDNA was probably acquired by an
ancient progenitor of the phylum and may be of algal origin
(Williamson et al. 1994). The precise location of these organellar
DNAs in the cell shows they are in separate compartments (Kohler et
al 1997).
[0004] Thus, there are potentially two organellar protein
synthesising systems of independent prokaryotic origin within the
malaria organism that could be susceptible to inhibition with
antibiotics. Although the malarial mitochondrion is the best
characterised of the organelles, its genetic content is highly
idiosyncratic, contributing only incomplete fragments of two rRNA
genes to the machinery required for protein synthesis. The circular
DNA from the putative plastid, on the other hand, is much more
conventional, producing transcripts of four complete rRNA genes,
some twenty tRNA genes, subunits of a typical plastid RNA
polymerase, and a number of ribosomal protein genes organised in
modified bacterial operons.
SUMMARY OF THE INVENTION
[0005] In sequencing the malarial plastid DNA, we found that it
contains a gene encoding a new EF-Tu protein homologous to the
EF-Tu proteins known in prokaryotes. Thus, the invention provides
an EF-Tu protein encoded on the plastid DNA of the malaria parasite
Plasmodium falciparum. The invention also provides DNA encoding the
protein.
[0006] The prokaryotic EF-Tu proteins are known to be important in
controlling the elongation cycle in protein synthesis, and it is
known that inhibition of the proteins by various compounds has an
antibiotic effect. In view of the sequence similarity between the
prokaryotic EF-Tu proteins and our newly-identified malarial
plastid EF-Tu protein, we proposed the theory that the antibiotic
compounds which inhibit the prokaryotic proteins may also inhibit
the malarial protein and therefore be useful as anti-malarials. We
tested such antibiotics (e.g. kirromycin and aurodox) for their
anti-malarial effect and found our theory was correct; the
antibiotics were found to be effective anti-malarials both in vitro
and in vivo. Thus, the invention provides a method of preventing or
treating infection of a patient with the malaria parasite
Plasmodium falciparum, which method comprises administering to the
patient a compound which inhibits the EF-Tu protein encoded on the
plastid DNA of said malaria parasite.
[0007] The knowledge provided by the invention of the EF-Tu protein
in the malaria plastid and the fact that its inhibitors are
effective anti-malarials allows the protein to be used in screening
for new anti-malarial compounds. Accordingly, the invention
includes a method of identifying an anti-malarial compound, which
method comprises
[0008] (i) contacting a test compound with the EF-Tu protein
encoded on the plastid DNA of the malaria parasite Plasmodium
falciparum; and
[0009] (ii) determining whether the compound binds to or inhibits
the protein, any such binding or inhibition being indicative that
the compound is an anti-malarial.
[0010] We also investigated the ability of antibiotics which bind
to other components of the prokaryotic protein synthesis machinery
to act as anti-malarial compounds. As a result of these
investigations, it was found that thiostrepton, which is known to
bind to the GTPase domain of the 23S ribosomal RNA of E. coli, is
also able to bind to GTPase domain of the 23S rRNA encoded on the
plastid of the malaria parasite Plasmodium falciparum (Pf 23S
rRNA.sub.pl). Accordingly, the invention provides a method of
identifying an anti-malarial compound, which method comprises
[0011] (i) contacting the compound with the 23S ribosomal RNA
encoded on the plastid DNA of the malaria parasite Plasmodium
falciparum (Pf 23S rRNA.sub.pl) or with a fragment of said RNA
containing the GTPase domain; and
[0012] (ii) determining whether the compound binds to said RNA or
said fragment, any such binding being indicative that the compound
is an anti-malarial.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 is a schematic illustration of the elongation cycle
that occurs during protein synthesis and shows the points in the
cycle at which various inhibitors operate.
[0014] FIG. 2A shows the amino acid sequence of the EF-Tu protein
according to the invention from the plastid of the malaria parasite
Plasmodium falciparum (pf). The sequence is aligned with sequences
of EF-Tu proteins from other organisms, namely E. coli ("ecoli"),
Anacystis nidulans ("anani"), Cyanophora paradoxa ("cypha") and
Cryptomonas phi ("cryph").
[0015] FIG. 2B shows the nucleotide sequence of the tufA gene that
encodes the EF-Tu protein according to the invention.
[0016] FIG. 3A shows a Southern blot of endonuclease-restricted
malarial genomic DNA hybridised with a PftufA-specific PCR product
as probe. A single band for the 35 kb plastid was obtained for each
restriction digest.
[0017] FIG. 3B shows cross-hybridisation between
endonuclease-restricted malarial genomic DNA and the yeast tufM
gene, indicating the possible presence of a malarial version of
tufM.
[0018] FIG. 4 shows the results of an experiment in which an
antisense RNA probe (about 230 nts) made by in vitro transcription,
corresponding to a portion of the tufA gene encoding domains I and
II of the predicted EF-Tu.sub.pl protein, was used in an RNase
protection assay to demonstrate the presence of tufA transcripts in
total RNA extracted from erythrocytic parasites during the course
of a single growth cycle (0-40 hrs).
[0019] FIG. 5 shows dose-response curves for the effects of fusidic
acid, mocimycin (kirromycin), thiostrepton and GE 2270 on
incorporation of .sup.3H-hypoxanthine and .sup.14C-isoleucine into
erythrocytic stages of P. falciparum grown in cultures over a 36
hour period. The Figure also shows a dose-response curve for the
effect of mocimycin on myeloma cells (a control).
[0020] FIG. 6 shows the effects of aurodox and mocimycin on the
growth of P. chabaudi in mice. The solid line is for aurodox, the
dotted line is for mocimycin and the dashed line is for no drug
controls. The arrows on the x-axis indicate days on which three 0.1
ml 100 mM ip injections were given.
[0021] FIG. 7 shows the sequence of the GTPase region of the
plastid 23S rRNAs of Plasmodium falciparum (Pf) and Toxoplasma
gondii (Toxo) (numbers based on E.coli), showing substitution sites
(circled) affecting the binding of thiostrepton. The alternative
nucleotides in Pf cytosolic 28S rRNA and Pf mitochondrial 23S rRNA
are indicated.
[0022] FIG. 8 shows thiostrepton titrations (means of duplicates,
bar=range) in a filter binding assay with transcripts of the GTPase
region of LSU rRNA.
[0023] A) Short 23S transcripts of P.falciparum (Pf) wild type
rRNA.sub.pl (open circle A1067) and mutated forms (open square
A1067U and filled triangle A1067G), as well as Pf 28S rRNA
transcripts (filled square), are compared with an optimized E.coli
control transcript (filled circle). For convenience, nucleotide
numbers correspond to E.coli.
[0024] B) T. gondii wild type rRNA.sub.pl transcript (filled
triangle) and mutated transcript (open triangle) compared with
control transcripts from P.falciparum rRNA.sub.pl (open circle) and
E.coli (filled circle).
[0025] FIGS. 9 and 10 show the structures of various antibiotics
usable in the invention.
[0026] FIG. 11 shows slot blots of RNA fractioned on sucrose
gradients. Pretreatment with anisomycin blocked the
puromycin-induced shift of the hybridization signal for
P.falciparum cytosolic 23S ribosomes but not the plastid 16S
ribosomes.
[0027] A-C. Blots hybridized with a probe for the cytosolic large
subunit (23S) rRNA. Anisomycin blocked the puromycin-induced
shift.
[0028] D-F. The same blots hybridized with a probe for the
plastid-encoded small subunit (16S) rRNA. Anisomycin did not block
the puromycin-induced shift.
[0029] FIG. 12 is a slot blot showing the puromycin-induced shift
of the hybridization signal for plastid mRNA specifying EF-Tu.
[0030] FIG. 13 contains immunoblots showing that binding of
antibiotics modifies migration of EF-Tu.GDP in native
polyacrylamide gels. Two segments of the same gel show A)
heterologously expressed Pf EF-Tu.sub.pl protein detected with a
malaria peptide-specific antibody and B) E.coli EF-Tu detected with
a specific antibody (Breidenbach et al 1990). Lanes without
antibiotics (1 and 5), lanes with 100 .mu.M antibiotic: GE2270A (2
and 6), enacyloxin lla (3 and 7), kirromycin (4 and 8). Arrows
indicate uncomplexed EF-Tu.
DETAILED DESCRIPTION OF THE INVENTION
[0031] The EF-Tu Protein
[0032] The function of the EF-Tu protein is in the elongation cycle
of protein synthesis. The cycle is illustrated in FIG. 1. EF-Tu
reacts with GTP and AA-tRNA to form an EF-Tu/AA-tRNA/GTP complex.
After binding to EF-Tu, the AA-tRNA component is transferred to the
ribosomal A site with the release of free EF-Tu-GDP complex and
phosphate. The GDP is released from EF-Tu and the EF-Tu is then
ready for another cycle.
[0033] EF-Tu is an exceedingly abundant protein in E. coli, present
in approximately as many copies as there are tRNA molecules. It can
bind every tRNA except for fMet-tRNA.
[0034] The malarial plastid EF-Tu has much sequence identify with
known EF-Tu proteins from other organisms (see FIG. 2A). We have
made a 3D-model structure for the malarial plastid EF-Tu protein
based on the crystal structures available for bacterial equivalents
(E. coli and T. thermophilus). This model showed that the bacterial
and malarial proteins are very similar indeed, strongly implying
that the malarial plastid EF-Tu is functional.
[0035] The sequence of the malarial plastid EF-Tu protein of the
invention may be that labelled "eftu_pf" in FIG. 2A, but variations
in this sequence are possible. The protein may, for example, have a
sequence identity with the sequence in FIG. 2A of 80% or more, 90%
or more, 95% or more or 99% or more.
[0036] The sequence of FIG. 2A may be modified by substitution,
deletion,-extension or insertion. A substitution, deletion or
insertion may involve one or more amino acids, typically from 1 to
5, from 1 to 10 or from 1 to 20 amino acids.
[0037] Such modified sequences must retain the functions of the
EF-Tu protein necessary for participation in the elongation cycle
of protein synthesis. In general, the physicochemical nature of the
sequence of FIG. 2A should be preserved; the amino acids of a
modified sequence should generally be of a similar charge, size and
hydrophobicity/hydrophilicity as those in the sequence of FIG. 2A.
Candidate substitutions are those in which an amino acid from one
of the following groups is replaced by a different amino acid from
the same group:
[0038] H, R and K
[0039] I, L, V and M
[0040] A, G, S and T
[0041] D and E.
[0042] The EF-Tu protein of the invention may be provided in
purified form. The protein may also be provided in pure form and in
isolated form. The protein may, for example, be provided in a
preparation in which it constitutes 10% or more, 40% or more, 80%
or more, 90% or more, 95% or more or 99% or more of the total
protein in the preparation by weight.
[0043] The protein will usually be obtained by expression of
recombinant DNA containing the protein, but may also be obtained by
biochemical purification of the protein from the malaria
parasite.
[0044] DNA Encoding the Malarial Plastid EF-Tu Protein
[0045] The DNA encoding the EF-Tu protein may have the sequence
shown in FIG. 2B, but variations in this sequence are possible. The
DNA molecule may, for example, have a sequence identity with the
sequence shown in FIG. 2B of 70% or more, 80% or more, 90% or more,
95% or more or 99% or more.
[0046] A recombinant DNA molecule encoding the EF-Tu protein of the
invention may be obtained using well-known and conventional
recombinant DNA techniques, such as those described in Sambrook et
al (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
[0047] Such DNA molecules may be obtained by making a library of
replicable expression vectors. The library may be created by
cloning all the DNA or, more preferably, the plastid DNA of the
malaria parasite into a parent vector. The library may be screened
for members containing the desired nucleic acid sequence, e.g. by
means of a DNA probe or antibody.
[0048] The term "replicable expression vector" is used herein to
mean a vector which contains the appropriate origin of replication
sequence for directing replication of the vector. The vector may
also contain the appropriate sequences for expression of the EF-Tu
protein. The sequences for expression of the protein will generally
include a transcription promotor and a translation initiator
operably linked to the coding sequence. The term "operably linked"
refers to a linkage in which the promotor and initiator are
connected in such a way to the coding sequence as to permit
expression of the protein. A vector may, for example, be a plasmid,
virus or phage vector. A vector may contain one or more selectable
markers, for example an ampicillin resistance gene in the case of a
bacterial vector or a neomycin resistance gene in the case of a
mammalian vector. A foreign gene sequence encoding the EF-Tu
protein inserted into a vector may be transcribed in vitro or the
vector may be used to transform a host cell.
[0049] According to one embodiment of the invention, there is
provided a host cell transformed with a vector encoding the EF-Tu
protein. A vector and host cell will be chosen so as to be
compatible with each other, and may be prokaryotic or eukaryotic. A
prokaryotic host may, for example, be E.coli in which case the
vector may, for example, be a bacterial plasmid or a phage vector.
A eukaryotic host may, for example, be a yeast (e.g. S.cerevisiae),
a chinese hamster ovary (CHO) cell or an insect cell (e.g.
Spodoptera frugiperda).
[0050] The invention includes a method of producing the EF-Tu
protein encoded on the plastid DNA of the malaria parasite
Plasmodium falciparum, which method comprises
[0051] (i) culturing a host cell containing a DNA molecule encoding
the protein under conditions such that the protein is expressed;
and
[0052] (ii) recovering the protein from the culture.
[0053] Antibodies to the Malarial Plastid EF-Tu Protein
[0054] The invention includes an antibody specific for the EF-Tu
protein of the invention. The antibody is preferably monoclonal,
but may also be polyclonal. The antibody may be labelled. Examples
of suitable antibody labels include radiolabels, biotin (which may
be detected by avidin or streptavidin conjugated to peroxidase),
alkaline phosphatase and fluorescent labels (e.g. fluorescein and
rhodamine). The term "antibody" is used herein to include both
complete antibody molecules and fragments thereof. Preferred
fragments contain at least one antigen binding site, such as Fab
and F(ab').sub.2 fragments. Humanised antibodies and fragments
thereof are also included within the term "antibody".
[0055] The antibody may be produced by raising antibody in a host
animal against an EF-Tu protein according to the invention or an
antigenic epitope thereof (hereinafter "the immunogen"). Methods of
producing monoclonal and polyclonal antibodies are well-known. A
method for producing a polyclonal antibody comprises immunising a
suitable host animal, for example an experimental animal, with the
immunogen and isolating immunoglobulins from the serum. The animal
may therefore be inoculated with the immunogen, blood subsequently
removed from the animal and the IgG fraction purified. A method for
producing a monoclonal antibody comprises immortalising cells which
produce the desired antibody. Hybridoma cells may be produced by
fusing spleen cells from an inoculated experimental animal with
tumour cells (Kohler and Milstein, Nature 256, 495-497, 1975).
[0056] An immortalized cell producing the desired antibody may be
selected by a conventional procedure. The hybridomas may be grown
in culture or injected intraperitoneally for formation of ascites
fluid or into the blood stream of an allogenic host or
immunocompromised host. Human antibody may be prepared by in vitro
immunisation of human lymphocytes, followed by transformation of
the lymphocytes with Epstein-Barr virus.
[0057] For the production of both monoclonal and polyclonal
antibodies, the experimental animal is suitably a goat, rabbit, rat
or mouse. If desired, the immunogen may be administered as a
conjugate in which the immunogen is coupled, for example via a side
chain of one of the amino acid residues, to a suitable carrier. The
carrier molecule is typically a physiologically acceptable carrier.
The antibody obtained may be isolated and, if desired,
purified.
[0058] Assays for Identifying Anti-malarial Compounds that Inhibit
the Malarial Plastid EF-Tu Protein
[0059] As mentioned above, the knowledge provided by the invention
of the EF-Tu protein in the malaria plastid and the fact that its
inhibitors are effective anti-malarials allows the protein to be
used in screening for new anti-malarial compounds.
[0060] Various different assay systems may be used to carry out the
screening, but all the assays have in common that the EF-Tu protein
of the invention is contacted with test compounds and the ability
of each test compound to bind to or inhibit the protein is
determined. Any such binding or inhibition is indicative that the
compound could be useful as an anti-malarial drug.
[0061] The screening assays will generally require one or more
controls. It will generally be desirable to include a positive
control in the form of a compound known to bind to or inhibit the
EF-Tu protein, so as to ensure that the assay system is responding
properly. Examples of suitable positive controls include kirromycin
(mocimycin) and aurodox (1-methylmocimycin), which we have shown
through our experiments to be effective anti-malarials and which
are known to inhibit prokaryotic EF-Tu. It will also generally be
desirable to include a negative control in the form of a sample
containing no test compound, so as to obtain a measurement of the
background signal in the assay. If a test compound gives a signal
in the assay above that of the background, this is indicative that
the compound has given a positive result and could be an
anti-malarial.
[0062] One convenient type of assay system is a "band shift"
system. This involves determining whether a test comopund advances
or retards the EF-Tu protein of the invention on gel
electrophoresis relative to the EF-Tu protein in the absence of
test compound. The mobility of GDP complexed EF-Tu is decreased
with GE2270A but increased with enacyloxin IIa or kirromycin.
[0063] Another convenient type of assay system is a competitive
binding system. Such a system may comprise
[0064] (i) incubating the EF-Tu protein of the invention with a
test compound and a labelled reference compound that is known to
bind the protein (e.g. kirromycin or aurodox);
[0065] (ii) determining the amount of the labelled reference
compound that is bound to the protein; and
[0066] (iii) comparing the amount of bound labelled reference
compound determined in step (ii) with the amount of said compound
that binds to the protein in the absence of the test compound;
[0067] wherein any reduction in the binding of the labelled
reference compound in the presence of the test compound compared to
the binding in the absence of the test compound shows that the test
compound is competing with the reference compound for binding to
the protein and indicates that the test compound could be an
anti-malarial.
[0068] The amount of the labelled reference compound bound to the
protein may be measured directly or indirectly. A direct
measurement may be carried out by removing assay mixture containing
the unbound labelled reference compound and measuring the amount of
label that is in the protein fraction. Alternatively, the amount of
labelled reference compound bound to the protein could be
determined indirectly by measuring the amount of label remaining in
the assay solution after removal of the protein fraction, which
will be inversely related to the amount that has bound to the
protein.
[0069] In a competitive binding assay system, the EF-Tu protein may
be immobilised on a solid support or may be in solution. The use of
immobilised protein has the advantage that, after the binding
reaction is complete, the protein/labelled reference compound
complex may be separated from the labelled reference compound that
remains in solution by simply removing the solution away from the
solid support. If, on the other hand, the protein is not
immobilised during the assay but rather is in solution, then it
will generally be necessary to devise a means for separating the
protein/labelled reference compound complex from the uncomplexed
reference compound before measuring the amount of label. Such
separation could be achieved, for example, by precipitating the
protein using an antibody to the protein or by using a non-specific
protein precipitation technique.
[0070] Suitable labels for use in the assay systems according the
invention are well-known in the art and include the labels set out
above that may be attached the antibodies of the invention.
[0071] Use of Compounds that Inhibit the Malarial Plastid EF-Tu
Protein as Anti-malarials
[0072] Compounds that inhibit the malarial plastid EF-Tu protein
may be used as anti-malarial compounds. Such compounds may be
identified using the screening assays described above.
[0073] We have already identified some such compounds. e.g. the
antibiotics kirromycin (mocimycin), aurodox (1-methylmocimcyin),
Efrotomycin (a glycoside of kirromycin), Enacyloxin IIa and GE2270.
Efrotomycin has previously been shown to have the desirable
properties of rapid oral absorption and prolonged plasma
half-life.
[0074] The antibiotics in the kirromycin series may be represented
by the general formula: 1
[0075] wherein R.sup.1 is hydrogen or a C.sub.1-C.sub.4 alkyl group
(e.g. methyl); R.sup.2 is hydrogen, C.sub.1-C.sub.4 alkyl or a
sugar group (e.g. a disaccharide); and R.sup.3 is hydrogen, OH or
C.sub.1-C.sub.4 alkyl.
[0076] Preferred antibiotics for use in the invention are as
follows: 2
[0077] The compounds may be used in either the treatment of an
existing infection by the malaria parasite or in the prevention of
such an infection from occurring in the first place. The dosage
regimen will ultimately be at the discretion of the physician, who
will take into account factors such as the nature of the compound,
the severity of any disease and the weight and age of the patient.
However, suitable routes of administration may include the oral
route, the rectal route, the intramuscular route and the
intravenous route. The oral route is preferred because this is
generally the most convenient route for a patient to take regular
doses of the compound without the assistance of a physician. A
typical dose would be from 1 to 1000 mg and such a dose may, for
example, be taken from 1 to 3 times daily.
[0078] In order to be administered to a patient, the compound will
be provided in the form of a pharmaceutical composition containing
the active compound and a pharmaceutically acceptable carrier or
diluent. Typical oral dosage compositions include tablets,
capsules, liquid solutions and liquid suspensions.
[0079] Compounds that Bind to the 23S rRNA Encoded on the Malaria
Parasite
[0080] We have found that the 23S rRNA encoded on the plastid of
the malaria parasite Plasmodium falciparum (Pf23S rRNA.sub.pl) is a
target for compounds with anti-malarial activity. More
specifically, we have found that the mechanism of action of the
antibiotic thiostrepton, which was known to have anti-malarial
activity, is through binding to the GTPase domain of the 23S rRNA
of the malaria plastid.
[0081] This information allows the design of assays for screening
for further anti-malarial compounds whose mechanism of action
operates through the 23S rRNA. These assays involve contacting each
of the test compounds with the 23S rRNA or a fragment thereof
containing the GTPase binding domain, and measuring any binding of
the test compounds to the rRNA or fragment. Any such binding is of
course indicative that the compound could be an anti-malarial.
[0082] We have already developed one assay for detecting binding to
the 23S rRNA. We made a short transcript from DNA encoding the 23S
rRNA of the malaria plastid corresponding to the GTPase domain
(about nucleotide 1051 to about nucleotide 1108) and found that the
transcript bound thiostrepton strongly.
[0083] The binding was specific. It depended to a large extent on
the presence of an A residue at the position corresponding to E.
coli position 1067. A transcript corresponding to the GTPase domain
of the E. coli 23S rRNA (which contained the A at position 1067)
was also shown to bind thiostrepton strongly. Mutation of A1067 to
either U or G in the malaria plastid transcript dramatically
reduced binding. A transcript corresponding to the GTPase domain of
the cytosolic malaria 28S rRNA also bound thiostrepton poorly.
[0084] A screening assay for further anti-malarial compounds can be
based on a competitive binding assay in which the ability of each
test compound to compete with thiostrepton for binding to the Pf23S
rRNA.sub.pl is measured. Such an assay comprises
[0085] (i) incubating the Pf 23S rRNA.sub.pl or a fragment thereof
containing the GTPase domain with the test compound and
thiostrepton as a reference compound (or another reference compound
known to bind to the rRNA or the fragment);
[0086] (ii) determining the amount of thiostrepton (or other
reference compound) that is bound to the rRNA or the fragment;
and
[0087] (iii) comparing the amount of thiostrepton (or other
reference compound) bound to the rRNA or the fragment with the
amount that is bound in the absence of the test compound;
[0088] wherein any reduction in the binding of the thiostrepton (or
other reference compound) in the presence of the test compound
compared to the binding in the absence of the test compound is
indicative that the test compound is competing for binding to the
rRNA and that the test compound could be an anti-malarial.
[0089] In a screening assay based on the invention for further
anti-malaria compounds, it would be necessary to use appropriate
controls. A good positive control would be to use a compound known
to compete with thiostrepton (or with the other reference compound)
to ensure that the assay is working properly; a positive result for
the known competitor in the assay would indicate that the assay had
worked correctly. It would also generally be desirable to use a
negative control comprising, for example, a sample in which no
thiostrepton or test compound is present; this would enable the
background signal in the assay to be determined and any signal
above the background would indicate binding to the 23S rRNA.
[0090] The following experiments serve to illustrate the
invention.
[0091] Experimental Section
[0092] Materials and Methods
[0093] Polysome preparation and puromycin shift--P.falciparum was
grown in blood cultures (Trager et al 1976) and ribosomes prepared
as described (Sherman et al 1975). Parasitized erythrocytes were
lysed for 1 hr on ice in a buffer containing 0.14% Nonidet P-40
(Trade Name), 25 mM KCl, 10 mM MgCl.sub.2, 380 mM-sucrose, 6.5 mM
.beta.-mercaptoethanol and 50 mM Tris HCl, pH 7.6. The lysate was
centrifuged.times.3 at 10,000 g for 10 min at 4.degree. C. to
remove genomic DNA and other cell debris before further
centrifugation at 105,000 g for 1 hr in an SW40 rotor (Trade Name,
Beckman) at 4.degree. C. The resulting pellet was resuspended in 25
mM KCl, 5 mM MgCl.sub.2 and 50 mM Tris HCl (pH 7.6) and homogenized
by hand (.times.50 strokes) with a glass dounce homogenizer
(Wheatstone, USA). The suspension was centrifuged at 10,000 g for
10 min at 4.degree. C. and the crude pellet discarded before
further centrifugation for 2 hr at 105,000 g in an SW55 rotor
(Beckman) at 4.degree. C. The pellet was resuspended in 10 mM Tris
HCl, 10 mM MgCl.sub.2, 100 mM KCl and homogenized again to give a
suspension of ribosomes.
[0094] Polysomes were fractioned on sucrose gradients (20-50% w/v)
prepared in 0.3M KCl, 3 mM MgCl.sub.2 and 1 mM dithiothreitol (DDT)
with 0.02M Tris HCl (pH 7.6)--referred to as "high salt" buffer;
centrifugation was at 30,000 g (Beckman Sw40 rotor) for 21 hr at
4.degree. C.
[0095] In an experiment with RNase (Cox 1969), total polysomes were
incubated with a range of concentrations of RNase (1-13 ng
ml.sup.-1 ribosomes, Boehringer) prior to centrifugation for 30 min
at 26.degree. C. In other experiments, polysomes were dissociated
to monosomes and subunits by the incorporation of puromycin; here
the total ribosome preparation was incubated for 20 min at
37.degree. C. with 2 mM puromycin in the "high salt" buffer to
which was added 2 mM GTP, 10 .mu.lml.sup.-1 RNAsin (39
U.mu.l.sup.-1, Promega) and 1 mM DTT. In some experiments,
ribosomes were incubated with both anisomycin (Sigma) and
puromycin. Anisomycin was added at 3 mM for 10 min at 37.degree. C.
followed by incubation with puromycin as above (Cundliffe et al
1974). After ribosome fractionation on the sucrose gradients, RNA
was extracted with phenol/chloroform/isoamyl alcohol (Chomczynski
it al 1987), precipitated in ethanol and blotted on to nylon
membranes (Gene Screen, Trade Name) using a slot-blot apparatus
(Scot-Labs). Hybridization was carried out with .sup.32P-labelled
DNA prepared from either cloned fragments of the 35 kb plDNA of P
falciparum, PCR products amplified from it, oligonucleotides based
on its sequence (Wilson et al 1996), or with PCR products based on
the sequence of Pf28S cytosolic rRNA (McCutchen et al 1988).
Hybridization signals were quantitated using a Molecular Dynamics
phosphor imager.
[0096] Antibiotics--Samples of Mocimycin (kirromycin), Aurodox
(N-methylated kirromycin), and Efrotomycin (a glycoside of
kirromycin) were used. Aurodox was dissolved in RPMI-Albumax medium
(Grande et al 1977), kirromycin was dissolved in RPMI made alkaline
by addition of 1M NaOH, and efrotomycin was dissolved in ethanol
before dilution in culture medium. Enacyloxin IIA was dissolved in
1% NaHCO.sub.3 prior to dilution in RPMI-Albumax medium. The
antibiotic GE2270A was dissolved in 100% DMSO before dilution in
culture medium. Fusidic acid (Sigma) was dissolved directly in
culture medium, and thiostrepton (Sigma) in 100% DMSO before
dilution in culture medium. A hemisuccinate form of thiostrepton
was prepared as a potassium salt, according to Bodanszky et al
1965. Incorporation of radiotracers by P.falciparum growing in
blood cultures in the presence and absence of drugs was carried out
as described (Strath et al 1993).
[0097] EF-Tu model--Pf EF Tu.sub.pl was modelled by homology with
the known 3D structures determined by X-ray crystallography of
EF-Tu.GTP (Berchtold et al 1993) and EF-Tu.GDP (Polekhina et al
1996) both from Thermus aquaticus. Modelling was carried out with
the WHAT-IF program package (Trade Name, Vriend 1990), as described
in Tews et al 1996. Alignments had to be adjusted manually because
of small gaps and insertions. An iterative procedure of the
automated model-building algorithm checked and corrected the
alignments until no errors were detectable. Threee insertions in
the Pf EF-Tu.sub.pl sequence had to be deleted: Leu 190, Pro263 and
Leu359-Val363. The final alignment with the T. aquaticus structure
had single residue gaps in the Pf sequence between Leu41 and Ser42
as well as residues Asn209 and lle210. Co-ordinates for the C
(alpha) backbone were copied from the known structure for
overlapping segments and the atoms for the amino acids Gly, Ala and
Pro were placed directly in their calculated positions. All
remaining residues were assigned to Ala before the order in which
side changes had to be placed was calculated by the algorithm
implemented by the program. Atoms were subsequently placed using a
position-dependent amino acid rotamer library. The model was
refined geometrically and re-numbered according to the P.falciparum
sequence.
[0098] Heterologous expression--The malarial plastid tufA gene was
amplified by PCR, cloned into the TA vector (Trade Name,
Invitrogen) and its sequence determined (Wilson et al 1996).
Re-cloning into the expression vector pGEX (Trade Name, Pharmacia)
was carried out with a PCR product generated using 5' and 3'
primers providing custom-made restriction sites. Transfectants in
E.coli (strains DH5 alpha, Sure. JM109) were found mostly to carry
deletions within the tufA sequence, but one clone in JM109
contained the complete insert (sequenced on a single strand). This
was expressed as a fusion protein of the expected size by induction
of mid-log phase cultures with 50 .mu.M isopropyl-.beta.-D-thio-
galactoside (IPTG) at 37.degree. C. or 27.degree. C. The insoluble
fusion protein was solubilized in 5M guanidinium HCl and refolded
by dilution (Lin et al 1991).
[0099] Antibody to an epitope of Pf EF-Tu--A rabbit polyclonal
antibody was prepared against a 13-mer synthetic peptide
IQKNKDYELIKSN from domain I of Pf EF-Tu coupled to polylysine beads
(Severn Biotech. Ltd). In Western blots (ECL protocol, Amersham),
this antibody did not cross-react with EF-Tu from E.coli, nor did
an antibody to E.coli EF-Tu react with the expressed malarial
protein.
[0100] Drug binding--Thiostrepton binding to short rRNA transcripts
generated in vitro was assayed according to Ryan et al 1991, as
modified by Clough et al 1997. A band shift method in native 12%
polyacrylamide gel (Cetin et al 1996) was used to demonstrate
complex formation between a resolubilized fraction of the expressed
Pf EF-Tu.sub.pl and various antibiotics. Before electrophoresis and
immunoblotting, samples were incubated on ice for 15 mins in 50 mM
imidazole acetate (pH 7.6), 10 mM NH.sub.4Cl, 10 mM MgCl.sub.2, 1
mM DDT and 100 .mu.M GDP, in a final volume of 20 .mu.l.
[0101] Results
[0102] Evidence for Plastid Protein Synthesis
[0103] Ribosomes from erythrocytic parasites were fractionated by
centrifugation on linear gradients (20-50% sucrose) and RNA was
extracted from fractions collected over the length of the
gradients. Slot blots of the RNA were hybridized with
.sup.32P-labelled DNA probes prepared from either cloned fragments
of Pf plDNA, PCR products based on its sequence, or kinased
oligonuceotides. As shown in FIG. 11 C&F, hybridization with
probes for the large.sub.(cytosolic) or small.sub.(plastid) subunit
rRNAs gave signals extending to the bottom of the gradient,
indicative of rRNA incorporated in polysomes. Supportive evidence
was obtained by limited digestion of the total ribosome preparation
with RNase (13 ng RNase/mg ribosomes for 30 min at 26.degree. C.)
before fractionation--this causes dissociation of the polysomes
(Cox 1969) and shifted the hybridization signal up the gradient
(data not shown). More specific evidence for a subset of polysomes
belonging to the plastid compartment was obtained by incubating
total ribosomes with 2 mM puromycin in the presence of GTP, 0.3M
KCl and 1 mM DDT prior to density gradient fractionation: puromycin
acts as an analogue of the 3' terminal adenosine of aminoacylated
tRNAs and is incorporated into nascent peptide chains, terminating
translation and dissociating polysomes (Gale et al 1981).
Incubation with puromycin caused a shift of both the cytosolic and
plastid rRNA hybridization signals up the gradient (FIG. 11,
B&E). The specificity of the puromycin-shift was confirmed by
pre-treating Pf ribosomes with the antibiotic anisomycin which
binds only to eukaryotic ribosomes and prevents puromycin
incorporation (Gale et al 1981). As shown in FIG. 11A, anisomycin
blocked the puromycin-induced shift of the hybridization signal for
Pf 28S cytosolic rRNA, whereas hybridization of the same blot with
a probe for Pf 16S rRNA.sub.pl showed the puromycin-shift of the
plastid subset of polysomes was not blocked (FIG. 11D).
[0104] Similar results were obtained with a probe for an mRNA
specified by the plDNA. FIG. 12 shows the puromycin-induced shift
of the hybridization signal for mRNA specifying EF-Tu.sub.pl.
[0105] To quantitate the relative proportions of the hybridization
signals generated by different species of RNA, slot blots were
hybridized with .sup.32P-labelled oligonucleotides, known amounts
of DNA being used as appropriate standards. The 28S cytosolic rRNA
was estimated to be 80-fold more plentiful than 16S rRNApl and
2000-fold more plentiful than the mRNA specifying EF-Tu.sub.pl
(data not shown). These results and the puromycin-shifts are
consistent with the presence of actively translating plastid
ribosomes in blood cultures of malaria parasites.
[0106] tufA Sequence
[0107] From a combination of cloned DNA fragments and PCR products
amplified from the 35 kb circular DNA of P.falciparum, we derived a
1.23 kb nt sequence whose predicted peptide (calculated M. Wt.
46,633) is homologous to the elongation factor EF-Tu (FIG. 2A). The
malarial gene lies 45 nts downstream from two ribosomal
protein-encoding genes, rps12 and rps7. In this respect, the
organization resembles the str operon on the plDNAs of the
flagellate protists Euglena gracilis (Montadon & Stutz, 1984;
Hallick et al. 1993) and Astasia longa (Seimelster et al. 1990), as
well as the non-chlorophyte alga Cryptomonas (Douglas, 1991), and
the cyanelle of Cryptomonas paradoxa (Kraus et al. 1990), the
intervening fus gene encoding EF-G in the str operon of bacteria
such as E.coli (Zengal and Lindahl BBA 1050, 317 (1990)) presumably
having been transferred to the nucleus. The short intergenic region
upstream of the PfpltufA gene does not contain an open reading
frame or putative leader sequence. At the nt level, the malarial
pltufA gene is extremely rich in adenine and thymine (A+T) residues
(79%) compared to related sequences in the database, a feature with
important consequences for computations intended to establish the
gene's phylogenetic relationships.
[0108] At the predicted peptide level, the malarial sequence is
very divergent from other recorded EF-Tu's (only 45% amino acid
identity with E.coli and 51% identity with Anacystis nidulans).
Nonetheless, several highly conserved regions are evident,
including the four segments of domain I involved in GTP binding. In
E.coli, the first three of these segments carry the consensus
elements G18HVDHGK24; D80CPG83; and N135KCD138. In the malarial
sequence there is only one substitution C136E. Most of the residues
defining the GDP binding pocket also are conserved (in E.coli G23,
N135, K136, D138, S173, L175), the only substitution in the
malarial sequence being M139L. In a less well conserved region
(amino acids 180-190, topologically close to the GTP binding
domain). The malarial sequence has an insertion typical of plastid
versions of EF-Tu that is not found in the E.coli gene, and is only
partially present in the mitochondrial equivalent (tufM) of
Saccharomyces cerevisiae (Nagata et al. 1983). Despite the gene's
high A+T content, the predicted malarial EF-Tu peptide is one of
the most highly conserved proteins encoded by the 35 kb circle;
however, it is potentially more basic (calculated pI=8.43) than the
versions present in bacteria or the yeast mitochondrion (Piechulla
& Kuntzel, 1983).
[0109] In view of the unknown functional status of the 35 kb
circular DNA, it was of interest to compare the predicted malarial
EF-Tu.sub.pl peptide with the unusual chloroplast form in the
Charophycean alga Colochaete orbicularis, as it has been suggested
that the latter may no longer be functional, there being multiple
tufA-like sequences in the nucleus (Baldauf et al. 1990). Baldauf
and colleagues pointed out that the C.orbicularis EF-Tu.sub.pl
amino acid sequence differs in twenty two sites that otherwise are
conserved in all but four of 27 other EF-Tu sequences. Despite the
malarial gene's extreme A+T content, the predicted EF-Tu peptide
has only 6 conservative amino acid substitutions in the same 22
residues (Table 1). This suggests that the functional domains
encoded by the tufA gene on the 35 kb circle have been maintained
under selective pressure.
1TABLE 1 AMINO ACID SEQUENCE COMPARISON OF CONSENSUS SITES
(MODIFIED FROM BALDAUF ET AL. 1990) Site* C cp eub all Pf 21-22 FS
VD VD VD VD 60-62 NMS GIT GIT GIT GIT 87 N D D D D 90 N K K K K 128
I V V V V 153 N E E E E 210 L I I I I 227 R D D D D 233 S G G G G
236 L T T T T 241 T R R R K 248 N K K K N 272 K E E E E 286 D N N N
N 301 K R R R R 372 E D D D D 393 V A A A S 401 I V V V I 405 I V V
V I * = Amino acids numbered as in FIG. 2A. C = Coleochaete
orbicularis chloroplast tuf A (Baldauf et al. 1990) cp =
cyanobacteria and chloroplast consensus eub = eubacteria,
cyanobacteria and chloroplast consensus all = eubacteria,
eukaryotes and archaebacteria consensus Pf = Plasmodium falciparum
35 kb circule tufA
[0110] When hybridized with a PftufA-specific PCR product under
stringent conditions, Southern blots of endonuclease-restricted
malarial genomic DNA gave a single band of the size predicted (FIG.
3A). At low stringency no other bands were revealed that might have
corresponded to the nucleus-encoded mitochondrial gene tufM (Nagata
et al, 1983; Wells et al. 1994). The likely presence of a malarial
equivalent was indicated, however, by cross-hydridization at low
stringency with a PCR product based on the yeast tufM gene (FIG.
3B).
[0111] An antisense RNA probe (.about.230 nts) made by in vitro
transcription, corresponding to a portion of the malarial tufA gene
encoding domains I and II of the predicted EF-Tu.sub.pl protein,
was used in an RNase protection assay to demonstrate the presence
of tufA transcripts in total RNA extracted from erythrocytic
parasites (FIG. 4).
[0112] Modelling of the Three-dimensional Structure of P.falciparum
EF-Tu.sub.pl
[0113] Despite the highly divergent amino acid composition of Pf
EF-Tu.sub.pl a computer model showed conservation of secondary
structure motifs in all three domains of the hypothetical protein.
The model is reliable, with good confidence in the overall folding
and also in the detail of the secondary structure compared with
T.aquaticus. Only minor differences were found in the length of
some structural motifs: in domain I there are small length
differences in 5 of the nine alpha helices and in 3 out of six
.beta. strands. The changes are more pronounced in domain II where
the first two .beta.-strands seem to be continuous in Pf, and an
extra .beta.-strand is formed by residues Gly245-Leu249. In domain
III, differences from T. aquaticus are again minor with a slightly
different positioning of two .beta.-strands.
[0114] The model for Pf EF-Tu.sub.pl.GTP showed that the
GTP-binding site is conserved as well as the whole lining towards
the GTP-binding pocket. There is also conservation on the interface
between the domains. These interfaces are exposed when EF-Tu. GTP
converts to the effective EF-Tu.GDP form. In this form of the
protein, conserved residues in the cleft between domains I and III
correspond to the site which other studies have shown kirromycin
binds.
[0115] Antibiotics
[0116] The effects of three classes of compounds on
intraerythrocytic parasites of P.falciparum in vitro, as well as on
P.chabaudi in vivo, are considered below. In assessing the
significance of these results it should be noted that in
prokaryotes, kirromycin, whose binding site is at the interface of
domains I and III of EF-Tu.GTP (Mesters et al. 1994), binds to the
ternary complex of tRNA and EF-Tu.GTP preventing the conformational
change required for release from the ribosome upon GTP hydrolysis,
whereas the drug has a different effect on eukaryotic cells. In the
latter, at 100 .mu.M, it blocks RNA synthesis without affecting DNA
or protein synthesis (Schmid et al. 1978).
[0117] Kirromycin: Kirromycin-resistant forms of bacterial EF-Tu
are modified at one of seven amino acids along the opposing
interfaces of domains I and III (Mesters et al 1994 and Abdulkarim
et al 1994) and Pf EF-Tu.sub.pl has a substitution at one of these
sites (A375S-E.coli numbers) that could potentially confer
resistance to kirromycin. To test this possibility, kirromycin
(Mocimycin), its methylated derivative Aurodox or its disaccharide
derivative Efrotomycin were incubated with erythrocytic stages of
P.falciparum grown in cultures over a 36 hr period. The
incorporation of both .sup.3H-hypoxanthine and .sup.14C-isoleucine
was inhibited in a dose-dependent fashion, maximum inhibition being
achieved at 100 .mu.M kirromycin (FIG. 5). Similar levels of
inhibition were obtained for all three compounds. In synchronized
cultures, inhibitory effects on ring stage parasites were observed
as early as five hours after exposure to Aurodox and were maximal
after 10 hrs exposure. Once maximal depression of incorporation had
been reached at any particular dose of drug, residual incorporation
continued at a uniform rate thereafter. Vital staining with
rhodamine 123, a fluorescent dye that concentrates within the
mitochondrion (Divo et al. 1985) confirmed the parasiticidal
effect, loss of specific mt staining being evident within 2-3 hours
(data not shown). Treatment of parasites with 1 mM Aurodox for 1.5
cell cycles, followed by removal of the antibiotic by washing and
follow-up for 2 weeks in vitro, indicated the effect was parasite
death rather than stasis. Blood cultures of P.falciparum were about
10 times more sensitive to the antibiotic than a gram-positive
bacterium (Corynebacterium spp) used in parallel bioassays.
[0118] On the basis of these findings, preliminary studies were
carried out on mice infected with P.chabaudi. In the first
experiment, mice were infected and inoculated on the same day
intraperitoneally with 0.1 ml of 100 mM Aurodox, a dose calculated
to mimic the maximal inhibitory effect observed in vitro. The
Aurodox-treated animals showed a lag in development of the
infection compared with untreated controls indicating partial
inactivation of the infectious inoculum (FIG. 6). In a single
experiment. Mocimycin w as found to be less effective in vivo than
Aurodox.
[0119] Enacyloxin IIa: Enacyloxin IIa (ExIIa) is a linear
antibiotic representing a new family of polyenic antibiotics
(Watanabe 1992) that bind to EF-Tu and block transfer to the
nascent peptide chain of aminoacylated-tRNA bound at the A site
(Cetin 1996). The profiles for inhibition of radiotracer
incorporation in blood cultures of P.falciparium incubated with Ex
IIa were similar to those with Mocimycin.
[0120] GE2270: This is a thiopeptide antibiotic in the same family
as thiostrepton. It binds to a different site on Ef-Tu than
kirromycin and locks the protein into a different conformation
(Landini 1996). This antibiotic was more inhibitory in blood
cultures than either kirromycin or thiostrepton (FIG. 5).
[0121] Fusidic acid: Fusidic acid, presently in clinical use as a
narrow spectrum antibiotic, was assessed as a potential
antimalarial by titration with P.falciparum in vitro, as described
above. Maximum inhibition of radiotracer incorporation was achieved
at a concentration of 200 .mu.M (FIG. 5). Preliminary experiments
with fusidic acid in mice infected with P.chabaudi found little
effect on parasitaemias, even at toxic dose levels of the drug.
[0122] Thiostrepton: Nucleotide (nt) sequences are available for
the GTPase domain of the 28S rRNA specified by the nucleus (Rogers
et al. 1996), as well as the 23S rRNAs specified by the mt and pl
large subunit rRNA genes of the human malaria pathogen Plasmodium
falciparum (Pf) (Feagin, 1992). These data indicate that the high
affinity binding site for the thiazolyl peptide antibiotic
thiostrepton, A.sub.1067 in E.coli (Thompson et al 1991, Ryan et al
1991 and Rosendahl et al 1994), is conserved in the GTPase domain
encoded by the plastid DNA, but modified to a G in both nuclear and
mitochondrial genomes (FIG. 7).
[0123] We have tested thiostrepton to ascertain whether it inhibits
Pf and found reproducible inhibition of uptake of
.sup.3H-hypoxanthine and .sup.14C-isoleucine in blood cultures (50%
inhibition at 3-5 .mu.M thiostrepton). Onset of inhibition of
protein synthesis by thiostrepton was more rapid (5 hrs) than by
tetracycline (8 hrs). Specificity was demonstrated by the lack of
effect of viomycin (data not shown), an unrelated antibiotic that
also can inhibit translocation (Kutay et al 1990).
[0124] Having established thiostrepton's activity, we asked "does
the antibiotic bind preferentially to the nuclear, mitochondrial or
plastid forms of Pf 28/23 S rRNA?". Evidence that the highest
affinity interaction of thiostrepton is with 23S rRNA.sub.pl was
obtained from an in vitro binding assay (Ryan et al 1991). Short
transcripts of wild type (wt) RNA corresponding to the GTPase
domain of Pf 23S rRNA.sub.pl (nts 987-1078) were transcribed in
vitro from a PCR product that included a T7 promoter sequence in
one of the primers. Mutated malarial rRNA sequences (E.coli numbers
A1067U and A1067G) were obtained by PCR methodology and transcribed
in the same way. Both wild type and modified transcript sequences
were verified prior to thiostrepton binding assays. A positive
control transcript was used based on the 23S rRNA sequence of
E.coli with a mutation (U1061A) that increases stability and
binding. FIG. 8A shows that the mutation Pf.sub.pl (E.coli number
A1067U) markedly reduced thiostrepton binding (.about.14% of wt).
An intermediate level of binding (.about.35% of wt) was obtained
with the mutation Pf.sub.pl (E.coli number A1067G). Thiostrepton
binding to a transcript corresponding to the GTPase domain (nt
1334-1427) of the cytosolic Pf 28S rRNA was .about.10% of that for
Pf 23S rRNA.sub.pl. These data show that the nts crucial for
thiostrepton binding to Pf23 S rRNA are as in E.coli, and that the
plastid form has the highest binding affinity.
[0125] In the same way, we tested a transcript corresponding to the
GTPase domain of the 23S rRNA.sub.pl of Toxoplasma gondii (Tg), a
related apicomplexan that is an important opportunistic pathogen in
patients with AIDS. In this case, the wild type sequence has a
substitution at a different site (E.coli number A1077U)--see FIG.
7, that inhibits binding by thiostrepton in E. coli (Ryan et al
1991). This was found also to be the case with a transcript derived
from a PCR product covering the GTPase domain of Tg.sub.pl 23S rRNA
(nt 926-1024) (FIG. 8B). Corrective mutation of the Tg.sub.pl
transcript (E. coli number U1077A) conferred a significant increase
(.times.5) in thiostrepton binding (FIG. 8B).
[0126] These thiostrepton binding studies constitute the first
direct evidence that components of the malarial plastid organelle
could be preferentially targeted by drugs. The results complement
earlier studies (Pfefferkon et al 1994 and Beckers et al 1995)
which inferred that toxoplasma's 23S rRNA.sub.pl might be the
target of the macrolide antiobiotic, clindamycin, acting at a
different effector site.
[0127] Drugs Bind to Heterologously Expressed EF-Tu
[0128] The material tufA gene in pGEX was expressed as an insoluble
fusion protein in E.coli JM 109. The protein was detected either
with antibodies to the GST tag or with antibodies to a specific
peptide sequence in domain I (IQKNKDYELIKSN) not found on E.coli
EF-Tu. Washed inclusion bodies were dissolved and refolded by
dilution (Lin et al 1991). This yielded a small amount of refolded
protein that migrated in native acrylamide gels as a spontaneously
cleaved product and we used this to show that the expressed protein
forms complexes with kirromycin and other drugs that bind to
different sites on EF-Tu. As shown in FIG. 13, the mobility (Mr) of
the expressed malarial protein was advanced or retarded in these
complexes in the same characteristic way described for E.coli EF-Tu
(Cetin et al 1996): the M.sub.r of the GDP form of the complex
decreased with GE2270A, but increased with enacyloxin IIa or
kirromycin. These results show that the heterologously expressed Pf
EF-Tu.sub.pl can adopt a native conformation and bind the classical
antibiotic inhibitors.
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Sequence Listing
[0183] (1) General Information
[0184] (i) APPLICANT:
[0185] (A) NAME: CLOUGH et al
[0186] (B) STREET: National Institute for Medical Research, Mill
Hill
[0187] (C) CITY: London
[0188] (E) COUNTRY: United Kingdom
[0189] (F) POSTAL CODE (ZIP) NW7 1AA
[0190] (ii) TITLE OF INVENTION: AN EF-TU PROTEIN ENCODED ON THE
PLASTID DNA OF THE MALARIA PARASITE AND PROTEIN SYNTHESIS
INHIBITORS EFFECTIVE AS ANTI-MALARIAL COMPOUNDS
[0191] (iii) NUMBER OF SEQUENCES: 2
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[0198] (2) Information for SEQ ID NO: 1
[0199] (i) SEQUENCE CHARACTERISTICS:
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