Method of screening for anti-malarial compounds

Details Method of screening for anti-malarial compounds Method of screening for anti-malarial compounds

1998-08-26

2001-07-31

Chin, Christopher L. (Department: 1641)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

C435S007220, C435S007930, C435S947000, C435S069100, C435S071100, C436S501000, C436S086000, C530S350000, C530S822000

Reexamination Certificate

active

06268160

ABSTRACT:

FIELD OF THE INVENTION
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
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.
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).
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
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.
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.
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
(i) contacting a test 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.
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
pl
). Accordingly, the invention provides a method of identifying an anti-malarial compound, which method comprises
(i) contacting the compound with the 23S ribosomal RNA encoded on the plastid DNA of the malaria parasite
Plasmodium falciparum
(Pf 23S rRNA
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.


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