AU753169B2 - Method for identifying chemical active agents and active agents for inhibiting the 1-desoxy-D-xylulose-5-phosphate biosynthetic pathway - Google Patents

Description

1 Process for identifying chemical active ingredients and active ingredients for inhibiting the l-desoxy-Dbiosynthesis pathway The invention relates to a process for identifying active ingredients which are suitable for treating parasitic diseases caused by unicellular or multicellular parasites. Medicine and the pharmaceutical industry are the areas of application of the invention.

The invention also relates to proteins, and fragments of proteins, also DNA-sequences which code these proteins or fragments of proteins, the use of these DNAsequences, these proteins or their fragments for identifying substances acting against unicellular or multicellular parasites, and the active ingredients identified in this way and their use for producing pharmaceutical compositions.

The term parasites includes unicellular parasites and multicellular parasites including Helminthes and b anthropoidea. These cause infectious diseases in humans and animals. In the context of this invention, the strictly scientific definition of parasites is to be used, i.e. unicellular parasites are to be take to mean Protozoa.

A large number of preparations against parasitic diseases already exist. The preparations available are already becoming useless for treating humans and animals due to fast developing resistance. As a result there are already many regions affected by malaria parasites which are resistant to standard medications such as chloroquine. Reports are also known about development of resistance to standard preparations (praziquantel) for treating bilharziosis. These developments of resistance and other factors have led to the fact that malaria and bilharziosis are already amongst the most frequent diseases in the Tropics. An estimated 300-500 million people are suffering from malaria. 2-2.5 million people die annually of malaria. New medications such a mefloquin are, furthermore, very expensive to produce and have many side effects. There is therefore a great need for pharmaceutical compositions for treating humans and animals.

In the past there were many attempts at developing chemotherapy compositions against parasites, in particular against malaria and bilharziosis pathogens.

One of these attempts is concerned with inhibiting socalled isoprenoid biosynthesis. Isoprenoids are molecules which are formed from individual isoprene units (isopentenyldiphosphate) and adopt important functions in the cell. These include sterols, ubiquinones and other molecules which are important for the parasite household. The process of proceeding was based, in this case, on a model which was established in fungi and mammalian cells. In fungi and in mammalian cells the sub-unit isopentenyldiphosphate is formed by condensation of three acetyl-CoA molecules to HMG-CoA.

HMG-CoA is then converted from the HMG-CoA-reductase to mevalonate which is then converted with mevalonate phosphate as an intermediate stage to I 3 isopentenyldiphosphate (see Fig. HMG-CoA-reductase inhibitors such as, for example Lovastatin, Simvastatin and Pravastatin have been used for inhibiting the growth of parasites. Although it was possible to obtain in vitro inhibition by using very high doses of Lovastatin and Simvastatin, in vivo inhibition failed. Treatment of Schistosome-infected mice with Lovastatin led to inhibition of the egg-laying by these worms, however, very high concentrations of Lovastatin had to be used to 10 destroy some of these worms in vivo.

Surprisingly it has been found that parasites, in particular plasmodia and trypanosomes (causes of malaria and sleeping sickness) have at least one further metabolic pathway to the synthesis of isoprenoids. This metabolic pathway is based on condensation of glyceraldehyde-3-phosphate and pyruvate to 1-desoxy-D- (DOXP). DOXP is then converted to 2-C-methyl-D-erythrose-4-phosphate which is then b 20 converted to 2-C-methyl-erythrithol-4-phosphate as intermediate stage to isopentenyldiphosphate. The enzymes DOXP-synthase and DOXP-reductoisomerase inter alia are involved in this metabolic pathway (see Fig.

In the past this metabolic pathway had only been described in plants, in algae and in some bacteria (Sprenger et al. PNAS, 94 (1997) 12857-62 und Kuzuyama et al. Tetrahedron Letters 39 (1998) 4509-12).

Inhibition of the DOXP-metabolic pathway described above, in particular the enzymes DOXP-synthase and DOXPreductoisomerase, by techniques known to the person 4 skilled in the art is suitable for preventing and treating infections caused by unicellular and multicellular parasites in humans and animals. As this metabolic pathway does not occur in humans, it is ideally suited as the targe for a selective chemotherapy of parasites. The enzyme and deoxyxylulose-5-phosphate-reductoisomerase are particularly suited as the target for chemotherapy. The inhibition of the enzyme phosphate reductoisomerase of malaria proved to be particularly low in side effects and suitable as man does not have substrates and their precursors, nor the product of the enzyme, nor the enzyme itself.

The present invention relates to processes for obtaining active ingredients which inhibit the DOXP-metabolic pathway and to these active ingredients for producing pharmaceutical compositions for the treatment and prophylaxis of infections diseases caused by unicellular or multicellular parasites.

It is the object of the invention to provide a new process for identifying active ingredients for the treatment of parasitic diseases in humans and animals. A further object is to develop a process for obtaining a medication which selectively destroys the pathogen and has few side effects.

According to a first embodiment of the invention there is provided a process for obtaining chemically active ingredients which are suitable for treating infectious diseases caused by unicellular or multicellular parasites, wherein proteins which are involved in the 1-deoxy-D-xylulose- 5-phosphate metabolic pathway, or similarly acting derivatives thereof, are brought into contact with 20 the active ingredients to be investigated for their activity with respect to parasites, and the active ingredients which inhibit the proteins or their derivatives are selected.

According to a second embodiment of the invention there is provided isolated, purified or recombinant protein from parasites, with or without activity which is involved in the 1-deoxy-D-xylulose-5-phosphate metabolic pathway and is 25 coded from the DNA-sequence shown in Fig. lb and 2b, or is coded from DNA-sequences which hybridise under stringent conditions with the DNA-sequences shown in Fig. lb or 2b or fragments larger than 200 pairs of bases of these DNA-sequences in the DNA region which codes for the mature protein.

According to a third embodiment of the invention there is provided isolated, purified or recombinant protein from parasites, with or without reductoisomerase activity, wherein said protein is involved in the metabolic pathway and is coded from the DNA-sequence shown in Fig. lb and 2b, or is SR coded from the DNA sequences which hybridise under stringent conditions with the DNAsequences shown in Fig. lb or 2b or fragments larger than 200 pairs of bases of those DNA- S sequences in the DNA region which codes for the mature protein.

R\ I BH]O I 665.doc:ael According to a fourth embodiment of the invention there is provided nucleic acid which codes a protein according to the second or third embodiment of the invention, wherein said nucleic acid is chosen from the group of the DNA sequences shown in Fig. la, lb, 2a, 2b, or of the complementary DNA sequences, nucleic acid sequences hybridising under stringent conditions with the sequences from or nucleic acid sequences which hybridise with one of the aforementioned sequences under stringent conditions.

According to a fifth embodiment of the invention there is provided DNA having a sequence selected from the group consisting of the sequence shown in Fig. la, the sequence shown in Fig.

1 b, the sequence shown in Fig. 2a, and the sequence shown in Fig. 2b.

According to a sixth embodiment of the invention there is provided a recombinant expression vector, containing DNA, which codes a protein according to the second or third embodiment of the invention and expresses the protein-coding DNA in a transformed micro-organism or a transformed eukaryotic cell, or in an animal or plant.

According to a seventh embodiment of the invention there is provided a host cell which, with a DNA coding a protein according to the second or third embodiment of the invention, is transfected and can produce said protein.

According to an eighth embodiment of the invention there is provided the use of DNA which S: codes for a protein according to the second or third embodiment of the invention for the transfection of a prokaryotic or eukaryotic organism According to a ninth embodiment of the invention there is provided the use of a protein from the 1-deoxy-D-xylulose-5-phosphate metabolic pathway according to the second or third embodiment of the invention as an antigen or immunogen for producing antibodies which link said protein.

According to a tenth embodiment of the invention there is provided isolated antibodies 25 against a protein from the 1-deoxy-D-xylulose-5-phosphate metabolic pathway according to the second or third embodiment of the invention which can be obtained by in vitro immunisation techniques or by immunising an animal with a protein according to the second or third embodiment of the invention and obtaining the antibodies from the serum or from the spleen cells of the immunised animal.

According to an eleventh embodiment of the invention there is provided the use of a protein according to the second or third embodiment of the invention for identifying antiparasitically acting substances.

R, According to a twelfth embodiment of the invention there is provided a protein according to the second or third embodiment of the invention when used for identifying an antiparasitically acting s substance.

[R:\LIBH]01123.doc:LUG According to a thirteenth embodiment of the invention there is provided the use of an antibody according to the tenth embodiment of the invention for identifying an antiparasitically acting substance.

According to a fourteenth embodiment of the invention there is provided an antibody according to the tenth embodiment of the invention when used identifying an antiparasitically acting substance.

According to a fifteenth embodiment of the invention there is provided a process for identifying nucleic acids which code a protein according to the second or third embodiment of the invention, wherein a sample to be investigated is incubated with a nucleic acid probe selected from the group consisting of the DNA sequence shown in Fig. la and 1b, or the sequence complementary thereto, nucleic acids, hybridising under stringent conditions with one of the sequences of wherein the nucleic acid probe is incubated with the nucleic acid of the sample and hybridisation is optionally detected via a further binding partner of the nucleic acid probe.

According to a sixteenth embodiment of the invention there is provided a test system using a protein according to the second or third embodiment of the invention for identifying an antiparasitically active substance.

According to a seventeenth embodiment of the invention there is provided the use of 3-(Nformyl-N-hydroxyamino)-propylphosphonate or 3-(N-acetyl-N-hydroxyamino)-propylphosphonate, for treating diseases caused by parasitic infections.

According to an eighteenth embodiment of the invention there is provided the use of 3-(Nformyl-N-hydroxyamino)-propylphosphonate or 3-(N-acetyl-N-hydroxyamino)-propylphosphonate in the manufacture of a pharmaceutical composition for treating infectious diseases caused by unicellular or multicellular parasites.

According to a nineteenth embodiment of the invention there is provided a method for the 25 treatment of infectious diseases caused by unicellular or multicellular parasites, comprising administering to a subject in need of said treatment a therapeutically effective amount of a compound selected from 3-(N-formyl-N-hydroxyamino)-propylphosphonate and 3-(N-acetyl-Nhydroxyamino)-propylphosphonate.

.0 0. According to a twentieth embodiment of the invention there is provided a compound selected from 3-(N-formyl-N-hydroxyamino)-propylphosphonate and 3-(N-acetyl-N-hydroxyamino)propylphosphonate when used for the treatment of infectious diseases caused by unicellular or multicellular parasites.

n R The process according to the invention and the active ingredients found are characterised in Sthat isoprenoid biosynthesis in the so-called 1-deoxy-D-xylulose-5-phosphate metabolic pathway is a 35 inhibited.

[R:\LIBH]01 123.doc:LJG None of the metabolic pathways described are present in humans and animals, only in plants, algae, some eubacteria and in parasites, such as, for example, malaria parasites; this treatment strategy stands out therefore as having few side effects.

The present invention further relates to enzymes involved in this metabolic pathway and fragments of these enzymes. These enzymes are proteins suitable for carrying out the process according to the invention for identifying active ingredients. The present invention further relates to DNA-sequences which code these enzymes or fragments of these enzymes.

The present invention relates to a process and antibodies for identifying the enzymes or their fragments and producing the enzymes or their fragments by means of recombinant technology.

The invention further relates to the use of these enzymes or their fragments or the use of the DNA-sequences which code these enzymes or fragments of these enzymes for identifying substances active against unicellular or multicellular pathogens.

0* 0 e 00 0 [R:\LIBHIOI I 23.doc:UG 6 The invention further relates to active ingredients discovered with the aid of the enzymes according to the invention.

The invention will be described in more detail hereinafter with the aid of the accompanying drawings, in which: Fig. la shows the nucleotide sequence of the gene coding the protein reductoisomerase from Plasmodium falciparum, Fig. lb shows the nucleotide sequence of the gene coding the 1-desoxy-D-xylulose-5-phosphate-synthase from Plasmodium falciparum, Fig. 2a shows the nucleotide sequence of the gene coding the from Plasmodium falciparum and the corresponding amino acid sequence, Fig. 2b shows the nucleotide sequence of the gene coding the 1-desoxy-D-xylulose-5-phosphate-synthase from Plasmodium falciparum and the corresponding amino acid sequence, Fig. 3a shows the amino acid sequence of the protein 1from Plasmodium falciparum, Fig. 3b shows the amino acid sequence of the protein 1from the parasites Plasmodium falciparum, Fig. 4a is a detail from the nucleotide sequence according to Fig. ib, Fig. 4b is a detail from the nucleotide sequence with the corresponding amino acid sequence according to Fig. 2b, Fig. 4c is a detail from the amino acid sequence according to Fig. 3b, Fig. 5 shows in vivo data for the parasitemia values after 4 days of treatment with 3 doses in each case of the substances: formyl, corresponding to 3-(N-formyl-N-hydroxylamino)propyl-phosphonic acid monosodium salt, and acetyl, corresponding to 3-(N-acetyl-N-hydroxylamino)propyl-phosphonic acid monosodium salt, Fig. 6a shows the inhibition of the growth of P.

falciparum after addition of 3-(N-formyl-Nhydroxylamino)-propyl-phosphonic acid monosodium salt (open circles) and 3-(N-acetyl-N-hydroxylamino)-propylphosphonic acid monosodium salt (closed circles) for the HB3 strain, Fig. 6b shows the inhibition of the growth of P.

falciparum after addition of 3-(N-formyl-N- 8 hydroxylamino)-propyl-phosphonic acid monosodium salt (open circles) and 3-(N-acetyl-N-hydroxylamino)-propylphosphonic acid monosodium salt (closed circles) for the A2 strain and Fig. 6c shows the inhibition of the growth of P.

falciparum after addition of 3-(N-formyl-Nhydroxylamino)-propyl-phosphonic acid monosodium salt (open circles) and 3-(N-acetyl-N-hydroxylamino)-propylphosphonic acid monosodium salt (closed circles) for the Dd2 strain, and Fig. 7 shows the classic acetate/mevalonate biosynthesis pathway in comparison to the alternative DOX-Pbiosynthesis pathway.

The coding genes of the enzymes DOXP-synthase and DOXPreductoisomerase were detected by genetic processes (Fig. la, ib, 2a, 2b). After enrichment by the polymerase chain conversion from the genome of P.

falciparum these genes were cloned in bacterial plasmids and their nucleotide sequence determined. The sequence data showed a high homology of these genes with the corresponding genes from algae, plants and bacteria. The very high homologies showed that the three genes code the enzymes DOXP-synthase and DOXP-reductoisomerase of P. falciparum.

After expression in heterologous systems the enzymes were purified as recombinant proteins and used for activity studies in cell-free systems. The activity of 9 the DOXP-synthase was measured by converting glyceraldehyde-3-phosphate and pyruvate to 1-desoxy-D- The activity of the DOXPreductoisomerase was measured by converting 1-desoxy-Dxylulose-5-phosphate to 2-C-methyl-D-erythritol-4phosphate in the presence of NADPH. Measurement of the change in the NADPH concentration is via a parameter variation. This process is known to the person skilled in the art.

The enzymes can be defined by the DNA-sequence coding them (Fig. la, ib, 2a, 2b) and the amino acid sequence derived therefrom (Fig. 3a and 3b). The enzymes of the individual parasites can, however, differ from parasite to parasite. Such variations of the amino acids are usually amino acid exchanges. There can, however, also be deletions, insertions and additions of amino acids to the total sequence. The enzymes according to the invention, both in size and type depending on the cell and cell type in which they are expressed, can be glycosylated or non-glycosylated.

The enzymes according to the invention or fragments of these enzymes are produced by expression of the DNA according to the invention in suitable expression systems, for example in bacteria, in particular in E.

coli, as prokaryotic expression system or in a eukaryotic expression system, in particular COS-cells or Dictyostelium discoideum.

With the aid of the nucleic acid sequence according to the invention, it is possible to look for the coding gene or its variants in the genome of any parasite, to identify these and to isolate the desired coding gene for the enzymes. Processes of this type and screening processes suitable for this purpose are known to the person skilled in the art.

As a result of the application of recombinant technology, it is possible to produce a multiplicity of variants of enzymes or fragments of enzymes. Derivatives of this type can be modified, for example, in one or more amino acids by substitution, deletion or addition.

The derivation can be, for example, by site directed mutagenesis. Variations of this type can easily be carried out by the person skilled in the art. It merely has to be ensured that the characteristic properties of the enzymes are retained. A further subject of this invention is therefore the enzymes, which are involved in the DOXP metabolic pathway, in particular DOXPsynthase and DOXP-reductoisomerase, which a) are the product of a prokaryotic or eukaryotic expression of an exogenous DNA, b) are coded from a sequence in Fig. la, ib, 2a and 2b, c) are coded from DNA-sequences which hybridise with the DNA-sequences shown in Fig. la, lb, 2a and 2b or fragments of these DNA sequences (see, for example, Fig. 4a and 4b) in the DNA region which codes the mature protein, or 11 d) are coded from DNA-sequences which would hybridise without degeneration of the genetic code with the sequences defined in b) to c) and code a polypeptide with the same amino acid sequence.

Enzymes are preferred which are coded from the nucleotides from Fig. la, lb, 2a and 2b or from DNAsequences which, due to the degeneration of the genetic code, would code a polypeptide with the same amino acid sequence.

The two enzymes according to the invention (sequence in Fig. 3a and 3b) can be seen as new prototypes of specific proteins, unicellular and multicellular parasites, in particular of the unicellular parasites.

This invention relates to nucleic acid sequences which code the enzymes and are selected from the group a) of DNA sequences shown in Fig. la, ib, 2a and 2b or their complementary sequences, b) nucleic acid sequences which hybridise with one of the sequences in a), c) nucleic acid sequences which would hybridise with one of the sequences mentioned in a) or b) without degeneration of the genetic code.

The invention also relates to enzymes from any parasites which essentially condense pyruvate and glyceraldehydes- 3-phosphate to 1-desoxy-D-xylulose-5-phosphate

(DOXP-

synthase) and convert l-desoxy-D-xylulose-5-phosphate to 12 2-C-methyl-D-erythritol-4-phosphate (DOXPreductoisomerase). These enzymes, similar to the enzymes from malaria parasites, can be obtained in that a cDNA library or genomic library of the corresponding parasites is screened by processes familiar to the person skilled in the art with a hybridising probe containing enzymes from sequences coding malaria parasites, or by the sequence comparison of the DNA and protein sequence for enzymes of malaria parasites with other parasite enzymes.

With the aid of the nucleic acids, enzymes according to the invention can be obtained in large quantities in a repeatable manner. The nucleic acid is integrated into suitable expression vectors by processes familiar to the person skilled in the art, for expression in prokaryotic and eukaryotic organisms. An expression vector of this type preferably contains an adjustable/inducible promoting agent. These recombinant vectors are then introduced by known processes into suitable host cells for expression and the transformed, transfected or transduced host cells are cultivated under conditions which allow an expression of the heterologous gene.

Suitable host cells include prokaryotic cells such as, for example, E. coli, and eukaryotic cells, in particular yeasts (for example Saccharomyces cervisiae, Schizosaccharomyces pombe, Pichia pastoris), insect cells, (for example cell lines of Drosophila melanogaster such as S2 cells, Spodoptera frugiperda, Trichoplusia ni), vertebrate animal cell lines, particularly teratocarcinoma cell lines such as CHO or COS cells and plant cell lines.

The enzymes according to the invention can also be expressed in transgenic plants and animals, (for example mice, sheep, goats, pigs, guinea pigs). Advantageously the expression system is to be arranged by techniques known to the person skilled in the art, in such a way that the enzymes produced are separated off with the milk of the animals or can be obtained from easily obtained plant parts (fruit, leaves, blossom, shoot and root parts).

Particularly suitable as expression vectors for vertebrate animal cell lines are systems derived from papilloma viruses (for example SV40), retro viruses, sindbis viruses, cytomegalo viruses and vaccinia viruses. Particularly suitable for insect cells is the baculo virus system, for plants, particularly suitable are cell systems based on the ti-plasmid of Agrobacterium tumefaciens and the bombardment of cells with particles covered with nucleic acid.

The expression of the enzymes according to the invention is particularly significant in slime fungi such as Dictyostelium discoideum, Polysphondylium pallidum and Physarum polycephalum as their cells can be cultivated economically in large quantities on simple media. The use of Dictyostelium discoideum offers the further advantage that this organism uses similar codons for the respective amino acids such as Plasmodium falciparum and particularly effective production of the enzymes according to the invention is thus achieved. Moreover, inducible promoting agents (for example due to lack of food) are known for expression vectors for Dictyostelium discoideum. As a result the recombinant enzyme yield can be further increased.

Particularly suitable for the expression of the enzymes according to the invention are host cells and organisms of the type which have no intrinsic enzymes which condense pyruvate and glyceraldehyde-3-phosphate to 1- (DOXP-synthase) and react to 2-C-methyl-Derythritol-4-phosphate (DOXP-reductoisomerase). This applies to archaebacteria, animals, fungi, slime fungi and some eubacteria. Detection and purification of the recombinant enzymes is substantially facilitated by the lack of these intrinsic enzyme activities. Moreover, it is possible for the first time to measure economically the activity and in particular the inhibition of the activity of the recombinant enzymes according to the invention by various chemicals and pharmaceutical compositions in raw extracts from the host cells.

The enzymes according to the invention are advantageously expressed in eukaryotic cells, when posttranslatory modifications and a native folding of the polypeptide chain is to be achieved. Moreover, as a function of the expression system, during the expression of genomic DNA-sequences, introns are eliminated by splicing the DNA and the enzymes are produced in the polypeptide sequence characteristic for the parasite.

Sequences coding introns can also be eliminated from the DNA-sequences to be expressed by recombinant DNA technology or inserted experimentally.

The protein can be isolated from the host cell or the culture supernatant of the host cell by the process known to the person skilled in the art. An in vitro reactivation of the enzymes may also be required.

To facilitate purification, the enzymes according to the invention or fragments of the enzymes can be expressed as fusion protein with various peptide chains. Oligohistidine sequences and sequences derived from the glutathione-S-transferase, thioredoxin or calmodulinbinding peptides are particularly suitable for this.

Fusions with thioredoxin derived sequences are particularly suitable for prokaryotic expression as the solubility of the recombinant enzymes is thus increased.

Furthermore, the enzymes according to the invention or part sequences of the enzymes can be expressed with peptide chains, known to the person skilled in the art, which are such that the recombinant enzymes are transported into the extra cellular milieu or into certain compartments of the host cells. As a result, purification and investigation of the biological activity of the enzymes can be facilitated.

With the expression of the enzymes according to the invention, it may prove expedient to change individual 16 codons. Specific exchange of bases in the coding region is also sensible when the codons used in the parasites are different from the codons used in the heterologous expression system to ensure optimum synthesis of the protein. Deletions of non-translated 5' or 3' portions are also often sensible, for example when a plurality of destabilising sequence motives ATTTA are present in the 3' region of the DNA. These should then be deleted in the preferred expression in eukaryons. Changes of this kind are deletions, additions or exchange of bases and also subject of the present invention.

The enzymes according to the invention can further be obtained under standardised conditions by techniques known to the person skilled in the art by in vitro translation. Systems which are suitable for this are rabbit reticulocytes and wheat germ extracts. Also, mRNA transcribed in vitro can be translated into xenopusoocytes.

By virtue of chemical synthesis, oligo- and polypeptides can be produced, the sequences of which are derived from the peptide sequence of the enzymes according to the invention. With suitable choice of the sequences, peptides of this type have features which are characteristic of the complete enzymes according to the invention. Peptides of this type can be produced in large quantities and are particularly suitable for studies on the kinetics of enzyme activity, the adjustment of enzyme activity, the three-dimensional structure of enzymes, the inhibition of enzyme activity

S

17 by various chemicals and pharmaceutical compositions and the binding geometry and binding affinity of various ligands.

A DNA with the nucleotides from the sequences shown in Fig. la, ib, 2a and 2b or a fragment according to Fig.

4a and 4b is preferably used for the recombinant production of enzymes according to the invention.

The invention also relates to processes for obtaining enzymes involved in the DOXP metabolic pathway, in particular the enzymes DOXP-synthase and DOXPreductoisomerase by isolating from the parasites. The enzymes are isolated from parasite extracts by chromatographic, electophoretic and other processes known to the person skilled in the art. The enzymes are found by measuring the respective enzymatic activity or reactivity with appropriate antibodies.

The detection of transformed, transfected or transduced host cells which recombinantly produce the enzymes and the purification of the protein are preferably by antibodies which bind to these enzymes. Antibodies of this type can be obtained easily with the aid of the enzymes according to the invention or parts of the enzymes as antigen or immunogen by known processes.

Homologous or cross-converting proteins of other parasites can be detected with the antibodies to proteins according to the invention, for example, by the Western blot test.

18 This invention also relates to methods for determining the enzymatic activity of the DOXP enzymes, in particular of the enzymes DOXP-synthase and DOXPreductoisomerase. This can be determined according to known instructions (Sprenger et al. PNAS, 94 (1997) 12857-62 und Kuzuyama et al. Tetrahedron Letters 39 (1998) 4509-12). In this process the condensation of pyruvate and glyceraldehyde-3-phosphate to 1-desoxy-Dxylulose-5-phosphate (DOXP-synthase) and the conversion of l-desoxy-D-xylulose-5-phosphate to 2-C-methyl-Derythritol-4-phospate (DOXP-reductoisomerase) is detected. This invention also relates to the use of these measuring processes for obtaining substances which inhibit the activity of the respected enzymes.

By the application of recombinant technology it is possible to produce a multiplicity of variations of enzymes or fragments of enzymes. Derivatives of this type can, for example, be modified in one or more amino acids by substitution, deletion or addition. The derivation can, for example, be by site directed mutagenesis. Variations of this type can easily be carried out by the person skilled in the art. It must merely be ensured that the characteristic features of the enzymes are retained.

With the help of the enzymes according to the invention and their homologues, new specific active ingredients against parasites can be found.

19 In particular, the detection processes described above can be used in appropriate test kits for screening for anti-parasitic activity of substances. These include processes known to the person skilled in the art and suitable for screening natural substances from flora and fauna, from plants, algae, bacteria or animals, and their derivatives, chemical libraries, also libraries which have been compiled by means of techniques known to the person skilled in the art, including combinatory chemistry. (Pindur et al. Pharmazie in unserer Zeit 26 (1997) 24-30; Broach et al. Nature 384 (1997) 14-6; Lack et al. Chimia 50 (1996) 445-7; Czarnik und Ellmann Accounts of Chemical Research 29 (1996); Chemical and Engineering News 74 (1996) 28-73; Lorin et al. Chemical Reviews 96 (1996) 555-600; Weber et al. Nachrichten aus Chemie, Technik und Laboratorium 42 (1994) 698-702).

The present invention also relates to the use of proteins or fragments of these proteins, including proteins or fragments of proteins with or without enzymatic activity in techniques known to the person skilled in the art for determining structures of protein, in particular the characterisation of binding sites suitable for the development of preparations with inhibiting effect on enzymatic activity.

Active ingredients obtained with the aid of proteins according to the invention are of great interest to medicine and veterinary medicine.

Active ingredients found with the aid of the proteins according to the invention are suitable, in favourable homoeothermic toxicity, for fighting pathogenic parasites which occur in humans and in animal husbandry and rearing in domestic, breeding, zoo, laboratory and animals for experimentation and pets. They are effective here against all or individual stages of development of the destructive parasites and against resistant and normally sensitive parasites. As a result of the fight against parasites, diseases, fatalities and reductions in performance (for example in the production of-meat, milk, wool, skins, eggs, etc) should be reduced, so the use of active ingredients allows easier and more costefficient animal husbandry.

By using these processes according to the invention including established assays, it could be shown that the activity of the DOXP-reductoisomerase is inhibited by 3- (N-acetyl-N-hydroxyamino)propylphosphonate and derivatives 3-(N-formyl-N-hydroxyamino)propylphosphonate (fosmidomycin). Both substances originate from a chemical library of acylhydroxylamino-alkylphosphonic acid derivatives. This group of compounds was described in the past as herbicidal and bactericidal (US 4693742, DE2733658). The efficiency of the system for obtaining anti-parasitic active ingredients has been shown here.

The results from the enzyme assays could be confirmed both in the malaria culture (see examples) and in the animal experiment (see examples). The inhibitors found by means of these enzyme assays were able to inhibit the 21 growth of malaria parasites in vitro and in vivo.

Treatment of animals over a time period of 8 days showed a healing of the animals. The acetyl form showed a three times greater efficacy than the formyl form. This result is very surprising, as substantially higher (up to 1000x) concentrations of 3-(N-acetyl-Nhydroxyamino)propylphosphonate were needed to inhibit the bacteria growth.

The process according to the invention is thus suitable for identifying active ingredients and the active ingredients according to the invention are suitable for the therapeutic and prophylactic treatment of infections in humans and animals caused by parasites, fungi or viruses. The compounds are suitable as prophylactics against, and for treatment of, infections, caused by pathogens of malaria and sleeping sickness as well as Chagas' disease, toxoplasmosis, amoebic dysentery, leishmanosis, trichomoniasis, pneumocystosis, balantidiosis, cryptosporidiosis, sarcocystosis, acanthamebiasis, naegleriasis, coccidiosis, giardiasis and lambliosis.

The processes according to the invention and the active ingredients according to the invention are particularly suitable for treating malaria, sleeping sickness and leishmanoses.

The active ingredients according to the invention are also suitable for inhibiting the metabolic pathway of bacteria and plants. Substances which are identified according to the invention as inhibitors of DOXP metabolic pathway are therefore also suitable for use as herbicides and for use in treating bacterial infections in humans and animals.

Domestic and breeding animals suitable for treatment include mammals such as, for example, cattle, horses, sheep, pigs, goats, camels, water buffalos, donkeys, rabbits, salt and freshwater fish as, for example, trout, carp and eels. Suitable laboratory animals and animals for experimentation include mice, rats, guinea pigs, golden hamsters, dogs, cats and pigs. Suitable pets include dogs and cats. Application can be both prophylactic and therapeutic. The application of active ingredients is direct or in the form of suitable preparations known to the person skilled in the art such as enteral, parenteral, dermal or nasal.

The active ingredients according to the invention can be used in combination with any anti-infective agents known to the person skilled in the art. These include substances which have an antibacterial, antiparasitic, antiviral or fungicidal effect. These include antiinfective agents which are listed in the red list and in the specialist literature (Allegemeine und spezielle Pharmakologie und Toxikololgie von Forth et al. BI- Wissenschaftsverlag, Mannheim 1998; Antibiotikatherapie von Simon und Stille, Schattauer-Verlag, Stuttgart 1993).

As some parasites have both the mevalonate metabolic pathway and the DOXP metabolic pathway, the invention also relates to the combination of inhibitors of the DOXP metabolic pathway with preparations which inhibit the fat metabolic pathway, including inhibitors of the synthesis or absorption of lipids, in particular, inhibitors of the mevalonate metabolic pathway. The inhibitors of the enzymes HMG-CoA-synthase and inhibitors of the HMG-CoA-reductase deserve particular mention. Included amongst the inhibitors of the HMG-CoAreductase are, in particular, Lovastatin and derivatives, Mevastatin and derivatives, Compactin and derivatives, Simvastatin and derivatives, Pravastatin and derivatives, Atorvastatin and derivatives, Fluvastatin and derivatives and Cerivastatin and derivatives.

Example 1 Expression cloning of the gene of P. falciparum coding the DOXP-reductoisomerase.

The gene coding the DOX-reductoisomerase of P.

falciparum was cloned by PCR amplification of the corresponding sequences of genomic DNA as matrix. To obtain genome DNA, the P. falciparum strain HB3 was cultivated by the Kerzentopf process (Tranger und Jensen (1976), Science 193, 673-675). As culture medium, RPMI 1640 (with HEPES and L-glutamine, Gibco) was supplemented with 10% of human serum, 0.3 ig/ml of Gentamicin and 0.1 mM of Hypoxanthine and a hematocrit of 5% adjusted with human erythrocytes. 15 culture dishes with 35 ml culture volume in each case were used with approximately 4% of parasitemia for the preparation of the DNA. The infected erythrocytes were harvested by centrifugation and washed twice in carrier buffer (57 mM NaCi, 58 mM KC1, 1 mM NaH 2

PO

4 7 mM K 2

HPO

4 11 mM NaHCO 3 14 mM glucose). The parasites were released from the erythrocytes by lysing the cell sediment with a ten-fold volume of 1% saponin solution in carrier buffer for minutes on ice (modified according to Kilejian (1979), Proc. Natl. Acad. Sci. USA 76, 4650-4653). The free parasites were washed twice by centrifugation (10 min, 10,000 rpm, 4 0 C) with a solution of 1% BSA in carrier buffer. The DNA preparation from the free parasites obtained took place according to standard procedures.

The parasites were then digested with proteinase K. The assay was then extracted four times with phenol/chloroform, the DNA solution was dialysed overnight against TE and then precipitated with isopropanol. The following primer was used for the PCR amplification: PfYAEMfor 5"-CTGAATTTCATATTACAAAATTAATAGATG-3" PfYAEMrev 5'-GTACTATGAAGAATTATGTTTGTTGTATAT-3' The following assay was used for the PCR conversion: 3 il 10 x PCR-buffer 2.4 Al 25 mM MgS04 2.4 Al 2.5 mM dNTP 2 pl DNA matrices (0,2 Ag/ml) 2 [Ll primer 1 (7.5 pM) 2 pl primer 2 (7.5 iM) 0.2 il tag-polymerase (5 U/Il) 16 il The amplification took place with the following profile: 3 cycles: 96°C 1 min 48°C 1 min 72°C 3 min 32 cycles: 95°C 40 sec 480C 1 min 72°C 3 min After the last cycle the assay was incubated for a further 10 minutes at 72°C to lengthen all the products.

The PCR product of four assays of this type were combined and purified with a 0.7% agarose gel. The elution of the DNA from the agarose blocks took place with the "kit for DNA extraction" (Millipore, Cat. No.

S667). The eluted DNA was precipitated with ethanol and absorbed in 10 Al H 2 0. The PCR product was then cloned according to the manufacturer's instructions with the TA cloning kit (in vitro gene). 20 mg of insert-DNA were used for a ligation assay. Colonies of bacteria bearing the desired recombinant plasmid were identified by analytical plasmid preparation and EcoR I-digestion of the plasmids. The cloned PCR products were then sequenced using standard, forward and reverse primers; the sequences were completed with the Walkings primer technique.

A PCR product, present in the corresponding orientation in the pCR2.1 vector, was re-cloned in the expression vector pBK-CMV (Stratagene) for expression in COS-7cells. The re-cloning took place via the intersections of the restriction enzymes Not I and BamH I, which occur in the polylinker of the two vectors. For the transfection of the COS-7-cells, the expression vector with the PCR product as insert was produced on a preparative scale by anion exchange chromatography (Qiagen).

All the methods used for the cloning are described in detail in J. Sambrook, E.F. Fritsch, T. Maniatis (1989), Molecular cloning: a laboratory manual, 2 nd edition, Cold Spring Habor Laboratory Press, Cold Spring Habor, USA.

The COS-7-cells were cultivated in DMEM medium with FCS under standard conditions. 30 ml of culture medium were allowed per cell culture bottle. Cells with approximately 50% confluency were used for the transfection, which had been split the day before. DOTAP (Boehringer) was used as transfection reagent. 40 il of DNA solution (0.5 Ag/ml) were mixed with 110 Al of 20 mM HEPES (pH 100 il of DOTAP were also mixed with 230 Al 20 mM HEPES (pH 7.4) in a polystyrene conversion vessel. Then the DNA solution was pipetted into the DOTP solution and incubated for 15 minutes at room temperature. Then the assay was mixed with 20 ml of culture medium and the medium of the COS-7-cells was replaced by this mixture. The next day the cells were transferred with fresh medium into new cell culture bottles. After a further 48 hour incubation the transfected COS-7-cells were harvested. The cells were scraped off for this purpose and washed 3 times by centrifugation in assay buffer (100 mM TrisHCl (pH 1 mM MnCl 2 The cells were suspended again in a minimal volume of assay buffer and digested by being frozen three times (in liquid nitrogen) and thawing. Cell fragments were centrifuged off in a 1.5 ml conversion vessel (13,000 rpm, 10 min, 4 0 C) and the supernatant used directly for measuring the enzyme activity or purifying the enzyme.

Example 2 Purification of the recombinant DOXP-reductoisomerase of P. falciparum.

The recombinant DOXP-reductoisomerase of P. falciparum expressed in COS-7-cells was purified for considerable homogeneity and more precise characterisation. The purification took place in an affinity chromatography.

and a gel permeation chromatography step. Antibodies against the DOXP-reductoisomerase of P. falciparum were produced for the production of a suitable affinity chromatography column. Portions were also chosen from the amino acid sequence derived from the DNA sequence, for which a particularly high antigen effect could be predicted. Appropriate peptides were synthesised and used for immunising rabbits. The quality of the antisera obtained was confirmed by its reactivity with the synthetic peptides and by Western blot tests. For the Western blot tests (BM Western Blotting Kit, Boehringer) extracts were used from the P. falciparum and recombinant COS-cells.

The antiserum for eliminating low-molecular amines was dialysed against PBS for producing the affinity chromatography column. The antibodies were bound to protein A-sepharose and covalently coupled by crosslinking with DMP (IgG Orientation Kit, Pierce). The protein extract was, as described in Example 1, obtained from 55 cell culture bottles with transfected COS-7cells and loaded on to the columns equilibrated with assay buffer. After excessive washing with assay buffer the column was eluted with elution buffer (100 mM GlycinHCl (pH 2.8) 0.4% CHAPS). The eluate was immediately neutralised with 1 M TrisHCl (pH The main fractions were identified by Western blot analysis.

Biotinylated antibodies were used for detection to avoid disruption by antibodies eluted from the column in small quantities. The main fractions were combined, dialysed against assay buffer and concentrated by ultrafiltration Kda, Amicon). Further purification took place by gel permeation chromatography (Superdex 200, Pharmacia) with assay buffer as input and elution buffer. The main fractions were identified as described above, combined and concentrated, reacted with 20% glycerol and frozen at -70 0 C. As a result of SDS-PAGE (12% acrylamide) under reducing conditions and silver staining (Gel Code Silver Stain Kit, Pierce), the cleaned DOXP-reductoisomerase of p. falciparum was shown as a unified band at 54 kDa.

Example 3 Determination of the activity of the purified enzyme and screening for inhibitors.

The DOXP-reductoisomerase activity of the purified enzyme was confirmed in an in vitro test system. 100 il of assay buffer with 0.3 mM NADPH, 0.3 mM DOXP and 10 Ag of recombinant enzyme were used for a typical test assay. The conversion was started by the addition of DOXP to the complete assay. The oxidation of NADPH was carried out photometrically with 340 nm in microquartz cuvettes at 37 0 C. This test system was used to show the inhibition of recombinant DOXP-reductoisomerase of P.

falciparum by various substances. After addition of 1 AM 3-(N-formyl-N-hydroxylamino)-propyl-phosphonic acid monosodium salt and 1 AM 3-(N-acetyl-N-hydroxylamino)propyl-phosphonic acid monosodium salt) to the conversion assay no change was observed to the absorption at 340 nm. Under these conditions, the DOXPreductoisomerase of P. falciparum was completely inhibited.

Example 4 Test of the effectiveness of the substances against malaria in vivo.

The various derivatives were tested by the modified Peters' test. The substances were applied in a quarter of the median lethal dose (LD50). In the test assay, mice were infected with Plasmodium vinckeii, the pathogen of mouse malaria. Once infection had been confirmed by blood examination, the 4 mice were treated.

6 mice which had not been treated were used as controls.

The treatment with 1-1000 mg/kg/d 3-(N-formyl-Nhydroxylamino)-propyl phosphonic acid monosodium salt over 3 days led to a destruction of the parasites in the blood of the mice. The treated group was free of living parasites after only 1 day. The control mice had to be destroyed on day 5 after infection with a parasitemia of 80%. The treated mice were still free of parasites 8 weeks after the end of the treatment. Further experiments showed an effectiveness of 50 mg/kg/d 3-(Nformyl-N-hydroxylamino)-propyl-phosphonic acid monosodium salt in mice with a parasitemia of 80%. These mice were also free of living parasites after 1 day.

Further results for 3-(N-formyl-N-hydroxylamino)-propylphosphonic acid monosodium salt and 3-(N-acetyl-Nhydroxylamino)-propyl-phosphonic acid monosodium salt are shown in Fig. Example Protection from malaria in the experiment with infected mice.

The effectiveness of the compounds in vivo against malaria was tested by using male mice (BALB/c-strain) weighing 20 to 25 g. One day before the infection, 4 mice were treated intraperitoneally with 50 mg/kg of 3- (N-formyl-N-hydroxylamino)-propyl-phosphonic acid monosodium salt. The mice were then infected with Plasmodium vinckeii. Mice which had not been pre-treated with the substance were used as controls. No infection could be detected in the treated mice, while the control mice were destroyed after 5 days with a parasitemia over The treated mice were free of parasites 8 weeks after the infection.

Example 6 In vitro inhibition of the growth of malaria parasites on the principle of the IC50 determination (the concentration at which the vitality of the parasites is reduced by half).

For determining the IC50 values, the malaria parasites are initially cultivated for a complete 48 hour cycle in the presence of inhibitors, in the next 24 hours the survival rate was measured by [3H]-hypoxanthine insertion. A dilution series of 3-(N-formyl-Nhydroxylamino)-propyl-phosphonic acid mono-sodium salt was presented on a micro-titre plate in concentrated by 10. Then 180 Al of parasite suspension in culture medium was added to each well. Asynchronous cultures with about 0.4% parasitemia and 2% hematocrit were used. The micro-titre plates were then incubated for 48 hours. Then 30 Ail [3H]-hypoxanthine were added to each well. After incubating for 24 hours the cells were harvested and the incorporated radioactivity was measured. The results with the HB3, A2 and Dd2 strains with known resistances against other malaria medications is shown in Fig. 6a, 6b and 6c. In both strains, an ICvalue of below 0.5 iM occurs. The resistances of these strains are: Plasmodium falciparum HB3 (Honduras) is resistant to Pyrimethamine.

Plasmodium falciparum Dd2 (Indochina) is resistant to chloroquine, quinine, pyrimethamine, cycloguanil and sulfadoxine.

Plasmodium falciparum A2 (Gambia) is resistant to chloroquine and cycloguanil.

No cross resistances were found with anti-malaria preparations.

Editorial Note 44816/99 The following sequence listing, pages 1-11, are part of the description.

WO 99/52938 PCT/EP99/02463 Sequence Listing Number of Sequences: 2 DETAILS OF SEQUENCE ID NO: 1 Plasmodium falciparum 1-desoxy-D-xylulose-5-phosphate reducto-isomerase (dxr) gen SEQUENCE CHARACTERISTICS: LENGTH: 1467 BASE PAIRS TYPE: nucleotide sequence PARENT: HB3 (ii) TYPE OF MOLECULE: DNA (iv) PRIMARY EXTRACTION: ORGANISM: Plasmodium falciparum (ix) CHARACTERISTIC NAME CODE: mRNA SITE: 1 1467 GEN dxr PRODUCT 1-desoxy-D-xylulose-5-phosphate reducto-isomerase (ix) CHARACTERISTIC NAME CODE:Gen SITE: 1 1467 GEN dxr (ix) CHARACTERISTIC NAME CODE: CDS SITE: 1 1467 GEN dxr FUNCTION: involved in the biosynthesis of isopentenyl diphosphate Start codon: 1 PRODUCT 1-desoxy-D-xylulose-5-phosphate reducto-isomerase PROTEIN: 488 amino acids ORGANISM: plasmodium falciparum; (Apicomplexa) PARENT: HB3 WO 99/52938 PCT/EP99/02463 (xi SEQUENCE DESCRIPTION: SEQ ID NO:I ATG AAG AAA TAT ATT TAT ATA TAT TTT TTC TTC ATC ACA ATA ACT ATT 48 Met Lys Lys Tyr Ile Tyr Ile Tyr Phe Phe Phe Ile Thr Ile Thr Ile 10 AAT GAT TTA GTA ATA AAT AAT ACA TCA AAA TGT GTT TCC ATT GAA AGA 96 Asn Asp Leu Val Ile Asn Asn Thr Ser Lys Cys Val Ser Ile Glu Arg 25 AGA AAA AAT AAC GCA TAT ATA AAT TAT GGT ATA GGA TAT AAT GGA CCA 144 Arg Lys Asn Asn Ala Tyr Ile Asn Tyr Glu Ile Glu Tyr Asn Glu Pro 40 GAT AAT AAA ATA ACA AAG AGT AGA AGA TGT AAA AGA ATA AAG TTA TGC 192 Asp Asn Lys Ile Thr Lys Ser Arg Arg Cys Lys Arg Ile Lys Leu Cys 55 AAA AAG GAT TTA ATA GAT ATT GGT GCA ATA AAG AAA CCA ATT AAT GTA 240 Lys Lys Asp Leu Ile Asp Ile Glu Ala Ile Lys Lys Pro Ile Asn Val 70 75 GCA ATT TTT GGA AGT ACT GGT AGT ATA GGT ACG AAT GCT TTA AAT ATA 288 Ala Ile Phe Glu Ser Thr Glu Ser Ile Glu Thr Asn Ala Leu Asn Ile 90 ATA AGG GAG TGT AAT AAA ATT GAA AAT GTT TTT AAT GTT AAA GCA TTG 336 Ile Arg Glu Cys Asn Lys Ile Glu Asn Val Phe Asn Val Lys Ala Leu 100 105 110 TAT GTG AAT AAG AGT GTG AAT GAA TTA TAT GAA CAA GCT AGA GAA TTT 384 Tyr Val Asn Lys Ser Val Asn Glu Leu Tyr Glu Gin Ala Arg Glu Phe 115 120 125 TTA CCA GAA TAT TTG TGT ATA CAT GAT AAA AGT GTA TAT GAA GAA TTA 432 Leu Pro Glu Tyr Leu Cys Ile His Asp Lys Ser Val Tyr Glu Glu Leu 130 135 140 AAA GAA CTG GTA AAA AAT ATA AAA GAT TAT AAA CCT ATA ATA TTG TGT 480 Lys Glu Leu Val Lys Asn Ile Lys Asp Tyr Lys Pro lie Ile Leu Cys 145 150 155 160 GGT GAT GAA GGG ATG AAA GAA ATA TGT AGT AGT AAT AGT ATA GAT AAA 528 Glu Asp Glu Glu Met Lys Glu Ile Cys Ser Ser Asn Ser Ile Asp Lys 165 170 175 ATA GTT ATT GGT ATT GAT TCT TTT CAA GGA TTA TAT TCT ACT ATG TAT 576 Ile Val Ile Glu lie Asp Ser Phe Gin Glu Leu Tyr Ser Thr Met Tyr 180 185 190 WO 99/52938 PCTIEP99/02463 GCA ATT ATG AAT AAT AAA ATA GTT GCG TTA GCT AAT AAA GAA TCC ATT 624 Ala Ile Met Asn Asn Lys Ile Val Ala Leu Ala Asn Lys Glu Ser Ile 195 200 205 GTC TCT GCT GGT TTC TTT TTA AAG AAA TTA TTA AAT ATT CAT AAA AAT 672 Val Ser Ala Glu Phe Phe Leu Lys Lys Leu Leu Asn Ile His Lys Asn 210 215 220 GCA AAG ATA ATA CCT GTT GAT TCA GAA CAT AGT GCT ATA TTT CAA TGT 720 Ala Lys lie lie Pro Val Asp Ser Glu His Ser Ala Ile Phe Gln Cys 225 230 235 240 TTA GAT AAT AAT AAG GTA TTA AAA ACA AAA TGT TTA CAA GAC AAT TTT 768 Leu Asp Asn Asn Lys Val Leu Lys Thr Lys Cys Leu Gin Asp Asn Phe 245 250 255 TCT AAA ATT AAC AAT ATA AAT AAA ATA TTT TTA TGT TCA TCT GGA GGT 816 |er Lys Ile Asn Asn lie Asn Lys Ile Phe Leu Cys Ser Ser Glu Glu 260 265 270 CCA TTT CAA AAT TTA ACT ATG GAC GAA TTA AAA AAT GTA ACA TCA GAA 864 Pro Phe Gin Asn Leu Thr Met Asp Glu Leu Lys Asn Val Thr Ser Glu 275 280 285 AAT GCT TTA AAG CAT CCT AAA TGG AAA ATG GGT AAG AAA ATA ACT ATA 912 Asn Ala Leu Lys His Pro Lys Trp Lys Met Glu Lys Lys Ile Thr lie 290 295 300 GAT TCT GCA ACT ATG ATG AAT AAA GGT TTA GAG GTT ATA GAA ACC CAT 960 Asp Ser Ala Thr Met Met Asn Lys Glu Leu Glu Val Ile Glu Thr His 305 310 315 320 TTT TTA TTT GAT GTA GAT TAT AAT GAT ATA GAA GTT ATA GTA CAT AAA 1008 *Phe Leu Phe Asp Val Asp Tyr Asn Asp Ile Glu Val lie Val His Lys 325 330 335 GAA TGC ATT ATA CAT TCT TGT GTT GAA TTT ATA GAC AAA TCA GTA ATA 1056 Glu Cys Ile lie His Ser Cys Val Glu Phe Ile Asp Lys Ser Val Ile 340 345 350 AGT CAA ATG TAT TAT CCA GAT ATG CAA ATA CCC ATA TTA TAT TCT TTA 1104 Ser Gin Met Tyr Tyr Pro Asp Met Gin Ile Pro Ile Leu Tyr Ser Leu 355 360 365 ACA TGG CCT GAT AGA ATA AAA ACA AAT TTA AAA CCT TTA GAT TTG GCT 1152 Thr Trp Pro Asp Arg Ile Lys Thr Asn Leu Lys Pro Leu Asp Leu Ala 370 375 380 CAG GTT TCA ACT CTT ACA TTT CAT AAA CCT TCT TTA GAA CAT TTC CCG 1200 Gin Val Ser Thr Leu Thr Phe His Lys Pro Ser Leu Glu His Phe Pro 385 390 395 400 WO 99/52938 PCT/EP99/02463 TGT ATT AAA TTA GCT TAT CAA GCA GGT ATA AAA GGA AAC TTT TAT CCA 1248 Cys Ile Lys Leu Ala Tyr Gin Ala Glu Ile Lys Glu Asn Phe Tyr Pro 405 410 415 ACT GTA CTA AAT GCG TCA AAT GAA ATA GCT AAC AAC TTA TTT TTG AAT 1296 Thr Val Leu Asn Ala Ser Asn Glu Ile Ala Asn Asn Leu Phe Leu Asn 420 425 430 AAT AAA ATT AAA TAT TTT GAT ATT TCC TCT ATA ATA TCG CAA GTT CTT 1344 Asn Lys Ile Lys Tyr Phe Asp Ile Ser Ser Ile Ile Ser Gin Val Leu 435 440 445 GAA TCT TTC AAT TCT CAA AAG GTT TCG GAA AAT AGT GAA GAT TTA ATG 1392 Glu Ser Phe Asn Ser Gin Lys Val Ser Glu Asn Ser Glu Asp Leu Met 450 455 460 AAG CAA ATT CTA CAA ATA CAT TCT TGG GCC AAA GAT AAA GCT ACC GAT 1440 Lys Gin Ile Leu Gin Ile His Ser Trp Ala Lys Asp Lys Ala Thr Asp 465 470 475 480 ATA TAC AAC AAA CAT AAT TCT TCA TAG 1467 Ile Tyr Asn Lys His Asn Ser Ser 485 DETAILS OF SEQUENCE ID NO: 2 Plasmodium falciparum 1-desoxy-D-xylulose-5-phosphate synthase (dxs) gen (iii) SEQUENCE CHARACTERISTICS: LENGTH: 3872 BASE PAIRS TYPE: nucleotide sequence PARENT: HB3 (ii) TYPE OF MOLECULE: DNA (iv) PRIMARY EXTRACTION: ORGANISM: Plasmodium falciparum (ix) CHARACTERISTIC NAME CODE: mRNA GEN dxs PRODUCT 1-desoxy-D-xylulose-5-phosphate synthase (ix) CHARACTERISTIC WO 99/52938 PCT/EP99/02463 NAME CODE:Gen SITE: 1 3872 GEN dxs (ix) CHARACTERISTIC NAME CODE: CDS GEN dxs FUNCTION: involved in the biosynthesis of isopentenyl diphosphate Start codon: 1 PRODUCT 1-desoxy-D-xylulose-5-phosphate synthase PROTEIN: 1205 amino acids ORGANISM: plasmodium falciparum; (Apicomplexa) PARENT: HB3 (xi) SEQUENCE DESCRIPTION SEQ ID NO:2 kGTAATATAC GTATAATATA TATATAATAT ATTCTTACGT ATGTATCATT TATGAATCAT AATAATATTC TAAATTTACC TTCCGTTTTT GCTCGATCTT CTCATTTTCG TTTCAGCTTT 120 TATCA ATG ATT TTT AAT TAT GTG TTT TTT AAG AAC TTT GTA CCA GTT GTT 170 Met Ile Phe Asn Tyr Val Phe Phe Lys Asn Phe Val Pro Val Val 1 5 10 CTA TAC ATT CTC CTT ATA ATA TAT ATT AAC TTA AAT GGC ATG AAT AAT 218 Leu Tyr Ile Leu Leu Ile Ile Tyr Ile Asn Leu Asn Gly Met Asn Asn 25 AAA AAT CAA ATA AAA ACA GAA AAA ATT TAT ATA AAG AAA TTG AAT AGG 266 Lys Asn Gin Ile Lys Thr Glu Lys Ile Tyr Ile Lys Lys Leu Asn Arg 40 fTTG TCA AGG AAA AAT TCG TTA TGT AGT TCT AAA AAT AAA ATA GCA TGC 314 Leu Ser Arg Lys Asn Ser Leu Cys Ser Ser Lys Asn Lys Ile Ala Cys 55 TTG TTC GAT ATA GGA AAT GAT GAT AAT AGA AAT ACG ACA TAT GGC TAT 362 Leu Phe Asp Ile Gly Asn Asp Asp Asn Arg Asn Thr Thr Tyr Gly Tyr 70 AAT GTG AAT GTT AAA AAT GAT GAT ATT AAT TCC TTA CTA AAA AAT AAT 410 Asn Val Asn Val Lys Asn Asp Asp Ile Asn Ser Leu Leu Lys Asn Asn 85 90 TAT AGT AAT AAA TTG TAC ATG GAT AAG AGG AAA AAT ATT AAT AAT GTA 458 Tyr Ser Asn Lys Leu Tyr Met Asp Lys Arg Lys Asn Ile Asn Asn Val 100 105 110 WO 99/52938 PCT/EP99/02463 ATT AGT ACT AAT AAA ATA TCT GGG TCC ATT TCA AAT ATT TGT AGT AGA 506 lie Ser Thr Asn Lys Ile Ser Gly Ser Ile Ser Asn Ile Cys Ser Arg 115 120 125 AAT CAA AAA GAA AAT GAA CAA AAA AGA AAT AAA CAA AGA TGT TTA ACT 554 Asn Gln Lys Glu Asn Glu Gin Lys Arg Asn Lys Gin Arg Cys Leu Thr 130 135 140 CAA TGT CAC ACT TAT AAT ATG TCA CAT GAA CAG GAC AAA CTA GCT AAT 602 Gin Cys His Thr Tyr Asn Met Ser His Glu Gin Asp Lys Leu Ala Asn 145 150 155 GAT AAT AAT AGG AAT AAT AAA AAG AAT TTT AAT TTA TTA TTT ATA AAT 650 Asp Asn Asn Arg Asn Asn Lys Lys Asn Phe Asn Leu Leu Phe Ile Asn 160 165 170 175 TAT TTT AAT TTG AAA CGA ATG AAA AAT TCT CTT CTA AAT AAA GAC AAT 698 7yr Phe Asn Leu Lys Arg Met Lys Asn Ser Leu Leu Asn Lys Asp Asn 180 185 190 TTC TTT TAC TGT AAA GAA AAA AAA TTG TCA TTT CTG CAT AAG GCC TAT 746 Phe Phe Tyr Cys Lys Glu Lys Lys Leu Ser Phe Leu His Lys Ala Tyr 195 200 205 AAA AAA AAA AAT TGC ACT TTT CAA AAT TAT AGT TTA AAA AGA AAA TCT 794 Lys Lys Lys Asn Cys Thr Phe Gin Asn Tyr Ser Leu Lys Arg Lys Ser 210 215 220 AAT CGT GAT TCA CAT AAA TTG TTT TCT GGA GAA TTT GAC GAT TAT ACA 842 Asn Arg Asp Ser His Lys Leu Phe Ser Gly Glu Phe Asp Asp Tyr Thr 225 230 235 AAT AAT AAT GCT TTA TAT GAA TCC GAA AAA AAA GAA TAC ATT ACA CTA 890 WAsn Asn Asn Ala Leu Tyr Glu Ser Glu Lys Lys Glu Tyr Ile Thr Leu 240 245 250 255 AAT AAT AAT AAT AAA AAT AAT AAT AAT AAA AAT AAT GAT AAT AAA AAT 938 Asn Asn Asn Asn Lys Asn Asn Asn Asn Lys Asn Asn Asp Asn Lys Asn 260 265 270 AAT GAT AAT AAT GAT TAT AAT AAT AAT AAT AGT TGT AAT AAT TTA GGA 986 Asn Asp Asn Asn Asp Tyr Asn Asn Asn Asn Ser Cys Asn Asn Leu Gly 275 280 285 GAG AGA TCC AAT CAT TAT GAT AAT TAT GGT GGA GAT AAT AAT AAT CCA 1034 Glu Arg Ser Asn His Tyr Asp Asn Tyr Gly Gly Asp Asn Asn Asn Pro 290 295 300 TGT AAT AAT AAT AAT GAC AAA TAT GAT ATA GGA AAA TAT TTC AAA CAG 1082 Cys Asn Asn Asn Asn Asp Lys Tyr Asp Ile Gly Lys Tyr Phe Lys Gin 305 310 315 WO 99/52938 PCT/EP99/02463 ATT AAT ACC TTT ATT AAT ATT GAT GAA TAT AAA ACT ATA TAT GGT GAT 1130 fie Asn Thr Phe Ile Asn lie Asp Glu Tyr Lys Thr Ile Tyr Gly Asp 320 325 330 335 GAA ATA TAT AAA GAA ATA TAT GAA CTA TAT GTA GAA AGA AAT ATT CCT 1178 Glu Ile Tyr Lys Glu Ile Tyr Glu Leu Tyr Val Glu Arg Asn Ile Pro 340 345 350 GAA TAT TAT GAA CGA AAA TAT TTT TCA GAA GAT ATT AAA AAG AGT GTC 1226 Glu Tyr Tyr Glu Arg Lys Tyr Phe Ser Glu Asp lie Lys Lys Ser Val 355 360 365 CTA TTT GAT ATA GAT AAA TAT AAT GAT GTC GAA TTT GAA AAA GCT ATA 1274 Leu Phe Asp Ile Asp Lys Tyr Asn Asp Val Glu Phe Glu Lys Ala Ile 370 375 380 AAA GAA GAA TTT ATA AAT AAT GGA GTT TAT ATT AAT AAT ATA GAT AAT 1322 Oys Glu Glu Phe Ile Asn Asn Gly Val Tyr Ile Asn Asn Ile Asp Asn 385 390 395 ACA TAT TAT AAA AAA GAA AAT ATT TTA ATA ATG AAA AAG ATA TTA CAT 1370 Thr Tyr Tyr Lys Lys Glu Asn Ile Leu Ile Met Lys Lys Ile Leu His 400 405 410 415 TAT TTC CCA TTA TTA AAA TTA ATT AAT AAT CCA TCA GAT TTA AAA AAG 1418 Tyr Phe Pro Leu Leu Lys Leu Ile Asn Asn Pro Ser Asp Leu Lys Lys 420 425 430 TTA AAA AAA CAA TAT TTA CCT TTA TTA GCA CAT GAA TTA AAA ATA TTT 1466 Leu Lys Lys Gin Tyr Leu Pro Leu Leu Ala His Glu Leu Lys Ile Phe 435 440 445 TTA TTT TTT ATT GTA AAT ATA ACA GGA GGT CAT TTT TCC TCT GTT TTA 1514 OLeu Phe Phe Ile Val Asn Ile Thr Gly Gly His Phe Ser Ser Val Leu W 450 455 460 AGC TCT TTA GAA ATT CAA TTA TTA TTA TTG TAT ATT TTT AAT CAA CCA 1562 Ser Ser Leu Glu lie Gin Leu Leu Leu Leu Tyr Ile Phe Asn Gin Pro 465 470 475 TAT GAT AAT GTT ATA TAT GAT ATA GGA CAT CAA GCA TAT GTA CAT AAG 1610 Tyr Asp Asn Val Ile Tyr Asp Ile Gly His Gin Ala Tyr Val His Lys 480 485 490 495 ATA TTG ACC GGA AGA AAA CTA TTA TTT CTA TCA TTA AGA AAT AAA AAA 1658 lie Leu Thr Gly Arg Lys Leu Leu Phe Leu Ser Leu Arg Asn Lys Lys 500 505 510 GGT ATT AGT GGA TTC CTA AAT ATT TTT GAA AGT ATT TAT GAT AAA TTT 1706 Gly Ile Ser Gly Phe Leu Asn lie Phe Glu Ser lie Tyr Asp Lys Phe 515 520 525 WO 99/52938 PCT/EP99/02463 GGG GCT GGT CAC AGT TCC ACT TCA TTA AGT GCT ATA CAA GGA TAT TAT 1754 Gly Ala Gly His Ser Ser Thr Ser Leu Ser Ala Ile Gin Gly Tyr Tyr 530 535 540 GAA GCC GAG TGG CAA GTG AAG AAT AAA GAA AAA TAT GGA AAT GGA GAT 1802 Glu Ala Glu Trp Gin Val Lys Asn Lys Glu Lys Tyr Gly Asn Gly Asp 545 550 555 ATA GAA ATA AGT GAT AAC GCA AAT GTC ACG AAT AAT GAA AGG ATA TTT 1850 Ile Glu Ile Ser Asp Asn Ala Asn Val Thr Asn Asn Glu Arg Ile Phe 560 565 570 575 CAA AAA GGA ATA CAC AAT GAT AAT AAT ATT AAC AAT AAT ATT AAT AAT 1898 Gin Lys Gly Ile His Asn Asp Asn Asn lie Asn Asn Asn lie Asn Asn 580 585 590 AAT AAT TAT ATC AAT CCT TCA GAT GTG GTA GGA AGA GAA AAT ACG AAT 1946 Asn Asn Tyr lie Asn Pro Ser Asp Val Val Gly Arg Glu Asn Thr Asn W 595 600 605 GTA CCA AAT GTA CGA AAT GAT AAC CAT AAC GTG GAT AAA GTA CAC ATT 1994 Val Pro Asn Val Arg Asn Asp Asn His Asn Val Asp Lys Val His Ile 610 615 620 GCT ATT ATA GGA GAT GGT GGT TTA ACA GGT GGA ATG GCA TTA GAA GCG 2042 Ala lie Ile Gly Asp Gly Gly Leu Thr Gly Gly Met Ala Leu Glu Ala 625 630 635 TTA AAT TAT ATT TCA TTC TTG AAT TCT AAA ATT TTA ATT ATT TAT AAT 2090 Leu Asn Tyr Ile Ser Phe Leu Asn Ser Lys lie Leu Ile Ile Tyr Asn 640 645 650 655 GAT AAC GGA CAA GTT TCT TTA CCA ACA AAT GCC GTA AGT ATA TCA GGT 2138 Asp Asn Gly Gin Val Ser Leu Pro Thr Asn Ala Val Ser Ile Ser Gly 0 660 665 670 AAT AGA CCT ATA GGT TCT ATA TCA GAT CAT TTA CAT TAT TTT GTT TCT 2186 Asn Arg Pro Ile Gly Ser Ile Ser Asp His Leu His Tyr Phe Val Ser 675 680 685 AAT ATA GAA GCA AAT GCT GGT GAT AAT AAA TTA TCG AAA AAT GCA AAA 2234 Asn Ile Glu Ala Asn Ala Gly Asp Asn Lys Leu Ser Lys Asn Ala Lys 690 695 700 GAG AAT AAC ATT TTT GAA AAT TTG AAT TAT GAT TAT ATT GGT GTT GTG 2282 Glu Asn Asn Ile Phe Glu Asn Leu Asn Tyr Asp Tyr lie Gly Val Val 705 710 715 AAT GGT AAT AAT ACA GAA GAG CTC TTT AAA GTA TTA AAT AAT ATA AAA 2330 Asn Gly Asn Asn Thr Glu Glu Leu Phe Lys Val Leu Asn Asn lie Lys 720 725 730 735 WO 99/52938 PCT/EP99/02463 GAA AAT AAA TTA AAA AGA GCT ACT GTT CTT CAT GTA CGT ACA AAA AAA 2378 Glu Asn Lys Leu Lys Arg Ala Thr Val Leu His Val Arg Thr Lys Lys 740 745 750 TCG AAT GAT TTT ATA AAT TCA AAG AGT CCA ATA AGT ATA TTG CAC TCT 2426 Ser Asn Asp Phe lie Asn Ser Lys Ser Pro Ile Ser Ile Leu His Ser 755 760 765 ATA AAG AAA AAT GAG ATT TTC CCT TTC GAT ACC ACT ATA TTA AAT GGA 2474 Ile Lys Lys Asn Glu Ile Phe Pro Phe Asp Thr Thr Ile Leu Asn Gly 770 775 780 AAT ATT CAT AAG GAG AAC AAG ATA GAA GAA GAG AAA AAT GTG TCT TCA 2522 Asn Ile His Lys Glu Asn Lys Ile Glu Glu Glu Lys Asn Val Ser Ser 785 790 795 TCT ACA AAG TAT GAT GTA AAT AAT AAG AAT AAT AAA AAT AAT GAT AAT 2570 0 er Thr Lys Tyr Asp Val Asn Asn Lys Asn Asn Lys Asn Asn Asp Asn o00 805 810 815 AGT GAA ATT ATA AAA TAT GAA GAT ATG TTT TCA AAA GAG ACG TTC ACA 2618 Ser Glu Ile Ile Lys Tyr Glu Asp Met Phe Ser Lys Glu Thr Phe Thr 820 825 830 GAT ATA TAT ACA AAT GAA ATG TTA AAA TAT TTA AAG AAA GAT AGA AAT 2666 Asp Ile Tyr Thr Asn Glu Met Leu Lys Tyr Leu Lys Lys Asp Arg Asn 835 840 845 ATA ATA TTC CTA TCT CCC GCT ATG TTA GGA GGA TCA GGA TTG GTT AAA 2714 Ile Ile Phe Leu Ser Pro Ala Met Leu Gly Gly Ser Gly Leu Val Lys 850 855 860 ATT AGT GAG CGT TAT CCA AAT AAT GTA TAT GAT GTA GGT ATA GCA GAA 2762 Ile Ser Glu Arg Tyr Pro Asn Asn Val Tyr Asp Val Gly Ile Ala Glu 865 870 875 CAA CAT TCT GTA ACT TTC GCA GCA GCT ATG GCA ATG AAT AAG AAA TTA 2810 Gin His Ser Val Thr Phe Ala Ala Ala Met Ala Met Asn Lys Lys Leu 880 885 890 895 AAA ATA CAA TTA TGT ATA TAT TCG ACC TTT TTA CAA AGA GCA TAT GAT 2858 Lys Ile Gin Leu Cys Ile Tyr Ser Thr Phe Leu Gin Arg Ala Tyr Asp 900 905 910 CAA ATT ATA CAT GAT CTT AAT TTA CAA AAT ATA CCT TTA AAG GTT ATA 2906 Gin Ile Ile His Asp Leu Asn Leu Gin Asn Ile Pro Leu Lys Val lie 915 920 925 ATT GGA AGA AGT GGA TTA GTA GGA GAG GAT GGG GCA ACA CAT CAA GGT 2954 Ile Gly Arg Ser Gly Leu Val Gly Glu Asp Gly Ala Thr His Gin Gly 930 935 940 WO 99/52938 PCT/EP99/02463 -ATh TAT GAT TTA TCT TAT CTT GGG ACA CTT AAC AAT GCA TAT ATA ATA 3002 lie Tyr Asp Leu Ser Tyr Leu Gly Thr Leu Asn Asn Ala Tyr Ile Ile 945 950 955 TCT CCA AGT AAT CAA GTT GAT TTG AAA AGA GCT CTT AGG TTT GCT TAT 3050 Ser Pro Ser Asn Gin Val Asp Leu Lys Arg Ala Leu Arg Phe Ala Tyr 960 965 970 975 TTA GAT AAG GAC CAT TCT GTG TAT ATA CGT ATA CCC AGA ATG AAC ATA 3098 Leu Asp Lys Asp His Ser Val Tyr Ile Arg Ile Pro Arg Met Asn Ile 980 985 990 TTA AGT GAT AAG TAC ATG AAA GGA TAT TTG AAC ATT CAT ATG AAA AAT 3146 Leu Ser Asp Lys Tyr Met Lys Gly Tyr Leu Asn Ile His Met Lys Asn 995 1000 1005 GAG AGC AAA AAT ATC GAT GTA AAC GTG GAT ATA AAC GAT GAT GTA GAT 3194 _,liu Ser Lys Asn Ile Asp Val Asn Val Asp Ile Asn Asp Asp Val Asp W 1010 1015 1020 AAA TAT AGT GAA GAA TAT ATG GAC GAT GAT AAT TTT ATA AAA TCG TTT 3242 Lys Tyr Ser Glu Glu Tyr Met Asp Asp Asp Asn Phe Ile Lys Ser Phe 1025 1030 1035 ATT GGA AAA TCT AGA ATT ATT AAA ATG GAT AAT GAA AAT AAT AAT ACA 3290 Ile Gly Lys Ser Arg Ile Ile Lys Met Asp Asn Glu Asn Asn Asn Thr 1040 1045 1050 1055 AAT GAA CAT TAT TCA AGC AGA GGA GAT ACA CAG ACA AAA AAA AAA AAA 3338 Asn Glu His Tyr Ser Ser Arg Gly Asp Thr Gin Thr Lys Lys Lys Lys 1060 1065 1070 GTT TGT ATC TTT AAC ATG GGT AGT ATG CTT TTT AAT GTA ATT AAT GCT 3386 Val Cys Ile Phe Asn Met Gly Ser Met Leu Phe Asn Val Ile Asn Ala 09 1075 1080 1085 ATA AAA GAA ATT GAA AAA GAA CAA TAT ATT TCA CAT AAT TAT TCT TTT 3434 Ile Lys Glu Ile Glu Lys Glu Gin Tyr Ile Ser His Asn Tyr Ser Phe 1090 1095 1100 TCA ATT GTT GAT ATG ATA TTT TTA AAT CCT TTA GAT AAA AAT ATG ATA 3482 Ser Ile Val Asp Met Ile Phe Leu Asn Pro Leu Asp Lys Asn Met Ile 1105 1110 1115 GAT CAT GTA ATA AAA CAA AAT AAA CAT CAA TAT TTA ATT ACT TAT GAA 3530 Asp His Val Ile Lys Gin Asn Lys His Gin Tyr Leu Ile Thr Tyr Glu 1120 1125 1130 1135 GAT AAT ACT ATA GGT GGT TTT TCT ACA CAT TTC AAT AAT TAT TTA ATA 3578 Asp Asn Thr Ile Gly Gly Phe Ser Thr His Phe Asn Asn Tyr Leu Ile 1140 1145 1150 \I WO 99/52938 PCT/EP99/02463 "GAA AAT AAT TAT ATT ACA AAA CAT AAC TTA TAT GTT CAT AAT ATT TAT 3626 Glu Asn Asn Tyr Ile Thr Lys His Asn Leu Tyr Val His Asn Ile Tyr 1155 1160 1165 TTA TCT AAT GAG CCA ATT GAA CAT GCA TCT TTT AAG GAT CAA CAA GAA 3674 Leu Ser Asn Glu Pro lie Glu His Ala Ser Phe Lys Asp Gln Gln Glu 1170 1175 1180 GTC GTC AAA ATG GAT AAA TGT AGT CTT GTC AAT AGA ATT AAA AAT TAT 3722 Val Val Lys Met Asp Lys Cys Ser Leu Val Asn Arg Ile Lys Asn Tyr 1185 1190 1195 CTT AAA .AT AAT CCT ACA TGA TGTAAGATAA ATATATATTT CTAAAATTAT 3773 Leu Lys Asn Asn Pro Thr 1200 1205 L7TTTTTTTTA TACTTTAATG TGTACAATAA AATATATATC TAAATATATT TTATTTGTAC 3833 GCTTTTTTTT TTTTTTTTTT AATTGTTATT TTTGTATAT 3872

Claims (44)

1. A process for obtaining chemically active ingredients which are suitable for treating infectious diseases caused by unicellular or multicellular parasites, wherein proteins which are involved in the 1-deoxy-D-xylulose-5-phosphate metabolic pathway, or similarly acting derivatives thereof, are brought into contact with the active ingredients to be investigated for their activity with respect to parasites, and the active ingredients which inhibit the proteins or their derivatives are selected.

2. The process according to claim 1, wherein the proteins are involved in at least one of the following steps converting glyceraldehyde and pyruvate to 1-deoxy-D-xylulose; converting glyceraldehyde-3-phosphate and pyruvate to form isopentenyldiphosphate; forming converting glyceraldehyde-3-phosphate and pyruvate to form phosphate; converting forming 2-C-methyl-D-erythritol-4-phosphate; converting 1-deoxy-D-xylulose-5-phosphate to 2-C-methyl-D-erythritol-4-phosphate; converting 2-C-methyl-D-erythritol-4-phosphate; converting 2-C-methyl-D-erythritol-4-phosphate to isopentenyldiphosphate.

3. The process according to claim 1 or 2, wherein the active ingredient inhibits the production of enzymes involved or co-factors involved, or promotes the degradation of enzymes involved or co-factors involved in the 1-deoxy-D-xylulose-5-phosphate pathway.

4. The process according to claim 3, wherein the active ingredient inhibits the conversion of the enzyme 1-deoxy-D-xylulose-5-phosphate-synthase or reductoisomerase.

Isolated, purified or recombinant protein from parasites, with or without 1-deoxy-D- activity which is involved in the metabolic pathway and is coded from the DNA-sequence shown in Fig. lb and 2b, or is coded from DNA-sequences which hybridise under stringent conditions with the DNA-sequences shown in Fig. lb or 2b or fragments larger than 200 pairs of bases of these DNA-sequences in the DNA region which codes for the mature protein.

6. Isolated, purified or recombinant protein from parasites, with or without 1-deoxy-D- activity, wherein said protein is involved in the 1-deoxy-D- metabolic pathway and is coded from the DNA-sequence shown in Fig. lb and 2b, or is coded from the DNA sequences which hybridise under stringent conditions with [R:\LIBH]01665.doc:ael 34 the DNA-sequences shown in Fig. lb or 2b or fragments larger than 200 pairs of bases of those DNA-sequences in the DNA region which codes for the mature protein.

7. Isolated, purified or recombinant protein according to claim 5 or 6, which is involved in the 1-deoxy-D-xylulose-5-phosphate metabolic pathway, wherein said proteins can be obtained from the culture supernatants of parasites or from the digested parasites by purification by chromatographic and electrophoretic techniques.

8. Protein according to any one of claims 5 to 7, wherein said protein is the product of a prokaryotic or eukaryotic expression of an exogenic DNA, is coded from the sequences shown in Fig. la, 1b, 2a, or 2b or is coded from DNA sequences which hybridise under stringent conditions with the DNA-sequences shown in Fig. la, 1b, 2a, or 2b or fragments of these DNA sequences in the DNA region which codes for the mature protein, or is coded from DNA sequences which would hybridise under stringent conditions with the sequences defined in

9. Protein according to any one of claims 5 to 8, consisting of the amino acid sequence of Fig. 2a, 2b, 3a, or 3b.

10. Protein according to any one of claims 5 to 9, wherein said protein is 1-deoxy-D- or

11. Nucleic acid which codes a protein according to any one of claims 5 to 10, wherein said nucleic acid is chosen from the group of the DNA sequences shown in Fig. la, 1b, 2a, 2b, or of the complementary DNA sequences, nucleic acid sequences hybridising under stringent 20 conditions with the sequences from or nucleic acid sequences which hybridise with one of .9. the aforementioned sequences under stringent conditions.

12. DNA having a sequence selected from the group consisting of the sequence shown in Fig. la, the sequence shown in Fig. 1b, the sequence shown in Fig. 2a, and the sequence shown in Fig. 2b. 25

13. A recombinant expression vector, containing DNA, which codes a protein according to any one of claims 5 to 10 and expresses the protein-coding DNA in a transformed micro-organism or a transformed eukaryotic cell, or in an animal or plant.

14. A host cell which, with a DNA coding a protein according to any one of claims 5 to is transfected and can produce said protein.

15. The host cell according to claim 14, selected from the group consisting of a prokaryotic host cell, eukaryotic host cell, animals and plants.

16. The host cell according to claim 14 or 15, which is E. coli or a mammalian cell line. R

17. Use of DNA which codes for a protein according to any one of claims 5 to 10 for the transfection of a prokaryotic or eukaryotic organism. [R:\L1BH]01I665.doc:ae1

18. The process according to any one of claims 1 to 4, wherein said protein is obtained from parasites or culture supernatants of parasite cultures by chromatographic and electrophoretic techniques.

19. The process according to any one of claims 1 to 4 or 18, wherein said protein is produced recombinantly by expression of the DNA which codes a protein according to any one of claims 5 to 10 in a suitable host cell and isolation of the protein from the host cell or from the culture supernatant of the host cell.

Use of a protein from the 1-deoxy-D-xylulose-5-phosphate metabolic pathway according to any one of claims 5 to 9 as an antigen or immunogen for producing antibodies which link said protein.

21. Isolated antibodies against a protein from the metabolic pathway according to any one of claims 5 to 10 which can be obtained by in vitro immunisation techniques or by immunising an animal with a protein according to any one of claims to 10 and obtaining the antibodies from the serum or from the spleen cells of the immunised animal.

22. Use of a protein according to any one of claims 5 to 10 for identifying antiparasitically S' acting substances. S*

23. A protein according to any one of claims 5 to 10 when used for identifying an antiparasitically acting substance. 20

24. Use of an antibody according to claim 21 for identifying an antiparasitically acting o.. substance.

An antibody according to claim 21 when used identifying an antiparasitically acting substance.

26. A process for identifying nucleic acids which code a protein according to any one of claims 5 to 10, wherein a sample to be investigated is incubated with a nucleic acid probe selected from the group consisting of the DNA sequence shown in Fig. la and lb, or the sequence complementary thereto, nucleic acids, hybridising under stringent conditions with one of the sequences of wherein the nucleic acid probe is incubated with the nucleic acid of the sample and hybridisation is optionally detected via a further binding partner of the nucleic acid probe.

27. The process according to claim 26, wherein the nucleic acid to be detected is amplified before detection.

28. A test system using a protein according to any one of claims 5 to 10 for identifying an R antiparasitically active substance. [R:\LIBH]0I 123.doc:UG

29. The system according to claim 28, wherein said antiparasitically active substance is 3- (N-formyl-N-hydroxyamino)-propylphosphonate or 3-(N-acetyl-N-hydroxyamino)- propylphosphonate.

Use of 3-(N-formyl-N-hydroxyamino)-propylphosphonate or 3-(N-acetyl-N- s hydroxyamino)-propylphosphonate, for treating diseases caused by parasitic infections.

31. Use of 3-(N-formyl-N-hydroxyamino)-propylphosphonate or 3-(N-acetyl-N- hydroxyamino)-propylphosphonate in the manufacture of a pharmaceutical composition for treating infectious diseases caused by unicellular or multicellular parasites.

32. The use according to claim 30 or 31, wherein said disease is selected from malaria, sleeping sickness and leishmaniosis.

33. The use according to claim 31, wherein the pharmaceutical composition also comprises one or a plurality of constituents from the group consisting of inhibitors of the fat metabolism pathway, cholesterol synthesis or cholesterol absorption.

34. The use according to claim 32, wherein the inhibitor of the fat metabolism is an HMG- CoA-reductase inhibitor or an HMG-CoA-synthase inhibitor.

The use according to claim 34, wherein said inhibitor is selected from the group consisting of Lovastatin, Mevastatin, Compactin, Simvastatin, Pravastatin, Atorvastatin, Fluvastatin and Cerivastatin.

36. A method for the treatment of infectious diseases caused by unicellular or multicellular parasites, comprising administering to a subject in need of said treatment a therapeutically effective amount of a compound selected from 3-(N-formyl-N-hydroxyamino)-propylphosphonate and 3-(N- acetyl-N-hydroxyamino)-propylphosphonate.

37. The method according to claim 36, wherein said disease is selected from malaria, sleeping sickness and leishmaniosis, S.:i 25

38. The method according to claim 36 or 37, wherein the compound is administered together with at least one inhibitor of the fat metabolism pathway, cholesterol synthesis or cholesterol absorption.

39. The method according to claim 38, wherein the inhibitor of the fat metabolism is an HMG-CoA-reductase inhibitor or an HMG-CoA-synthase inhibitor.

40. The method according to claim 39, wherein said inhibitor is selected from the group consisting of Lovastatin, Mevastatin, Compactin, Simvastatin, Pravastatin, Atorvastatin, Fluvastatin and Cerivastatin.

41. A compound selected from 3-(N-formyl-N-hydroxyamino)-propylphosphonate and 3- N-acetyl-N-hydroxyamino)-propylphosphonate when used for the treatment of infectious diseases 35 aused by unicellular or multicellular parasites. [R:\LLBH]01I 123.doc:UG 37

42. The compound when used according to claim 41, wherein said disease is selected from malaria, sleeping sickness and leishmaniosis,

43. The compound when used according to claim 41 or 42, wherein the compound is used together with at least one inhibitor of the fat metabolism pathways, cholesterol synthesis or cholesterol absorption.

44. The compound when used according to claim 43, wherein the inhibitor of the fat metabolism is an HMG-CoA-reductase inhibitor or an HMG-CoA-synthase inhibitor. The compound when used according to claim 44, wherein said inhibitor is selected from the group consisting of Lovastatin, Mevastatin, Compactin, Simvastatin, Pravastatin, Atorvastatin, Fluvastatin and Cerivastatin. Dated 30 April, 2002 Jomaa Hassan Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON 0 .g. [R:\LIBH]01I 123.doc:UG