Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Protozoa – media therefor
Reexamination Certificate
2000-03-08
2003-04-29
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Protozoa, media therefor
C536S023200, C435S194000, C435S320100, C435S419000, C435S252300, C435S254110, C435S258400
Reexamination Certificate
active
06555358
ABSTRACT:
SUMMARY OF THE INVENTION
The present invention relates to a novel protein kinase which may be used as a chemotherapeutic target for antiprotozoal agents. The invention further concerns a method for identifying potential antiprotozoal agents using the novel kinase, as well as a method for treating protozoal infections using a substance that inhibits the action of said kinase.
BACKGROUND OF THE INVENTION
Parasitic protozoa are responsible for a wide variety of infections in man and animals. Many of the diseases are life threatening to the host, and in animal husbandry, can cause considerable economic loss. For example, malaria remains a significant health threat to humans despite massive international attempts to eradicate the disease; trypanosomiasis such as Chagas disease caused by
Trypanosoma cruzi
and African sleeping sickness caused by
T. brucei
are not uncommon in South America and Africa, respectively; and opportunistic infections in immuno-compromised hosts caused by
Pneuniocystis carinii, Toxoplasma gondii,
Cryptosporidium sp. are becoming increasingly significant in the developed countries.
Coccidiosis, a widespread disease of domesticated animals, is caused by protozoal infection. In the poultry industry, coccidiosis is responsible for high levels of morbidity and mortality in the bird population and may result in extreme economic losses. The infectious agents are protozoa of the genus Eimeria. Some of the most significant avian Eimeria species include
E. tenella, E. acervulina, E. necatrix, E. brunetti
and
E. maxima.
In some protozoal diseases, such as Chagas disease, there is no satisfactory treatment; in others, drug-resistant strains of the protozoa may develop. Accordingly, there exists a continued need to identify new and effective anti-protozoal drugs. However, antiparasitic drug discovery has been, for the most part, a random and laborious process through biological screening of natural products and synthetic compounds against a panel of parasites. This process can be greatly facilitated and made more specific if a biochemical target of antiprotozoal drugs can be identified, and incorporated into the screening process.
cGMP dependent protein kinases (PKG) catalyze the phosphorylation of specific protein substrates. In the absence of cGMP the activity of these enzymes is very low. In mammalian cells there are two types of PKG, a soluble (PKG 1) and a membrane bound form (PKG2). Multiple splice variants of the soluble protein have been identified. PKGs are known to control many cellular processes in higher animals. Mammalian PKG1 is most abundant in smooth muscle, platelets and cerebellum. Targeted disruption of PKG1 in mice generated phenotypes clearly associated with smooth muscle, namely severe intestinal and vascular dysfunctions. PKG2 expression is highest in the small intestine, several regions of the brain (particularly the hypothalamus) and lung. Transgenic mice lacking PKG2 display a dwarfed phenotype caused by defects in ossification at the growth plates and also have intestinal secretion dysfunctions. PKGs have also been identified in Dictyostelium, Paramecium, Tetrahymena and Ascaris.
DETAILED DESCRIPTION OF THE INVENTION
In one aspect the present invention provides a novel protozoal cGMP dependent protein kinase (PKG) and a polynucleotide sequence encoding the PKG polypeptide. In one embodiment, the invention provides a PKG purified fraction of
Eimeria tenella
consisting essentially of the amino acid sequence of SEQ ID NO: 11, and the polynucleotide sequence encoding the polypeptide of SEQ ID NO: 11. More specifically, the polynucleotide sequences is as set forth in SEQ ID NO: 10. In another embodiment, the invention provides a substantially pure PKG of
Toxoplasma gondii
consisting essentially of the amino acid sequence of SEQ ID NO: 13, and the polynucleotide sequence encoding the polypeptide of SEQ ID NO: 13. More specifically, the polynucleotide sequences is as set forth in SEQ ID NO: 12.
In another aspect the present invention provides a method for identifying compounds having antiprotozoal activity comprising:
(a) contacting protozoal PKG with, (i) a known amount of a labeled compound that interacts with a PKG and, (ii) a known dilution of a test compound or a natural product extract; and
(b) quantitating the percent inhibition of interaction of said labeled compound induced by said test compound.
In another aspect the present invention provides a method for identifying compounds having antiprotozoal activity comprising:
(a) contacting, an intact host or protozoal cell with a test compound or a natural product extract;
(b) disrupting said cell to obtain a biochemical fraction possessing PKG catalytic activity; and
(c) determining the level of PKG activity in said biochemical fraction.
The methods of the invention provides a facile and specific assay to screen compounds as potential antiprotozoal drugs.
Polypeptides
The novel PKG of the present invention is of protozoal origin; in particular the enzyme is present in, but not restricted to, protozoa of the apicomplexan family, and more specifically in Eimeria sp. The native
Eimeria tenella
PKG of the present invention is a protein of about 120 kDa, and having about 1,003 amino acids. The native PKG from
Toxoplasma gondii
is approximately 115 kDa and having about 994 amino acids. The PKG of the present invention includes a crude extract of the soluble protein, a PKG purified fraction isolated from a protozoan parasite (native enzyme), affinity purified native PKG (purified from a soluble extract using cGMP, substrate based peptides, inhibitor based molecules or antibodies) as well as a PKG produced by recombinant DNA technology (recombinant expressed enzyme). The term “PKG purified fraction” as used herein, refers to a PKG polypeptide which is free of most other proteins, lipids, carbohydrates, nucleic acids, or other materials with which it is naturally associated. One skilled in the art can purify PKG using standard techniques for protein purification. The PKG purified fraction will yield a major band on a reducing polyacrylamide gel. The sequence of the polypeptide can be determined by amino acid sequencing.
The protozoal PKGs of the present invention include a polypeptide of SEQ ID NO: 11, and a polypeptide of SEQ ID NO:13, as well as functional polypeptides and fragments thereof. As used herein, the term “functional polypeptides and fragments” refers to a polypeptide which possesses PKG activity. Minor modifications of the PKG primary amino acid sequence may result in proteins which have substantially equivalent activity as compared to the PKG polypeptide described herein. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. All of the polypeptides produced by these modifications are included herein as long as the enzymatic activity of PKG is present. Further, deletion of one or more amino acids can also result in a modification of the structure of the resultant molecule without significantly altering its kinase activity. This can lead to the development of a smaller active molecule which may have broader utility. For example, it is possible to remove amino or carboxyl terminal amino acids which may not be required for kinase activity. Smaller peptides containing the biological activity of PKG are included in the invention.
The PKG polypeptide of the invention also includes conservative variations of the polypeptide sequence which do not substantially alter the biological activity of the protein. The term “conservative variation” as used herein denotes the replacement of an amino acid residue by another, biologically similar residue. Examples of conservative variations include the substitution of one hydrophobic residue such as isoleucine, valine or leucine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like. The term “conservative variation” also includes the use of a substituted amino acid in place
Donald Robert
Gurnett Anne
Harris Georgianna
Liberator Paul A.
Rattray Sandra J.
Giesser Joanne M.
Kohli Vineet
Prouty Rebecca E.
Steadman David J.
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