Methods of diagnosing parasitic infections and of testing drug susceptibility of parasites

The CTD kinase of sporozoan parasites displays a specificity distinct from the analogous activity in mammalian cells. Methods of diagnosing blood borne Plasmodium parasites, and of testing the susceptibility of Plasmodium parasites to anti-malarial drugs, are based on this specificity.

FIELD OF THE INVENTION 
The present invention relates to a protein kinase that hyperphosphorylates 
RNA polymerase II general, and particularly relates to this enzyme in 
parasites. The present invention further relates to substrate analogs and 
inhibitors of this enzyme and to methods of diagnosing and treating 
parasite-caused diseases using such analogs and inhibitors. 
BACKGROUND OF THE INVENTION 
In a large variety of eukaryotic species the largest subunit of nuclear RNA 
polymerase II (RPII) contains a region known as the C-terminal domain 
("CTD"). The CTD of human beings and other mammals such as mice consists 
of 52 repeats of the consensus heptamer Tyr-Ser-Pro-Thr-Ser-Pro-Ser, while 
the CTDs of most lower eukaryotes consist of fewer repeats of the same 
consensus sequence. The CTD of the yeast Saccharomyces cerevisiae, for 
example, contains 26 repeats of this heptamer, the CTD of the fruit fly 
Drosophila contains 45 repeats, and the malarial parasite Plasmodium 
falciparum contains 17 repeats. The repeating heptamers may not match the 
consensus sequence exactly, for example, in Saccharomyces cerevisiae 17 of 
the 26 repeats exactly match the consensus heptamer 
Tyr-Ser-Pro-Thr-Ser-Pro-Ser, while in the CTD of Drosophila, only two of 
the 45 repeats are exact matches. A CTD region is not found in the 
homologous subunits of RNA polymerases I or III, or in the prokaryotic 
.beta.' subunit. 
While the repetitive CTD domain is conserved among a wide range of 
eukaryotic organisms, some eukaryotic RNA polymerase II contains a 
carboxy-terminus extension (CTE) rather than a CTD region. For example, 
the largest subunit of Trypanosoma brucei RNA polymerase II has a 
carboxy-terminus extension (CTE) consisting of 228 amino acids which is 
rich in serine and proline. 
The CTD is essential for viability, as yeast or mouse cells containing RNA 
polymerase II from which all or most of the repeats have been removed do 
not grow. A notable feature of the CTD is that it is subject to 
hyperphosphorylation. A consequence of hyperphosphorylation is that the 
mobility in SDS gels of the largest RNA polymerase II subunit is markedly 
reduced. The mobility-shifted, hyperphosphorylated largest subunit is 
referred to as IIo, whereas the unphosphorylated subunit is referred to as 
IIa. 
SUMMARY OF THE INVENTION 
A first object of the present invention is a method for combatting a 
sporozoan infection in a mammalian subject by administering a sporozoan 
CTD kinase inhibitory binding ligand in an amount effective to decrease 
the number of parasites present in the subject over that which would occur 
without such treatment. 
A further object of the present invention is a method of detecting 
sporozoan parasites in a mammalian subject. A biological sample is 
collected from the subject and contacted with (a) a sporozoan CTD kinase 
substrate equivalent and (b) a phosphate donor; and any 
hyperphosphorylation of the substrate equivalent is detected. 
Hyperphosphorylation indicates the presence of parasites in the sample. 
A further object of the present invention is a method of testing the 
susceptibility of Plasmodium parasites to an anti-malarial drug. A test 
blood sample and a control blood sample are collected from a mammalian 
subject infected with the Plasmodium parasite; the anti-malarial drug in 
question is added to the test sample in an amount effective to eradicate 
susceptible Plasmodium parasites; and a sporozoan CTD kinase substrate 
equivalent and a phosphate donor are added to the samples. Reduced 
hyperphosphorylation in the test blood sample compared to the control 
blood sample indicates susceptibility of the parasite to the anti-malarial 
drug tested. 
A further aspect of the present invention is a method for determining 
whether a host-parasite combination is amenable to anti-parasite treatment 
using CTD kinase inhibitors. The CTD region on the RNA polymerase II of 
the parasite is identified, as is the CTD region on the RNA polymerase II 
of the host. A parasite CTD kinase that specifically phosphorylates said 
parasite CTD region is identified, as is a host CTD kinase that 
specifically phosphorylates the host CTD region. The specificity of the 
kinases is compared to determine whether it is distinct from that of the 
host. If the parasite CTD kinase is distinct from the host CTD kinase, the 
host-parasite pair is suitable to anti-parasite treatment using CTD kinase 
inhibitors directed to the parasite CTD kinase which will inhibit the 
parasite CTD kinase but will not appreciably inhibit the host CTD kinase. 
A further aspect of the present invention is a method for screening for 
anti-parasite compounds effective for anti-parasite therapy in a given 
parasite-host combination, where the parasite CTD kinase has a specificity 
distinct from that of the host. The parasite CTD kinase that specifically 
phosphorylates the parasite CTD region is identified. A combinatorial 
library is then screened for inhibitor molecules of the parasite CTD 
kinase, which do not inhibit host CTD kinase. 
A further aspect of the present invention is a method of diagnosing a 
Plasmodium infection in a mammalian subject. A sample of blood is obtained 
from a mammalian subject and treated to lyse red blood cells and any 
Plasmodium cells present. Kinases are separated from the lysed sample and 
assayed with a known substrate of Plasmodium CTD kinase to determine 
whether the kinases phosphorylate the substrate. Phosphorylation of the 
substrate indicates the presence of Plasmodium organisms in the blood 
sample. 
A further aspect of the present invention is a peptide having an amino acid 
sequence (Tyr-Ser-Pro-Thr-Ser-Pro-Lys).sub.n, 
(Tyr-Ala-Pro-Thr-Ala-Pro-Lys).sub.n, (Tyr-Ser-Pro-Thr-Ser-Pro-Arg).sub.n, 
or (Tyr-Ser-Pro-Thr-Ala-Pro-Arg).sub.n ; where n is from one to one 
hundred, and where the peptide is capable of inhibiting sporozoan CTD 
kinase and incapable of inhibiting mammalian CTD kinase. 
A further aspect of the present invention is a fusion protein comprising a 
peptide having amino acid sequence (Tyr-Ser-Pro-Thr-Ser-pro-Lys).sub.n, 
(Tyr-Ala-Pro-Thr-Ala-Pro-Lys).sub.n, (Tyr-Ser-Pro-Thr-Ser-Pro-Arg).sub.n, 
or (Tyr-Ser-Pro-Thr-Ala-Pro-Arg).sub.n ; where n is from one to one 
hundred. 
The foregoing and other objects and aspects of the present invention are 
explained in detail in the specification set forth below.

DETAILED DESCRIPTION OF THE INVENTION 
Amino acid sequences disclosed herein are presented in the amino to carboxy 
direction, from left to right. The amino and carboxy groups are not 
presented in the sequence. Amino acid residues are represented herein by 
three letter code, in accordance with 37 CFR Section 1,822 and established 
usage. See, e.g., Patent In User Manual, 99-102 (Nov. 1990) (U.S. Patent 
and Trademark Office, Office of the Assistant Commissioner for Patents, 
Washington, D.C. 20231); Genes and Genomes, Singer & Berg (Eds.), 
University Science Books, Mill Valley, Calif, 1991, at p. 60. 
As used herein, "ligand" refers to a molecule that is recognized by a 
particular receptor protein. With reference to the present invention, a 
CTD kinase ligand is a molecule, such as a peptide, that is bound by CTD 
kinase. As used herein, an "inhibitory ligand" or an "inhibitory binding 
ligand" is a ligand which binds to and inhibits the normal activity of the 
receptor protein. "Receptor" refers to a molecule that has an affinity for 
a given ligand. 
As used herein, the term "parasite" refers to an organism that must reside 
in a host organism during at least some portion of its life, in order to 
complete its life cycle. As used herein, "protozoan parasite" refers to 
single-celled parasites, including but not limited to Sporozoa (the class 
of protozoans which includes the genera Plasmodium, Eimeria, Isospora and 
Toxoplasma). The genus Plasmodium includes species which are the causal 
agents of malaria in humans and other mammalian hosts. Malaria in humans 
is caused, for example, by P. falciparum, P. vivax, P. malariae, P. ovale, 
P. tenue, and variants of these species. Other Plasmodium species include 
P. berghei (found primarily in rodents) and P. kochi and P. pitheci (found 
primarily in monkeys and apes). Monkeys and apes infected with Plasmodium 
species have been used experimentally as animal models for human malaria; 
P. berghei allows the use of rodents as animal models. The protozoan 
family Trypanosomatidae (class Zoomastiga, order Kinetoplastida) includes 
the genera Leishmania and Trypanosoma. Pathogenic forms of Trypanosoma 
cause trypanosomiasis in man as well as a number of other diseases in 
domestic animals. T. cruzi causes Chagas' disease in man; dog, cats, rats 
and certain monkeys can also be infected with T. cruzi. 
Multiple forms and stages of a parasite may be present within a host (e.g., 
cysts, gametocytes, trophozooites, etc.), and it will be understood by one 
skilled in the art that the present invention as directed to detection or 
treatment will appertain to those forms of the parasite which require 
active CTD kinase. 
As used herein, CTD refers to the carboxyl-terminal repeat domain of the 
largest subunit of RNA polymerase II as found, for example, in mammals, 
yeast, Drosophila and protozoans. 
A. CTD Kinase 
The CTD region is reported as essential for viability, as yeast or mouse 
cells containing RNA polymerase II from which all or most of the repeats 
have been removed do not grow. Allison et al, Mol. Cell Biol., 8, 321 
(1988); Nonet, Sweetser & Young, Cell, 50, 909 (1987); Bartolomei, Mol. 
Cell. Biol., 8, 330 (1988); Zehring et al, Proc. Natl. Acad. Sci., 85, 
3698 (1988). The hyperphosphorylation of CTD is thought to play an 
important role in initiating transcription and in other aspects of RNA 
polymerase II function (see Weeks et al., Genes & Development 7, 2329-2344 
(December 1993)). 
A protein kinase that hyperphosphorylates the CTD of human RNA polymerase 
II has been identified from the yeast Saccharomyces cerevisiae, and has 
been purified and characterized. Lee and Greenleaf, Proc. Natl. Acad. Sci. 
USA 86, 3624-3628, (1989). The CTD of Saccharomyces cerevisiae RNA 
polymerase II closely resembles that of mammals except that it is shorter 
(26 repeats of the heptamer Tyr-Ser-Pro-Thr-Ser-Pro-Ser (SEQ ID NO:1), 
rather than 52 repeats as in humans). Using bacterially produced 
CTD-containing fusion proteins as substrates, it has been shown that the 
yeast CTD kinase can efficiently hyperphosphorylate human CTD, and 
conversely, a similar kinase activity in human cell extracts 
hyperphosphorylates yeast CTD. Lee and Greenleaf, Proc. Natl. Acad. Sci. 
USA, 86, 3624 (1989). In both cases, a characteristic feature of the CTD 
kinase activity was the marked mobility shift induced in the substrate 
fusion proteins upon phosphorylation. It has also been shown that 
mammalian CTD can functionally replace yeast CTD in living yeast cells 
(see Allison et al., Mol. Cell. Biol., 8, 321-329 (1988)). 
Additional CTD kinases that have been purified are a template-associated 
protein kinase from HeLa cells (Dvir et al., J. Biol. Chem., 268, 10440 
(1993)), CTD kinases KI, KII and KIII from Aspergillis (Stone and 
Reinberg, J. Biol. Chem., 267, 6353 (1992)), kinases CTDK1 and CTDK2 from 
HeLa cells (Payne and Dahmus, J. Biol. Chem., 268, 80 (1993)), and 
cdc2-containing CTD kinases E1 and E2 from mouse (Zhang and Corden, J. 
Biol. Chem., 266, 2290 (1991)). Human general transcription factor IIH is 
reported to phosphorylate the C-terminal domain of RNA polymerase II (Lu 
et al., Nature, 358, 641 (1992), and yeast RNA polymerase II transcription 
factor b appears to be associated with CTD kinase (Gileadi et al, Science, 
257, 1389 (1992). Other CTD kinase activities have been identified in 
extracts from mammalian (Stevens and Maupin, Biochem. Biophys. Res. 
Commun., 159, 508 (1989); Legagneux et al., Eur. J. Biochem., 193, 121 
(1990)) and plant cells (Guilfoyle, Plant Cell, 1, 827 (1989)). 
The gene for the largest, catalytic subunit of the yeast CTD kinase (CTK1) 
has been cloned, sequenced and manipulated. The CTK1 protein was found to 
be a member of the cdc2 kinase family, and its protein kinase-homologous 
domain exhibited approximately 40% identity with known cdc2 proteins, 
including S. cerevisiae CDC28. It has been shown that CTD kinase is 
essential for normal growth of cells; furthermore, in cells with a mutated 
ctk1 gene, the CTD of the Pol II largest subunit is abnormally 
phosphorylated (Lee and Greenleaf, Gene. Expr. 1, 149-167, (1991)). 
The nucleotide and predicted amino acid sequences of the Plasmodium 
falciparum RPII gene, including the CTD, are given in Liet al., Nucleic 
Acids Res. 17, 9621-9636 (1989). The CTD sequences of Plasmodium 
falciparum and Plasmodium berghei are compared in Giesecke et al., 
Biochem. Biophys. Res. Commun. 180, 1350-1355 (1991). The CTD of P. vivax 
has been sequenced and found to be similar to that of P. falciparum (J. M. 
Lee, unpublished data, 1994). The Plasmodium CTD contains a variant of the 
human repeat heptad (Tyr-Ser-Pro-Thr-Ser-Pro-Ser (SEQ ID NO:1)), having a 
Lysine residue in place of the final Serine residue 
(Tyr-Ser-Pro-Thr-Ser-Pro-Lys (SEQ ID NO:2)). Because each repeat contains 
a Lys residue, the Plasmodium CTD is a highly positively charged entity. 
This contrasts to the relatively uncharged nature of yeast, Drosophila and 
mammalian CTDs. 
The RNA polymerase II of certain other protozoan parasites in addition to 
Plasmodium are known or suspected to contain C-terminal repeat domains. 
Toxoplasma species and coccidia (i.e., members of the order Coccidia) such 
as Eimeria and Isospora are taxonomically the closest relatives of 
Plasmodium, and therefore would be expected by those in the art to contain 
CTDs. 
Other parasitic protozoans contain a C-terminal extension (CTE) domain, 
rather than a CTD domain. CTE domains contain a single amino acid sequence 
rather than repeating amino acid sequences, and are rich in 
phosphorylatable residues. The largest subunit of Trypanosoma brucei RNA 
polymerase II, for example, has a carboxy-terminus extension (CTE) 
consisting of 228 amino acids and rich in serine and proline. Evers et 
al., Cell, 56, 585 (1989); Smith et al, Cell, 56, 815 (1989). The CTE of 
Schistosoma mansoni has been sequenced and found to contain a multiplicity 
of phosphorylatable residues (J. M. Lee, unpublished data, 1994). While 
the phosphorylatable area of a repeating domain is more readily apparent 
due to the repetition in sequence, using techniques known in the art the 
phosphorylatable area of a CTE domain and the peptide substrate can be 
determined. For example, once the sequence of a given CTE region is 
determined and an associated CTE kinase identified, a peptide library or 
other combinatorial library could be screened to find peptide substrates 
and inhibitors of the CTE kinase. 
The present invention is based on the identification of the CTD kinase of 
Plasmodium falciparum and the discovery that Plasmodium CTD kinase 
displays a specificity distinct from the analogous activity in human 
cells. The specificity differences between parasite and host CTD kinases 
can be used to design selective peptide or peptide mimetic inhibitors of 
the CTD kinase of parasites such as Plasmodium species and other sporozoan 
CTD kinase. Such inhibitors function as pharmacological inhibitors of CTD 
phosphorylation, and thus as anti-parasitic, and particularly 
anti-malarial, agents. The identification of distinct CTD kinase 
specificity in other parasite-host paris will likewise lead to the design 
of CTD kinase inhibitors directed to the given parasite, for use in 
anti-parasite treatments and in diagnosis. 
B. Inhibitory Analogs and Mimetics 
Analogs of CTD kinase ligands are an aspect of the present invention. As 
used herein, an "analog" is a chemical compound similar in structure to a 
first compound, and having either a similar or opposite physiologic action 
as the first compound. With particular reference to the present invention, 
CTD kinase ligand analogs are those compounds which, while not having the 
amino acid sequences of native CTD ligands, are capable of binding to CTD 
kinase. Such analogs may be peptide or non-peptide analogs, including 
nucleic acid analogs, as described in further detail below. 
The regular spacing of proline residues is highly conserved among CTDs of 
various species. Due to the repetitiveness and the high proline content of 
the CTD, it is predicted that the CTD adopts an unusual conformation. 
Several potential secondary structures have been proposed on the basis of 
modeling studies. See Matsushima, Creutz and Kretsinger, Proteins, 7, 
125-155 (1990). 
In protein molecules which interact with a receptor, the interaction 
between the protein and the receptor must take place at surface-accessible 
sites in a stable three-dimensional molecule. By arranging the critical 
binding site residues in an appropriate conformation, peptides which mimic 
the essential surface features of the present RNA polymerase CTD regions 
may be designed and synthesized in accordance with known techniques. 
Methods for determining peptide three-dimensional structure and analogs 
thereto are known, and are sometimes referred to as "rational drug design 
techniques". See, e.g., U.S. Pat. No. 4,833,092 to Geysen; U.S. Pat. No. 
4,859,765 to Nestor; U.S. Pat. No. 4,853,871 to Pantoliano; U.S. Pat. No. 
4,863,857 to Blalock; (applicants specifically intend that the disclosures 
of all U.S. Patent references cited herein be incorporated by reference 
herein in their entirety). See also Waldrop, Science, 247, 28029 (1990); 
Rossmann, Nature, 333, 392-393 (1988); Weis et al., Nature, 333, 426-431 
(1988); James et al., Science, 260, 1937 (1993) (development of 
benzodiazepine peptidomimetic compounds based on the structure and 
function of tetrapeptide ligands). 
In general, those skilled in the art will appreciate that minor deletions 
or substitutions may be made to the amino acid sequences of peptides of 
the present invention without unduly adversely affecting the activity 
thereof. Thus, peptides containing such deletions or substitutions are a 
further aspect of the present invention. In peptides containing 
substitutions or replacements of amino acids, one or more amino acids of a 
peptide sequence may be replaced by one or more other amino acids wherein 
such replacement does not affect the function of that sequence. Such 
changes can be guided by known similarities between amino acids in 
physical features such as charge density, hydrophobicity/hydrophilicity, 
size and configuration, so that amino acids are substituted with other 
amino acids having essentially the same functional properties. For 
example: Ala may be replaced with Val or Ser; Val may be replaced with 
Ala, Leu, Met, or Ile, preferably Ala or Leu; Leu may be replaced with 
Ala, Val or Ile, preferably Val or Ile; Gly may be replaced with Pro or 
Cys, preferably Pro; Pro may be replaced with Gly, Cys, Ser, or Met, 
preferably Gly, Cys, or Ser; Cys may be replaced with Gly, Pro, Ser, or 
Met, preferably Pro or Met; Met may be replaced with Pro or Cys, 
preferably Cys; His may be replaced with Phe or Gln, preferably Phe; Phe 
may be replaced with His, Tyr, or Trp, preferably His or Tyr; Tyr may be 
replaced with His, Phe or Trp, preferably Phe or Trp; Trp may be replaced 
with Phe or Tyr, preferably Tyr; Asn may be replaced with Gln or Ser, 
preferably Gln; Gln may be replaced with His, Lys, Glu, Asn, or Ser, 
preferably Asn or Ser; Ser may be replaced with Gln, Thr, Pro, Cys or Ala; 
Thr may be replaced with Gln or Ser, preferably Ser; Lys may be replaced 
with Gln or Arg; Arg may be replaced with Lys, Asp or Glu, preferably Lys 
or Asp; Asp may be replaced with Lys, Arg, or Glu, preferably Arg or Glu; 
and Glu may be replaced with Arg or Asp, preferably Asp. Once made, 
changes can be routinely screened to determine their effects on function 
with enzymes. 
Non-peptide mimetics of the peptides of the present invention are also an 
aspect of this invention. Non-protein drug design may be carried out using 
computer graphic modeling to design non-peptide, organic molecules able to 
bind to CTD kinase. See, e.g., Knight, BIO/Technology, 8, 105 (1990). 
Itzstein et al, Nature, 363, 418 (1993) (peptidomimetic inhibitors of 
influenza virus enzyme, sialidase). Itzstein et al modeled the crystal 
structure of the sialidase receptor protein using data from x-ray 
crystallography studies and developed an inhibitor that would attach to 
active sites of the model; the use of nuclear magnetic resonance (NMR) 
data for modeling is also known in the art. See also Lam et al, Science, 
263, 380 (Jan. 1994) regarding the rational design of bioavailable 
nonpeptide cyclic ureas that function as HIV protease inhibitors. Lam et 
al used information from x-ray crystal structure studies of HIV protease 
inhibitor complexes to design nonpeptide inhibitors. 
The modeling of a protein kinase structure using the known structure of 
other kinases is reported by Knighton et al., Science, 258, 130 (1992) 
(smooth muscle myosin light chain kinase catalytic core modeled using 
crystallography data of cyclic AMP-dependent protein kinase catalytic 
subunit and a bound pseudosubstrate inhibitor). See also Marcote et al., 
Mol. Cell. Biol., 13, 5122 (1993) (crystallography data of cyclic AMP 
dependent protein kinase used to model Cdc2 protein kinase); Knighton et 
al., Science, 253, 407 (1991); Knighton et al., Science, 253, 414 (1991); 
DeBondt et al., Nature, 363, 595 (1993) (crystal structure of human CDK2 
kinase determined). 
Analogs may also be developed by generating a library of molecules, 
selecting for those molecules which act as ligands for a specified target, 
and identifying and amplifying the selected ligands. See, e.g., Kohl et 
al., Science, 260, 1934 (1993) (synthesis and screening of tetrapeptides 
for inhibitors of farnesyl protein transferase, to inhibit ras oncoprotein 
dependent cell transformation). Techniques for constructing and screening 
combinatorial libraries of oligomeric biomolecules to identify those that 
specifically bind to a given receptor protein are known. Suitable 
oligomers include peptides, oligonucleotides, carbohydrates, 
nonoligonucleotides (e.g., phosphorothioate oligonucleotides; see Chem. 
and Engineering News, page 20, 7 Feb. 1994) and nonpeptide polymers (see, 
e.g., "peptoids" of Simon et al., Proc. Natl. Acad. Sci. USA, 89, 9367 
(1992)). See also U.S. Pat. No. 5,270,170 to Schatz; Scott and Smith, 
Science, 249, 386-390 (1990); Devlin et al., Science 249, 404-406 (1990); 
Edgington, BIO/Technology, 11, 285 (1993). Peptide libraries may be 
synthesized on solid supports, or expressed on the surface of 
bacteriophage viruses (phage display libraries). Known screening methods 
may be used by those skilled in the art to screen combinatorial libraries 
to identify CTD kinase ligands. Techniques are known in the art for 
screening synthesized molecules to select those with the desired activity, 
and for labelling the members of the library so that selected active 
molecules may be identified. See, e.g., Brenner and Lerner, Proc. Natl. 
Acad. Sci. USA, 89, 5381 (1992) (use of genetic tag to label molecules in 
a combinatorial library); PCT US93/06948 to Berger et al., (use of 
recombinant cell transformed with viral transactivating element to screen 
for potential antiviral molecules able to inhibit initiation of vital 
transcription); Simon et al., Proc. Natl. Acad. Sci. USA, 89, 9367, (1992) 
(generation and screening of "peptoids", oligomeric N-substituted 
glycines, to identify ligands for biological receptors); U.S. Pat. No. 
5,283,173 to Fields et al., (use of genetically altered Saccharomyces 
cerevisiae to screen peptides for interactions). 
As used herein, "combinatorial library" refers to collections of diverse 
oligomeric biomolecules of differing sequence, which can be screened 
simultaneously for activity as a ligand for a particular target. 
Combinatorial libraries may also be referred to as "shape libraries", 
i.e., a population of randomized polymers which are potential ligands. The 
shape of a molecule refers to those features of a molecule that govern its 
interactions with other molecules, including Van der Waals, hydrophobic, 
electrostatic and dynamic. 
Nucleic acid molecules may also act as ligands for receptor proteins. See, 
e.g., Edgington, BIO/ Technology, 11, 285 (1993). U.S. Pat. No. 5,270,163 
to Gold and Tuerk describes a method for identifying nucleic acid ligands 
for a given target molecule by selecting from a library of RNA molecules 
with randomized sequences those molecules that bind specifically to the 
target molecule. A method for the in vitro selection of RNA molecules 
immunologically cross-reactive with a specific peptide is disclosed in 
Tsai, Kenan and Keene, Proc. Natl. Acad. Sci. USA, 89, 8864 (1992) and 
Tsai and Keene, J. Immunology, 150, 1137 (1993). In the method, an 
antiserum raised against a peptide is used to select RNA molecules from a 
library of RNA molecules; selected RNA molecules and the peptide compete 
for antibody binding, indicating that the RNA epitope functions as a 
specific inhibitor of the antibody-antigen interaction. 
C. Proteins and Peptides 
The term CTD kinase "substrate equivalent" as used herein refers to 
proteins or peptides that bind CTD kinase and are phosphorylated in a 
manner similar to native CTD regions. These substrate equivalents may be 
fusion proteins containing the CTD region or may be formed by modifying 
the reactive groups within the substrate molecule's natural amino acid 
sequence or modifying the N-terminal amino and/or the C-terminal carboxyl 
group, and include salts formed with acids and/or bases, particularly 
physiologically acceptable inorganic and organic acids and bases. A 
particular embodiment is a peptide containing repeats of the consensus 
heptamer of the CTD region, or analogs of the consensus repeat which 
retain the ability to bind CTD kinase. Also included are substrate 
molecules with modified carboxyl and/or amino groups on the substrate to 
produce esters or amides, or amino acid protecting groups such as 
N-t-butoxycarbonyl. Preferred modifications are those which provide a more 
stable, active peptide which will be less prone to enzymatic degradation 
in vivo. It will be appreciated by one skilled in the art that a 
"substrate equivalent" may also function as an "inhibitory ligand" as 
described above, as a molecule which binds to CTD kinase will 
competitively inhibit the binding of RNA polymerase CTD regions with the 
CTD kinase receptor. The present invention encompasses peptides and 
analogs which bind to the CTD kinase and are phosphorylated by the enzyme, 
as well as those which bind but are not phosphorylated. 
The proteins and peptides of the invention may be made in accordance with 
techniques known in the art. Using accepted techniques of chemical 
synthesis, the peptide is built up either from the N-terminus or, more 
typically, the C-terminus using either single amino acids or preformed 
peptides containing two or more amino acid residues. Particular techniques 
for synthesizing peptides include (a) classical methods in which peptides 
of increasing size are isolated before each amino acid or preformed 
peptide addition, and (b) solid phase peptide synthesis in which the 
peptide is built up attached to a resin such as a Merrifield resin. In 
these synthetic procedures, groups on the amino acids will generally be in 
protected form using standard protecting groups such as t-butoxycarbonyl. 
If necessary, these protecting groups are cleaved once the synthesis is 
complete. Other modifications may be introduced during or after the 
synthesis of the peptide. 
Peptides and fusion proteins of the present invention may also be produced 
through recombinant DNA procedures. Nucleotide sequences for DNA sequences 
which code for peptides or fusion proteins of the present invention 
(useful as intermediates for making the same) can be determined with any 
table setting forth the genetic code. See, e.g., R. Old and S. Primrose, 
Principles of Gene Manipulation, 346 (3d ed. 1985). 
The peptides of the present invention include peptides consisting of 
repeats of short amino acid sequences. The number of repeats in a peptide 
will depend on its intended use. In general, peptides intended for in vivo 
use will consist of up to about twenty-five repeats of an amino acid 
sequence of about ten or fewer amino acids. Peptides intended for in vitro 
use may consist of any number of repeats, such as up to about 100 repeats 
of an amino acid sequence of about ten or fewer amino acids. 
The production of recombinant DNA, vectors, host cells, and proteins by 
genetic engineering techniques is well known. See, e.g., U.S. Pat. No. 
4,761,371 to Bell et al. at Col. 6 line 3 to Col. 9 line 65; U.S. Pat. No. 
4,877,729 to Clark et al. at Col. 4 line 38 to Col. 7 line 6; U.S. Pat. 
No. 4,912,038 to Schilling at Col. 3 line 26 to Col. 14 line 12; and U.S. 
Pat. No. 4,879,224 to Wallner at Col. 6 line 8 to Col. 8 line 59. 
(Applicants specifically intend that the disclosures of all U.S. Patent 
references cited herein be incorporated by reference herein in their 
entirety). 
DNA sequences encoding desired proteins may be recovered by use of the 
polymerase chain reaction (PCR) procedure and splicing by overlap 
extension (SOE), as is known in the art. See U.S. Pat. Nos. 4,683,195 to 
Mullis et al. and 4,683,202 to Mullis. 
The proteins may be synthesized in host cells transformed with vectors 
containing DNA encoding the proteins. A vector is a replicable DNA 
construct. Vectors are used herein either to amplify DNA encoding the 
protein and/or to express DNA which encodes the protein. An expression 
vector is a replicable DNA construct in which a DNA sequence encoding the 
protein is operably linked to suitable control sequences capable of 
effecting the expression of the protein in a suitable host. The need for 
such control sequences will vary depending upon the host selected and the 
transformation method chosen. Generally, control sequences include a 
transcriptional promoter, an optional operator sequence to control 
transcription, a sequence encoding suitable mRNA ribosomal binding sites, 
and sequences which control the termination of transcription and 
translation. Amplification vectors do not require expression control 
domains. All that is needed is the ability to replicate in a host, usually 
conferred by an origin of replication, and a selection gene to facilitate 
recognition of transformants. 
Vectors useful for practicing the present invention include plasmids, 
viruses (including phage), retroviruses, and integratable DNA fragments 
(i.e., fragments integratable into the host genome by homologous 
recombination). The vector replicates and functions independently of the 
host genome, or may, in some instances, integrate into the genome itself. 
Suitable vectors will contain replicon and control sequences which are 
derived from species compatible with the intended expression host. 
Transformed host cells are cells which have been transformed or 
transfected with the protein vectors constructed using recombinant DNA 
techniques. Transformed host cells ordinarily express the protein, but 
host cells transformed for purposes of cloning or amplifying the protein 
DNA need not express the protein. 
DNA regions are operably linked when they are functionally related to each 
other. For example: a promoter is operably linked to a coding sequence if 
it controls the transcription of the sequence; a ribosome binding site is 
operably linked to a coding sequence if it is positioned so as to permit 
translation. Generally, operably linked means contiguous and, in the case 
of leader sequences, contiguous and in reading phase. 
Suitable host cells include prokaryotes, yeast cells or higher eukaryotic 
cells. Prokaryotes include gram negative or gram positive organisms, for 
example Escherichia coli (E. coli) or Bacilli. Higher eukaryotic cells 
include established cell lines of mammalian origin as described below. 
Exemplary host cells are E. coli W3110 (ATCC 27,325), E. coli B, E. coli 
X1776 (ATCC 31,537), and E. coli 294 (ATCC 31,446). Pseudomonas species, 
Bacillus species, and Serratia marcesans are also suitable. 
A broad variety of suitable microbial vectors are available. Generally, a 
microbial vector will contain an origin of replication recognized by the 
intended host, a promoter which will function in the host and a phenotypic 
selection gene such as a gene encoding proteins conferring antibiotic 
resistance or supplying an autotrophic requirement. Similar constructs 
will be manufactured for other hosts. E. coli is typically transformed 
using pBR322. See Bolivar et al., Gene 2, 95 (1977). pBR322 contains genes 
for ampicillin and tetracycline resistance and thus provides easy means 
for identifying transformed cells. 
Expression vectors should contain a promoter which is recognized by the 
host organism. This generally means a promoter obtained from the intended 
host. Promoters most commonly used in recombinant microbial expression 
vectors include the beta-lactamase (penicillinase) and lactose promoter 
systems (Chang et al., Nature 275, 615 (1978); and Goeddel et al., Nature 
281, 544 (1979)), a tryptophan (trp) promoter system (Goeddel et al., 
Nucleic Acids Res. 8, 4057 (1980) and EPO App. Publ. No. 36,776) and the 
tac promoter (H. De Boer et al., Proc. Natl. Acad. Sci. USA 80, 21 
(1983)). While these are commonly used, other microbial promoters are 
suitable. Details concerning nucleotide sequences of many have been 
published, enabling a skilled worker to operably ligate them to DNA 
encoding the protein in plasmid or viral vectors (Siebenlist et al., Cell 
20, 269 (1980)). The promoter and Shine-Dalgarno sequence (for prokaryotic 
host expression) are operably linked to the DNA encoding the desired 
protein, i.e., they are positioned so as to promote transcription of the 
protein messenger RNA from the DNA. 
Eukaryotic microbes such as yeast cultures may be transformed with suitable 
protein-encoding vectors. See, e.g., U.S. Pat. No. 4,745,057. 
Saccharomyces cerevisiae is the most commonly used among lower eukaryotic 
host microorganisms, although a number of other strains are commonly 
available. Yeast vectors may contain an origin of replication from the 2 
micron yeast plasmid or an autonomously replicating sequence (ARS), a 
promoter, DNA encoding the desired protein, sequences for polyadenylation 
and transcription termination, and a selection gene. An exemplary plasmid 
is YRp7, (Stinchcomb et al., Nature 282, 39 (1979); Kingsman et al., Gene 
7, 141 (1979); Tschemper et al., Gene 10, 157 (1980)). This plasmid 
contains the trpl gene, which provides a selection marker for a mutant 
strain of yeast lacking the ability to grow in tryptophan, for example 
ATCC No. 44076 or PEP4-1 (Jones, Genetics 85, 12 (1977)). The presence of 
the trpl lesion in the yeast host cell genome then provides an effective 
environment for detecting transformation by growth in the absence of 
tryptophan. 
Suitable promoting sequences in yeast vectors include the promoters for 
metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. 
Chem. 255, 2073 (1980) or other glycolytic enzymes (Hess et al., J. Adv. 
Enzyme Reg. 7, 149 (1968); and Holland et al., Biochemistry 17, 4900 
(1978)), such as enolase, glyceral-dehyde-3-phosphate dehydrogenase, 
hexokinase, pyruvate decarboxylase, phosphofructokinase, 
glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, 
triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. 
Suitable vectors and promoters for use in yeast expression are further 
described in R. Hitzeman et al., EPO Publn. No. 73,657. 
Cultures of cells derived from multicellular organisms are a desirable host 
for recombinant protein synthesis. In principal, any higher eukaryotic 
cell culture is workable, whether from vertebrate or invertebrate culture, 
including insect cells. Propagation of such cells in cell culture has 
become a routine procedure. See Tissue Culture, Academic Press, Kruse and 
Patterson, editors (1973). Examples of useful host cell lines are VERO and 
HeLa cells, Chinese hamster ovary (CHO) cell lines, and WI138, BHK, COS-7, 
CV, and MDCK cell lines. Expression vectors for such cells ordinarily 
include (if necessary) an origin of replication, a promoter located 
upstream from the gene to be expressed, along with a ribosome binding 
site, RNA splice site (if intron-containing genomic DNA is used), a 
polyadenylation site, and a transcriptional termination sequence. 
The transcriptional and translational control sequences in expression 
vectors to be used in transforming vertebrate cells are often provided by 
viral sources. For example, commonly used promoters are derived from 
polyoma, Adenovirus 2, and Simian Virus 40 (SV40). See, e.g., U.S. Pat. 
No. 4,599,308. The early and late promoters are useful because both are 
obtained easily from the virus as a fragment which also contains the SV40 
viral origin of replication. See Fiers et al., Nature 273, 113 (1978). 
Further, the protein promoter, control and/or signal sequences, may also 
be used, provided such control sequences are compatible with the host cell 
chosen. 
An origin of replication may be provided either by construction of the 
vector to include an exogenous origin, such as may be derived from SV40 or 
other viral source (e.g. Polyoma, Adenovirus, VSV, or BPV), or may be 
provided by the host cell chromosomal replication mechanism. If the vector 
is integrated into the host cell chromosome, the latter may be sufficient. 
Host cells such as insect cells (e.g., cultured Spodoptera frugiperda 
cells) and expression vectors such as the baculovirus expression vector 
(e.g., vectors derived from Autographa californica MNPV, Trichoplusia ni 
MNPV, Rachiplusia ou MNPV, or Galleria ou MNPV) may be employed in 
carrying out the present invention, as described in U.S. Pat. Nos. 
4,745,051 and 4,879,236 to Smith et al. In general, a baculovirus 
expression vector comprises a baculovirus genome containing the gene to be 
expressed inserted into the polyhedrin gene at a position ranging from the 
polyhedrin transcriptional start signal to the ATG start site and under 
the transcriptional control of a baculovirus polyhedrin promoter. 
Rather than using vectors which contain viral origins of replication, one 
can transform mammalian cells by the method of cotransformation with a 
selectable marker and the chimeric protein DNA. An example of a suitable 
selectable marker is dihydrofolate reductase (DHFR) or thymidine kinase. 
See U.S. Pat. No. 4,399,216. Such markers are proteins, generally enzymes, 
that enable the identification of transformant cells, i.e., cells which 
are competent to take up exogenous DNA. Generally, identification is by 
survival of transformants in culture medium that is toxic, or from which 
the cells cannot obtain critical nutrition without having taken up the 
marker protein. 
D. Subjects 
The molecules of the present invention are useful in inhibiting the CTD 
kinase activity (and hence the RNA polymerase II activity) of parasites, 
and may be used in treating hosts with parasitic infections where the 
activity of the host CTD kinase is distinct from that of the parasite. 
Potential hosts include both mammals and avians such as chickens. 
Particularly preferred as subjects are mammalian hosts with protozoan 
infections, and more particularly, mammalian hosts with sporozoan 
infections such as Plasmodium infections. The present invention may also 
be useful in treating infections by metazoan parasites, where the activity 
of the host CTD kinase is distinct from that of the metazoan parasite. 
Such metazoan parasites include, but are not limited to, parasites of the 
genera Schistosoma, Onchocerca, Loa and Dracunculus. 
The peptides, fusion proteins and other molecules of the present invention 
may be prepared per se or in the form of pharmaceutically acceptable salts 
thereof. Pharmaceutically acceptable salts are those that retain the 
desired biological activity of the parent compound and do not impart 
undesired toxicological effects. For example, acid addition salts of 
acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, 
bisulfate, butyrate, citrate, camphorate, camphorsulfonate, 
cyclopentanepropionate, digluconate, dodecylsulfonate, ethanesulfonate, 
fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, 
hexanoate, hydrochloride, hydrobromide, hydroiodide, 
2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 
2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, 
persulfate, 3-phenylproprionate, picrate, pivalate, propionate, succinate, 
tartrate, thiocyanate, tosylate, and undecanoate. Base salts include 
ammonium salts, alkali metal salts such as sodium and potassium salts, 
alkaline earth metal salts such as calcium and magnesium salts, salts with 
organic bases such as dicyclohexylamine salts, N-methyl-D-glucamine, and 
salts with amino acids such as arginine, lysine, and so forth. Molecules 
of the present invention may also be formulated to increase membrane 
permeability, such as by using lipid vesicle delivery systems, or by the 
incorporation of hydrophobic cleavable protective groups, as is known in 
the art. 
Pharmaceutical formulations of the instant invention comprise the desired 
molecules in a pharmaceutically acceptable carrier, such as sterile, 
pyrogen-free water or sterile pyrogen-free phosphate-buffered saline 
solution. The molecules described above being the active ingredient in 
these compositions, they should be included in an amount effective to 
accomplish the intended treatment. The precise amount to be administered 
to a subject is determined in a routine manner, and will vary depending on 
the subject, condition being treated, severity of the condition, and route 
of administration. The effectiveness of a dosing regimen may be 
ascertained by measures known in the art, including but not limited to 
amelioration of clinical symptoms or laboratory signs, reduction in the 
number of parasites, and a decrease in the rate of increase or the rate of 
reproduction of the parasite. When used as an anti-parasite treatment, the 
molecules of the present invention may be used in conjunction with other 
anti-parasitic treatments. 
For the preparation of these compositions, use can be made of 
pharmaceutical carriers adapted for all conventional forms of 
administration, for example, tablets, capsules, dragees, syrups, 
solutions, suspensions, aerosols and the like. As injection medium, it is 
preferred to use water which contains the additives usual in the case of 
injection solutions, such as stabilizing agents and/or buffers. 
Any suitable route of administration may be employed in carrying out the 
methods of the present invention, including but not limited to oral 
administration, intranasal or inhalation administration, intravenous 
injection, intraarterial injection, intraperitoneal injection, 
intramuscular injection, and subcutaneous injection. 
Subjects to be treated by the methods disclosed herein are preferably 
mammalian subjects, such as human, cat, dog, rodent and horse subjects. 
Thus the present invention has both medical and veterinary applications. 
As used herein, the term "combatting" or "treating" parasitic infections 
means decreasing the numbers of parasites present in the mammalian host 
tissues or blood over that which would occur without such treatment. This 
decrease may occur by killing one or more developmental stages of the 
parasite, suppressing replication of the parasite, or interfering with the 
maturation of the parasite in vivo. See, e.g., Chemotherapy of Malaria, 2d 
Edition, L. J. Bruce-Chwatt et al (eds.), (Chapter Two: Fundamental 
Aspects of Chemotherapy of Malaria), World Health Organization, Geneva 
(1986). 
The present invention also provides a method to detect the presence of 
parasites in blood or other tissues from subjects, where the activities of 
the host and parasite CTD kinases are distinct. In such case, the 
hyperphosphorylation of parasite CTD kinase substrate equivalent in a 
sample of tissue indicates the presence of active CTD kinase of the 
parasite in question, and hence the presence of the parasite. Such a 
method can be used, for example, to test human blood for the presence of 
malarial parasites, or to test rodent blood for the presence of 
experimental P. berghei infections. The tissue type selected as the sample 
will vary depending on the parasite being assayed and the form of the 
parasite being assayed; the selection of tissue samples will be apparent 
to one skilled in the art. For example, blood samples may be assayed for 
the presence of Plasmodium parasites, while muscle tissue samples may be 
assayed for the presence of onchocercid parasites such as Onchocerca 
species. It will likewise be apparent to those skilled in the art that the 
sample may require treatment by known methods to make the parasite CTD 
kinase available to the added substrate; such treatments may include, for 
example, centrifugation, lysing of cells, and fractionation. 
Further, the present invention provides a method of testing the 
susceptibility of Plasmodium parasites to an anti-malarial drug. In 
clinical practice, failure of a subject to respond to drug treatment for 
Plasmodium infection may be due to inadequate dosage or to resistance of 
the parasite to the particular drug. Drug resistance varies among 
Plasmodium species and strains, and is a major factor in limiting the 
success of anti-malarial drug treatment. In vitro field tests for 
assessing drug resistance have been developed. For example, a test and a 
control sample of blood may be collected, and the test sample treated with 
the drug in question (for example, chloroquine). Because the maturation of 
susceptible parasites in vitro is inhibited by drugs such as chloroquine, 
the extent of inhibition caused by the drug is assessed by comparing the 
degree of maturation in the two samples. See Chemotherapy of Malaria, 2d 
Edition, L. J. Bruce-Chwatt et al (eds.), (Chapter Five: Drug Resistance 
in Malaria; Annex 6, In Vitro Tests for Susceptibility of P. falciparum to 
Chloroquine and Mefloquine), World Health Organization, Geneva (1986). 
Using the present invention, an exemplary method comprises collecting a 
test blood sample and a control blood sample from a mammalian subject 
infected with a Plasmodium parasite, contacting the test blood sample with 
the anti-malarial drug in question, contacting both blood samples with a 
sporozoan CTD kinase substrate equivalent and a source of phosphorus, and 
detecting and comparing the hyperphosphorylation of the substrate peptide 
in the blood samples, where significantly decreased hyperphosphorylation 
in the test sample indicates that the parasite is susceptible to the test 
drug. 
Further, the present invention provides a method for determining whether a 
given host-parasite combination is amenable to anti-parasite treatment 
using CTD kinase inhibitors. In this method, the CTD regions on the host 
and the parasite RNA polymerase II are identified, and the CTD kinases 
which act to hyperphosphorylate these CTD regions are also identified. It 
is then determined whether the two CTD kinases have distinct 
specificities, such that parasite CTD kinase inhibitors will inhibit the 
parasite CTD kinase, but will not appreciably inhibit the host CTD kinase. 
Where detection of hyperphosphorylation of CTD regions is employed in the 
present methods, any suitable source of phosphorus may be used, including 
phosphate donors such as adenosine triphosphate (ATP), uridine 
triphosphate (UTP), guanosine triphosphate (GTP), cytidine triphosphate 
(CTP), adenosine diphosphate (ADP), uridine diphosphate (UDP), guanosine 
diphosphate (GDP), and cytidine diphosphate (CDP). To facilitate the 
detection of hyperphosphorylation the phosphorus molecule may be labelled 
by methods known in the art, including the use of radiolabelled 
phosphorus. 
Further, the present invention provides a method for screening for 
anti-parasite compounds effective in anti-parasite treatment for a known 
host-parasite combination, where the CTD kinase of the parasite has a 
specificity distinct from that of the host CTD kinase. The parasite CTD 
kinase is isolated and a combinatorial library is screen for molecules 
which bind to the parasite CTD kinase. The structures of these ligands are 
then determined, and the ligands are screened using assay procedures known 
in the art to determine those which inhibit the parasite CTD kinase but 
which do not appreciably inhibit the host CTD kinase. The combination 
libraries may comprise peptides, oligonucleotides, "peptoids," or other 
non-peptide molecules. 
The following examples are provided to illustrate the present invention, 
and should not be construed as limiting thereof. In these examples, CTD 
means carboxy terminal domain; hCTD indicates a fusion protein carrying 
precise repeats of the human CTD; pCTD indicates a fusion protein carrying 
precise repeats of the plasmodium CTD; GST means 
glutathione-S-transferase; MBP means maltose binding protein; PMSF means 
phenylmethylsulfonyl fluoride; .mu.Ci means microCurie; .mu.l means 
microliter; ml means milliliter; .mu.M means microMolar; mM means milli 
Molar; mg means milligram; PAGE means polyacrylamide gel electrophoresis; 
kBq means kilo Bequerel. 
EXAMPLE 1 
Production of Fusion Proteins 
Fusion protein constructs that carried a CTD region composed of precise 
repeats of either the human-or Plasmodium-type CTD were designed, and are 
hereinafter referred to hCTD and pCTD, respectively. Synthetic 
oligonucleotides encoding the respective CTDs were polymerized and cloned 
into expression vectors based on beta-galactosidase, 
glutathione-S-transferase (GST), or maltose binding protein (MBP), as is 
known in the art. See, e.g., Ruther and Muller-Hill, EMBO J, 2, 1791 
(1983), Smith and Johnson, Gene, 67, 31 (1988); Maina et al, Gene, 74, 365 
(1988). See also Lee and Greenleaf, Proc. Natl. Acad. Sci. USA, 86, 3624 
(1989) (production of yeast CTD fusion proteins); and Lee J. M., A protein 
kinase that phosphorylates the C-terminal repeat domain of the largest 
subunit of RNA polymerase II, Ph.D. Thesis, Department of Biochemistry, 
Duke University, Durham, N.C. (1989). Each was then expressed as a fusion 
protein in Escherichia coli and purified, according to techniques known in 
the art. The resulting fusion proteins were used as a CTD kinase 
substrate. The fusion proteins are termed, for example, MBP-hCTD for 
maltose binding protein/human CTD fusion protein. 
To produce fusion proteins containing precise repeats of a heptamer the 
process described in Lee J. M., Ph.D. Thesis, Department of Biochemistry, 
Duke University, Durham, N.C. (1989), 44-46, was used. For example, to 
produce a beta-galactosidase fusion protein containing the peptide 
(Tyr-Ser-Pro-Thr-Ser-Pro-Ser).sub.n (SEQ ID NO:1), two oligonucleotides 
were synthesized for one repeat of the heptamer sequence and a self 
ligation strategy was employed as described in Lee. High frequency codons 
of E. coli were chosen for each amino acid (Grantham et al., 1981), and 
arranged to both make them in-frame with beta-galactosidase of plasmid 
pUR290 and to introduce a stop codon at the end of the repeats. The two 
oligonucleotides were mixed, kinased by polynucleotide kinase, and ligated 
by standard procedures (see, e.g., Maniatis et al., Molecular Cloning: A 
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N. 
Y. (1982)). Self-ligated DNA was selected for length using a nondenaturing 
polyacrylamide gel. The ends of DNA were made flush by filling with Klenow 
enzyme. The plasmid pUR290 was cut with BamHI, filled-in with Klenow 
enzyme, and the two DNAs were mixed and ligated. After transformation into 
E. coli, colonies were screened for fusion protein production. The fusion 
protein (betaGal-hCTD) was purified as described in Lee (Ph.D. Thesis, 
Department of Biochemistry, Duke University, Durham, N.C. (1989), chapter 
II). To construct the corresponding MBP-hCTD fusion protein, the DNA 
fragment encoding the repeats was subcloned (in frame with MBP) into 
plasmid pMALcR1 (New England Biolabs pMAL system; see also Maina et al., 
Gene, 74, 365 (1988)). This and other MBP fusion proteins were purified 
using an amylose column (New England Biolabs). 
Fusion proteins with other repeat sequences (e.g., SEQ ID NO:2, SEQ ID 
NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7) were made 
similarly, using either the pMAL system for MBP fusions or the pGEX system 
for GST fusions (Pharmacia; and see Smith and Johnson, Gene, 67, 31 
(1988)). 
EXAMPLE 2 
Assay of CTD Kinase Activity 
Assay conditions were patterned after those described in Lee and Greenleaf, 
Proc. Natl. Acad. Sci. USA 86, 3624-3628 (1989), and Lee and Greenleaf, 
Gene. Expr. 1, 149-167, (1991). Analysis was by polyacrylamide gel 
electrophoresis and autoradiography, or by polyacrylamide gel 
electrophoresis and phosphorimager analysis. 
Standard reaction mixtures (20 .mu.1) contained fusion protein (final 
concentration of 0.15mg/ml for intact protein), Tris-HCl (25mM, pH 7.8), 
MgCl.sub.2 (10 mM), NaF (5 mM), PMSF (1 mM), and [.gamma.-.sup.32 P]ATP 
(300 .mu.M, 1-3 .mu.Ci; 1 .mu.Ci=37 kBq). After incubation for 15 minutes 
at room temperature, reactions were terminated by the addition of 
NaDodSO.sub.4 sample buffer. Phosphorylated products were analyzed by 
NaDodSO.sub.4 /6% PAGE followed by autoradiography. 
EXAMPLE 3 
Plasmodium CTD Kinase Activity 
Plasmodium parasites were grown using the method of Trager and Jensen 
(Cultivation of erythrocytic and exoerythrocytic stages of Plasmodia, In 
Malaria, vol. 2, J. P. Kreier (Ed.), Academic Press, New York (1980)). 
Parasites were homogenized and separated into nuclear and cytoplasmic 
fractions. See Grall et al., Exp. Parasitology, 75, 10 (1992) (parasite 
isolation by percoll enrichment); Price et al., J. Biol. Chem., 262, 3244 
(1987) (preparation of nuclear extract); and Lee and Greenleaf, Proc. 
Natl. Acad. Sci. USA, 86, 3624 (1989) (purification of yeast kinase). 
Assays were conducted as described in Example 2, above. 
Results are shown in FIG. 1, where lanes 1 and 2 contain cytoplasmic 
fraction, lanes 3 and 4 contain nuclear fractions; + indicates the 
presence of MBP-pCTD fusion protein (lanes 2 and 4) and - indicates the 
absence of the MBP-pCTD fusion protein (lanes 1 and 3). The nuclear 
extract contained a CTD kinase activity that generated a 
hyperphosphorylated, mobility-shifted MBP-pCTD fusion protein (FIG. 1, 
lane 4 at arrowhead); this is the kinase activity of interest. Activities 
that phosphorylate the fusion protein without causing a mobility shift are 
found in both the cytoplasmic and nuclear fractions (FIG. 1, lanes 2 and 4 
at arrow). These activities may phosphorylate the MBP portion of the 
fusion protein or the CTD portion, but apparently at a low level. The 
intensity of the unshifted band is potentially misleading in the 
autoradiogram of FIG. 1 because only a small fraction of the fusion 
protein is shifted in mobility (stained gel, not shown); thus the .sup.32 
P/protein ratio is much higher for the shifted band. 
EXAMPLE 4 
Plasmodium CTD Kinase Activity 
The specificity of the Plasmodium CTD kinase was compared with that of a 
corresponding activity found in human (HeLa) cells. A Plasmodium extract 
was prepared from infected human blood cells (see Mol. Biochem. 
Parasitolog, 50, 17, 1992; see also Blood, 74, 471). This Plasmodium 
extract was thus contaminated with human proteins. The crude extract was 
partially fractionated by processing it through a phosphocellulose (P11, 
Whatman) column. Each fraction was assayed for activity and active 
fractions were pooled and applied to a DEAE-cellulose (DE52, Whatman) 
column. The enzyme was recovered and applied to a Mono S (FPLC, Pharmacia) 
column for further purification. 
The purified extract was incubated with either the MBP-hCTD or the MBP-pCTD 
substrate as described in Example 2, above. Results are shown in FIG. 2, 
where lanes 1-3 contain Plasmodium extract, lanes 4-6 contain HeLa 
extract; lanes 1 and 4 had no fusion protein added, lanes 2 and 5 had 
MBP-hCTD added, and lanes 3 and 6 had MBP-pCTD added. The Plasmodium 
extract hyperphosphorylated the -pCTD substrate and generated a 
mobility-shifted product (FIG. 2, lane 3, at arrowhead). In contrast, the 
HeLa extract did not generate a mobility-shifted -pCTD product (FIG. 2, 
lane 6), although it did phosphorylate the fusion protein without causing 
a mobility shift (FIG. 2, arrow, "MBP-pCTD"). The HeLa extract contained, 
as expected, an activity that efficiently phosphorylated and shifted the 
MBP-hCTD substrate (FIG. 2, lane 5, upper arrowhead); this was the 
counterpart of the parasite activity of interest. 
In experiments conducted essentially as described above, purified yeast 
enzyme also was active in mobility shifting the human-type CTD substrate, 
whereas no mobility shifting activity was detected toward the Plasmodium 
-type CTD (data not shown). The Plasmodium CTD did not substitute for the 
yeast CTD in vivo (J. Lee, unpublished data, data not shown). 
In the Plasmodium extract used to produce FIG. 2, an activity was found 
that phosphorylated the --hCTD substrate and shifted its mobility (FIG. 2, 
lane 2, indicated by upper arrowhead on right margin). Because the 
experimental preparation used was significantly contaminated with human 
constituents, however, it was inferred that the mobility shift seen for 
the human-type CTD fusion protein in this experiment was due to 
contaminating human CTD kinase. This inference is supported by the next 
experiment. 
EXAMPLE 5 
Specificity of CTD Kinase Activity 
The specificity of the CTD kinase activity was tested using a very pure 
sample of parasites as the starting material for extract preparation. See 
Choi & Mikkelson, Exp. Parasitology, 73, 93-100 (1991) for discussion of 
parasite purification; see Price, J. Biol. Chem. for details of extract 
preparation. CTD kinase was extracted as described in Example 3, above. 
Using this extract it was found that the Plasmodium enzyme has a substrate 
specificity distinct from that of the human enzyme as shown in FIG. 3 
(where lanes 1-3 contained Plasmodium extract; 4-6 lanes contained HeLa 
extract; lanes 1 and 4 contained no fusion protein; lanes 2 and 5 
contained GST-hCTD; lanes 3 and 6 contained GST-pCTD). The parasite CTD 
kinase activity mobility shifted a Plasmodium-type CTD substrate (FIG. 3, 
lane 3, arrowhead; unshifted position indicated by "GST-pCTD" at right 
margin), but did not phosphorylate a human-type substrate (FIG. 3, 
GST-hCTD, lane 2). A complementary specificity was displayed by HeLa 
extract, which phosphorylated and shifted the GST-hCTD fusion protein 
(FIG. 3, lane 5; arrowhead on right margin) but did not shift the GST-pCTD 
fusion protein (lane 6). 
The above results indicate that the Plasmodium parasite and its human host 
contain distinct CTD kinases with selectivity for hyperphosphorylating the 
homologous CTD. By analogy with results demonstrating the essential nature 
of the yeast CTD kinase, it can be inferred that the Plasmodium CTD kinase 
is essential for normal growth of the parasite. Because the parasite 
enzyme has a specificity distinct from that of the human enzyme, the 
Plasmodium CTD kinase represents a pharmacological target in mammalian 
hosts. 
EXAMPLE 6 
Plasmodium CTD Kinase Inhibitors 
Several fusion proteins with variant repeat domains as potential inhibitors 
of plasmodium CTD kinase were prepared and assayed. Positions in the 
repeats are referred to in accordance with sequence, numbered as follows: 
EQU Tyr.sub.1 Ser.sub.2 Pro.sub.3 Thr.sub.4 Ser.sub.5 Pro.sub.6 Ser.sub.7 (SEQ 
ID NO:1) 
As the difference in human CTD (Ser.sub.7) and Plasmodium CTD (Lys.sub.7), 
plays a role in determining Plasmodium CTD kinase specificity, derivatives 
based on the Plasmodium sequence Tyr-Ser-Pro-Thr-Ser-Pro-Lys (SEQ ID NO:2) 
were prepared and tested. The four repeat sequence analogs tested are 
shown in TABLE 1. 
TABLE 1 
______________________________________ 
Consensus: 
pCTD (Tyr--Ser--Pro--Thr--Ser--Pro--Lys)n 
(SEQ ID NO: 2) 
______________________________________ 
Analogs: 
Name Sequence 
______________________________________ 
A2 (Tyr--Ala--Pro--Thr--Ser--Pro--Lys).sub.13 
(SEQ ID 
NO: 3) 
A5 (Tyr--Ser--Pro--Thr--Ala--Pro--Lys).sub.13 
(SEQ ID 
NO: 4) 
A2A5 (Tyr--Ala--Pro--Thr--Ala--Pro--Lys).sub.13 
(SEQ ID 
NO: 5) 
R7 (Tyr--Ser--Pro--Thr--Ser--Pro--Arg).sub.13 
(SEQ ID 
NO: 6) 
______________________________________ 
Each analog was generated as a GST fusion protein with the repeats attached 
to the C-terminus of GST (as is known in the art) and were thus known as 
"GST-R7", etc. 
Results for the GST-A5.sub.13 analog and the GST-R7.sub.13 analog are shown 
in FIG. 4 (where lanes 1-5 contained varying amounts of GST-A5 and lanes 
6-8 contained varying amounts of GST-R7; lane 1 contained no fusion 
protein substrate and lanes 2-8 contained MBP-pCTD). The Plasmodium 
nuclear extract phosphorylated and mobility-shifted the MBP-pCTD fusion 
protein substrate as expected (FIG. 4, lane 2; arrow=unshifted, 
arrowhead=shifted). Little effect on substrate phosphorylation was seen 
when an increasing amount of the GST-A5.sub.13 analog was added to a set 
of reactions with a constant amount of substrate (FIG. 4, lanes 3-5). In 
contrast, adding increasing amounts of the GST-R7.sub.13 analog reduced 
the amount of mobility-shifted product (FIG. 4, lanes 6-8, arrowhead); at 
an equiweight ratio of GST-R7.sub.13 analog-to-substrate there was 
significant inhibition of the Plasmodium CTD kinase activity responsible 
for generating the shifted band (FIG. 4, lane 8). 
In other experiments it was found that the GST-R7.sub.13 analog did not 
similarly inhibit the corresponding human CTD kinase activity (data not 
shown). 
Additional analogs listed in Table 1 (GST-A2.sub.13 and GST-A2A5.sub.13) 
were similarly tested. The GST-A2A5.sub.13 analog displayed inhibitory 
activity at very high concentrations; a ratio of approximately 10:1 
analog-to-substrate was required before inhibition was noted. These 
results indicated that changing Lys.sub.7 to Arg.sub.7 altered the 
affinity for the enzyme such that the analog functioned as a competitive 
inhibitor of the Plasmodium, but not the human, CTD kinase. 
Further, these experiments indicated that it is the Ser at position 5 that 
is phosphorylated, since the GST-A5.sub.13 analog was not labeled (FIG. 4, 
lanes 3-5) whereas the GST-R7.sub.13 analog was labeled (FIG. 4, the major 
band in lanes 6,7 & 8 marked R7). Thus the R7 analog acted as an inhibitor 
but was also a substrate, as it contains serine residues and is 
phosphorylated. A2A5 is slightly inhibitory. Replacing lysine (L7) with 
arginine (R7) was found to increase the peptide affinity for the enzyme, 
while replacing the serine at the fifth position with alanine (A5) 
prevented phosphorylation. 
These data indicate that substrate analog GST-R7.sub.13 inhibits the 
Plasmodium CTD kinase but not the human CTD kinase. 
EXAMPLE 7 
Additional Plasmodium CTD Kinase Inhibitor 
The analog A5R7 is prepared for use as a Plasmodium CTD kinase inhibitor. 
Lys.sub.7 is substituted for Arg.sub.7 to increase the peptide affinity 
for the enzyme, as was noted with the use of GST-R7.sub.13 analog (Example 
6). The results of Example 6 also indicated that the Ser at position 5 is 
phosphorylated, and therefore alanine is substituted for Ser.sub.5 to 
prevent phosphorylation. The resulting A5R7 peptide 
(Tyr-Ser-Pro-Thr-Ala-Pro-Arg; SEQ ID NO:7) acts as an inhibitory peptide 
for Plasmodium CTD kinase, and is not phosphorylated. 
EXAMPLE 8 
Synthetic Peptide Inhibitors of Plasmodium CTD Kinase 
As noted above, a fusion protein containing repeats of the Plasmodium CTD 
sequence Tyr-Ser-Pro-Thr-Ser-Pro-Lys (SEQ ID NO:2) is a substrate for the 
Plasmodium enzyme. Synthetic peptide variants of this inhibitor will also 
function as inhibitors, in parallel with the above results for fusion 
proteins. In view of studies on inhibitors of other protein kinases (see 
e.g., Knighton et al., Science 253, 414-420 (1991) and references 
therein), it may be necessary to prepare and screen an extensive set of 
sequence variants and test the variants for inhibitory activity, using 
techniques known in the art. 
A set of variant peptides are synthesized in accordance with techniques 
known in the art, such as chemical synthesis, or through recombinant DNA 
procedures, as discussed above. Peptides are then characterized as to 
inhibition of Plasmodium CTD kinase using, for example, assays as 
described above. Results of these tests guide the design of the next set 
of peptides. Repeats of this iterative process lead to the design of 
effective inhibitors. 
EXAMPLE 9 
Peptide Mimetics as Inhibitors of Plasmodium CTD Kinase 
Non-peptide mimetics are synthesized based on the knowledge of effective 
peptide inhibitors of protozoan CTD kinase. The results and information 
generated during the process of identifying peptide inhibitors is used to 
guide the synthesis of peptide mimetics. Non-protein drug design may be 
carried out using computer graphic modeling. The crystal structure of the 
CTD kinase is modeled using data from x-ray crystallography studies of 
other protein kinases or nuclear magnetic resonance (NMR) imaging. Two 
protein kinases whose crystal structures are known are the catalytic 
subunit of cAMP-dependent protein kinase (see Knighton et al, Science, 
253, 407 (1991) and the cyclin-dependent kinase 2 (CDK2) (see De Bondt et 
al., Nature, 363, 595 (1993). Modeling of protein kinase structure based 
on the known structure of other kinases by homology is discussed in 
Knighton et al., Science, 258, 130 (1992) and Marcote et al., Mol. Cell. 
Biol., 13, 5122 (1993). The structures of related proteins are used as a 
template in computer modeling of CTK1. An inhibitor able to attach to the 
active sites of the model is then developed and assayed as above. 
The foregoing is illustrative of the present invention and is not to be 
construed as limiting thereof. The invention is defined by the following 
claims, with equivalents of the claims to be included therein. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 6 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
TyrSerProThrSerProSer 
15 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
TyrSerProThrSerProLys 
15 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
TyrAlaProThrSerProLys 
15 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
TyrSerProThrAlaProLys 
15 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
TyrAlaProThrAlaProLys 
15 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
TyrSerProThrSerProArg 
15 
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