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Title:  Pharmacological targeting of mRNA cap formation for treatment of parasitic infections

United States Patent:  6,844,167

Issued:  January 18, 2005

Inventors:  Shuman; Stewart (504 E. 63rd St., Apt. 9R, New York, NY 10021); Ho; Chong Kiong (310 E. 66th St., Apt. 2A, New York, NY 10021)

Appl. No.:  167831

Filed:  June 12, 2002

Abstract

This invention provides the genes encoding the RNA triphosphatase and RNA guanylyltransferase of the malaria parasite Plasmodium falciparum and the catalytically active recombinant RNA triphosphatase and RNA guanylyltransferase enzymes. These enzymes form the basis of activity inhibition assays to identify molecules that specifically target the formation of the mRNA 5' cap in unicellular eukaryotic parasites.

Description of the Invention

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of biochemical pharmacology and drug discovery. More specifically, the present invention relates to the novel mRNA capping enzymes Pgt1 and Prt1 from Plasmodium falciparum, the agent of malaria, and methods of screening for antimalarial and antiprotozoal compounds that inhibit mRNA cap formation.

2. Description of the Related Art

Malaria extracts a prodigious toll each year in human morbidity (400 million new cases) and mortality (1 million deaths). The malaria parasite is transmitted when humans are bitten by the Anopheles mosquito. Of the four species of Plasmodium parasites that cause human malaria--Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, and Plasmodium falciparum--it is P. falciparum that is principally responsible for fulminant disease and death. Malaria treatment and prevention strategies have been steadily undermined by the spreading resistance of the Plasmodium pathogen to erstwhile effective drugs and of the mosquito vector to insecticides [1]. Thus, there is an acute need for new malaria therapies.

It is anticipated that the Plasmodium falciparum genome project [2] will uncover novel targets for therapy and immunization. The most promising drug targets will be those gene products or metabolic pathways that are essential for all stages of the parasite life cycle, but either absent or fundamentally different in the human host and the arthropod vector. Such targets can be identified either by whole-genome comparisons or by directed analyses of specific cellular transactions. In those instances where Plasmodium differs from metazoans, comparisons to other unicellular organisms may provide insights into eukaryotic phylogeny.

Processing of eukaryotic mRNA in vivo is coordinated temporally and physically with transcription. The earliest event is the modification of the 5' terminus of the nascent transcript to form the cap structure m7GpppN. The cap is formed by three enzymatic reactions: (i) the 5' triphosphate end of the nascent RNA is hydrolyzed to a diphosphate by RNA 5' triphosphatase; (ii) the diphosphate end is capped with GMP by GTP:RNA guanylyltransferase; and (iii) the GpppN cap is methylated by AdoMet:RNA (guanine-N7) methyltransferase [3].

RNA capping is essential for cell growth. Mutations of the triphosphatase, guanylyltransferase, or methyltransferase components of the yeast capping apparatus that abrogate catalytic activity are lethal in vivo. Genetic and biochemical experiments highlight roles for the cap in protecting mRNA from untimely degradation by cellular 5' exonucleases and in recruiting the mRNA to the ribosome during translation initiation.

The physical and functional organizations of the capping apparatus differ in significant respects in metazoans, fungi, and viruses. Mammals and other metazoa encode a two-component capping system consisting of a bifunctional triphosphatase-guanylyltransferase polypeptide and a separate methyltransferase polypeptide. Fungi encode a three-component system consisting of separate triphosphatase, guanylyltransferase, and methyltransferase gene products. Viral capping systems are quite variable in their organization; poxviruses encode a single polypeptide containing all three active sites, whereas phycodnaviruses encode a yeast-like capping apparatus in which the triphosphatase and guanylyltransferase enzymes are encoded separately [4].

The guanylyltransferase and methyltransferase components of the capping apparatus are mechanistically conserved between metazoans and budding yeast. In contrast, the structures and catalytic mechanisms of the mammalian and fungal RNA triphosphatases are completely different [5]. The triphosphatase components of many viral mRNA capping enzymes are mechanistically and structurally related to the fungal RNA triphosphatases and not to the host cell triphosphatase [4, 6, 7]. Thus, cap formation and cap-forming enzymes, especially RNA triphosphatase, are promising targets for antifungal and antiviral drug discovery.

A plausible strategy for antimalarial drug discovery is to identify compounds that block Plasmodium-encoded capping activities without affecting the capping enzymes of the human host or the mosquito vector. For this approach to be feasible, the capping enzymes of the malaria parasite must be identified.

Little is known about the organization of the mRNA capping apparatus in the many other branches of the eukaryotic phylogenetic tree. RNA guanylyltransferase has been studied in the kinetoplastids Trypanosoma and Crithidia [8] but the triphosphatase and methyltransferase components have not been identified.

RNA Guanylyltransferase--Transfer of GMP from GTP to the 5' diphosphate terminus of RNA occurs in a two-step reaction involving a covalent enzyme-GMP intermediate [3]. Both steps require a divalent cation cofactor.

(i) E+pppG<>E-pG+PPi

(ii) E-pG+ppRNA<>GpppRNA+E

The GMP is covalently linked to the enzyme through a phosphoamide (P--N) bond to the epsilon-amino group of a lysine residue within a conserved KxDG element (motif I) found in all known cellular and DNA virus-encoded capping enzymes. Five other sequence motifs (III, IIIa, IV, V, and VI) are conserved in the same order and with similar spacing in the capping enzymes from fungi, metazoans, DNA viruses, and trypanosomes [3].

H.ang.kansson et al. [9] have determined the crystal structure of the Chlorella virus guanylyltransferase in the GTP-bound state and with GMP bound covalently. The protein consist of a larger N-terminal domain (domain 1, containing motifs I, III, IIIa, and IV) and a smaller C-terminal domain (domain 2, containing motif VI) with a deep cleft between them. Motif V bridges the two domains. Motifs I, III, IIIa, IV, and V form the nucleotide binding pocket. The crystal structure reveals a large conformational change in the GTP-bound enzyme, from an "open" to a "closed" state, that brings motif VI into contact with the beta and gamma phosphates of GTP and reorients the phosphates for in-line attack by the motif I lysine.

Identification of essential amino acids has been accomplished by site-directed mutagenesis of Ceg1 the RNA guanylyltransferase of Saccharomyces cerevisiae. The guanylyltransferase activity of Ceg1p is essential for cell viability. Hence, mutational effects on Ceg1 function in vivo can be evaluated by simple exchange of mutant CEG1 alleles for the wild type gene. The effects of alanine substitutions for individual amino acids in motifs I, III, IIIa, IV, V, and VI have been examined. Sixteen residues were defined as essential and structure-activity relationships at these positions were subsequently determined by conservative replacements [10]. Many of the essential Ceg1 side chains correspond to moieties which, in the Chlorella virus capping enzyme crystal structure, make direct contact with GTP.

RNA Triphosphatase--There are at least two mechanistically and structurally distinct classes of RNA 5' triphosphatases: (i) the divalent cation-dependent RNA triphosphatase/NTPase family (exemplified by Saccharomyces cerevisiae Cet1 and Cth1, Candida albicans CaCet1, Schizosaccharomyces pombe Pet1, Chlorella virus Rtp1, baculovirus LEF-4, and vaccinia virus, D1), which require three conserved collinear motifs (A, B, and C) for activity [4,6,7,11-14], and (ii) the divalent cation-independent RNA triphosphatases, e.g., the metazoan cellular mRNA capping enzymes, the baculovirus phosphatase BVP, and the human enzyme PIR1, which require a HCxxxxxR(S/T) phosphate-binding motif [15-17].

Metazoan capping enzymes consist of an N-terminal RNA triphosphatase domain and a C-terminal guanylyltransferase domain. In the 497-amino acid mouse enzyme Mce1, the two catalytic domains are autonomous and nonoverlapping [15]. The metazoan RNA triphosphatases belong to a superfamily of cysteine phosphatases that includes protein tyrosine phosphatases, dual specificity protein phosphatases, and phosphoinositide phosphatases. The metazoan RNA triphosphatases contain a HCxxxxxR(S/T) signature motif (referred to as the P loop) that defines the cysteine phosphatase superfamily. Metazoan RNA triphosphatases catalyze the cleavage of the .gamma. phosphate of 5' triphosphate RNA via a two-step pathway. First, a cysteine thiolate nucleophile of the enzyme (the conserved cysteine of the P loop) attacks the .gamma. phosphorus to form a covalent protein-cysteinyl-S-phosphate intermediate [16] and release the diphosphate-terminated product. Then the covalent intermediate is hydrolyzed to liberate inorganic phosphate. The metazoan RNA triphosphatases do not require a metal cofactor.

Saccharomyces cerevisiae Cet1 exemplifies the class of divalent cation-dependent RNA triphosphatase enzymes, which includes the RNA triphosphatase encoded by the pathogenic fungus Candida albicans, the fission yeast Schizosaccharomyces pombe, and the RNA triphosphatase components of the capping systems of poxviruses, baculoviruses, and Chlorella virus PBCV-1. This triphosphatase family is defined by three conserved collinear motifs (A, B, and C) that include clusters of acidic and basic amino acids that are essential for Cet1 catalytic activity [6,12].

Purified recombinant Cet1 catalyzes the magnesium-dependent hydrolysis of the .gamma. phosphate of triphosphate-terminated RNA to form a 5' diphosphate end. Cet1 also displays a robust ATPase activity in the presence of manganese or cobalt, but magnesium, calcium, copper, and zinc are not effective cofactors for ATP hydrolysis [6]. Cet1 displays broad specificity in converting rNTPs and dNTPs to their respective diphosphates. The manganese- and cobalt-dependent NTPase activity of Cet1 resembles the manganese- or cobalt-dependent NTPase activities of the of the other members of this family, including baculovirus LEF-4, C. albicans CaCet1, S. cerevisiae Cth1, S. pombe Pct1, and Chlorella virus Rtp1 [4,11-14].

Crystal Structure of Fungal RNA Triphosphatase--The biologically active triphosphatase derivative Cet1(241-539) was crystallized and its structure determined at 2.05 .ANG. resolution [5]. Consistent with solution studies, Cet1 crystallized as a dimer. The striking feature of the tertiary structure is the formation of a topologically closed tunnel composed of 8 antiparallel .beta. strands. The active site resides within this hydrophilic "triphosphate tunnel". The interior of the tunnel contained a single sulfate ion coordinated by two arginine and two lysine side chains. Insofar as sulfate is a structural analog of phosphate, it is likely that the side chain interactions of the sulfate reflect contacts made by the enzyme with the .gamma. phosphate of the triphosphate-terminated RNA and nucleoside triphosphate substrates.

The proteins most closely related to Cet1 at the primary structure level are CaCet1, Pct1, and Cth1. CaCet1 is the RNA triphosphatase component of the capping apparatus of Candida albicans. Pct1 is the RNA triphosphatase component of the capping apparatus of Schizosaccharomyces pombe [14]. Cth1 is a nonessential S. cerevisiae protein with divalent cation-dependent RNA triphosphatase/NTPase activity that may participate in an RNA transaction unrelated to capping [12].  The residues conserved in all four fungal enzymes are localized predominantly in the interior of the tunnel.

Cet1 triphosphatase activity is strictly dependent on a divalent cation cofactor. The hydrolysis of 5' triphosphate RNA termini is optimal in the presence of magnesium, whereas NTP hydrolysis specifically requires manganese or cobalt. The location of a metal-binding site on the enzyme was determined by X-ray diffraction of Cet1(241-539) crystals that had been soaked in manganese chloride [5]. Manganese is coordinated with octahedral geometry to the sulfate inside the tunnel, to the side chain carboxylates of three glutamates, and to two waters. The three glutamates that comprise the metal-binding site of fungal RNA triphosphatase are located in motifs A and C, which define the metal-dependent RNA triphosphatase family. Substitution of any one of the three glutamates by alanine or glutamine inactivates Cet1. The motif A and C glutamates are also essential for the activities of vaccinia virus RNA triphosphatase, baculovirus RNA triphosphatase, C. albicans CaCet1, S. pombe Pct1, and S. cerevisiae Cth1. Thus, it is likely that motifs A and C comprise the metal binding site in all members of this enzyme family.

The structure of Cet1(241-539) with bound sulfate and manganese is construed to reflect that of the product complex of enzyme with the hydrolyzed .gamma. phosphate [5]. The structure suggests a catalytic mechanism whereby acidic side chains located on the floor of the tunnel coordinate an essential divalent cation that in turn coordinates the .gamma. phosphate. The metal ion would activate the .gamma. phosphorus for direct attack by water and stabilize a pentacoordinate phosphorane transition state in which the attacking water is apical to the .beta. phosphate leaving group. Interactions between the sulfate and basic side chains located on the walls of the tunnel would contribute to the coordination of the 5' phosphates in the ground state and the stabilization of the negative charge on the .gamma. phosphate developed in the transition state. A key mechanistic distinction between the fungal-type RNA triphosphatases and the metazoan-type RNA triphosphatases is that the fungal-type enzymes do not form a covalent phosphoenzyme intermediate.

The prior art is deficient in the lack of methods that teach a person having ordinary skill in this art how to screen for a compound that inhibits cap formation by the enzymes of unicellular eukaryotic parasites such as Plasmodia. The prior art is also deficient in an identification and characterization of the enzymes comprising the mRNA capping apparatus of Plasmodia. In particular, the RNA triphosphatase component of the mRNA capping apparatus has not been identified and characterized in any unicellular eukaryotic parasite. The biochemical properties of an RNA triphosphatase from a unicellular eukaryotic parasite are not known. Hence, a mechanistic and structural comparison between the RNA triphosphatase of the parasite and the RNA triphosphatase of the metazoan host organism, which could underscore the potential of RNA triphosphatase as a therapeutic target for parasitic infections, is not possible. The present invention fulfills this longstanding need in the art.

SUMMARY OF THE INVENTION

The present invention facilitates the discovery of drugs that target an essential aspect of gene expression--the formation of the mRNA 5' cap m7GpppN--in unicellular eukaryotic parasites.

The invention discloses the amino acid sequences of the Plasmodium falciparum RNA triphosphatase and RNA guanylyltransferase, which catalyze the first and second steps of mRNA cap formation, respectively. The invention also provides for expression vectors and recombinant Plasmodium falciparum RNA triphosphatase and RNA guanylyltransferase.

The invention further encompasses in vitro screening methods to identify candidate inhibitors of the catalytic activity of RNA guanylyltransferase or the RNA 5' triphosphatase of unicellular eukaryotic parasites. These methods are simple, quantitative, and adaptable to calorimetric, spectrophotometric, or fluorescence detection assays that are suited to high-throughput screening for inhibitors of the RNA triphosphatase of Plasmodia and other unicellular eukaryotic parasites.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to the identification of compounds that inhibits the growth of Plasmodium falciparum and other unicellular eukaryotic parasites by virtue of the effects of said compounds on the capping of parasite mRNA.

The present invention provides isolated DNAs encoding a RNA guanylyltransferase and a RNA 5' triphosphatase from Plasmodium falciparum, vectors for expression of recombinant RNA guanylyltransferase and RNA 5' triphosphatase, and purified RNA guanylyltransferase and RNA 5' triphosphatase having amino acid sequences of SEQ ID No. 1 and 2 respectively.

It is well known in the art that the amino acid sequence of a protein is determined by the nucleotide sequence of the DNA that encodes the protein. Because of the degeneracy of the genetic code (i.e., for most amino acids, more than one nucleotide triplet (codon) codes for a single amino acid), different nucleotide sequences can code for a particular amino acid, or polypeptide. Thus, the polynucleotide sequences of the subject invention also encompass those degenerate sequences that encode the polypeptides of the subject invention, or a fragment or variant thereof. Accordingly, any nucleotide sequence (mutated from the sequences disclosed herein) which encodes the mRNA capping enzymes described herein comes within the scope of this invention and the claims appended hereto.

Also, as described herein, fragments or mutated versions of the mRNA capping enzymes are an aspect of the subject invention so long as such fragments or mutated versions retain the biochemical activity so that such fragments or mutated versions are useful in the methods described herein. As used herein, "fragment," as applied to a polypeptide, will ordinarily be at least 10 residues, more typically at least 20 residues, and preferably at least 30 (e.g., 50) residues in length, but less than the entire, intact sequence. Fragments can be generated by methods known to those skilled in the art, e.g,., by enzymatic digestion of naturally occurring or recombinant protein, by recombinant DNA techniques using an expression vector that encodes a defined fragment, or by chemical synthesis. As used herein, "mutated version," as applied to a polypeptide, will ordinarily be an altered form of the polypeptide in which one or more amino acids are substituted by different amino acids or by modified amino acids. Mutated versions can be generated by methods known to those skilled in the art, e.g., by chemical modification of naturally occurring or recombinant protein, by recombinant DNA techniques using an expression vector that encodes a defined fragment, or by chemical synthesis. The ability of a candidate fragment or mutated version to exhibit a characteristic of the mRNA capping enzymes can be readily assessed by a person having ordinary skill in this art by using the methods described herein.

In one embodiment of the present invention, there is provided a method of screening for a compound that inhibits the catalytic activity of Plasmodium RNA guanylyltransferase, comprising the steps of: a) contacting said Plasmodium RNA guanylyltransferase with guanosine triphosphate and a divalent cation cofactor in the presence or absence of a test compound; and detecting formation of a covalent enzyme-GMP intermediate. A lack of formation of an enzyme-GMP intermediate or a reduction in the formation of said intermediate indicates inhibition of said Plasmodium RNA guanylyltransferase by said test compound. Preferably, the divalent cation cofactor is manganese or magnesium. Detection of an enzyme-GMP intermediate may be by any method readily known to those having ordinary skill in this art; preferable methods include radioisotope assay and fluorescence assay. A representative Plasmodium RNA guanylyltransferase is the RNA guanylyltransferase from Plasmodium falciparum disclosed herein.

In another embodiment of the present invention, there is provided a method of screening for a compound that inhibits the catalytic activity of Plasmodium RNA guanylyltransferase, comprising the steps of: a) contacting said Plasmodium RNA guanylyltransferase with guanosine triphosphate and a divalent cation cofactor and a diphosphate-terminated RNA in the presence or absence of a test compound; and detecting formation of a GMP-capped RNA. A lack of formation of a GMP-capped RNA or a reduction in the formation of said GMP-capped RNA indicates inhibition of said Plasmodium RNA guanylyltransferase by said test compound. Preferably, the divalent cation cofactor is manganese or magnesium. Although detection of a GMP-capped RNA may be by any method readily known to those having ordinary skill in this art, preferable methods include radioisotope assay and fluorescence assay. A representative Plasmodium RNA guanylyltransferase is the RNA guanylyltransferase from Plasmodium falciparum disclosed herein, i.e., Plasmodium guanylyltransferase has the amino acid sequence of SEQ ID No. 1, is a fragment of the guanylyltransferase with the amino acid sequence of SEQ ID No. 1, or is a mutated version of the guanylyltransferase with the amino acid sequence of SEQ ID No. 1.

In yet another embodiment of the present invention, there is provided a method of screening for a compound that inhibits the catalytic activity of unicellular eukaryotic parasite RNA 5' triphosphatase, comprising the steps of: a) contacting said parasite RNA 5' triphosphatase with a 5' triphosphate-terminated RNA or a nucleoside triphosphate and a divalent cation cofactor in the presence or absence of a test compound; and detecting hydrolysis of said 5' triphosphate-terminated RNA or nucleoside triphosphate. A lack of hydrolysis of said 5' triphosphate-terminated RNA or nucleoside triphosphate or a reduction in the hydrolysis of said 5' triphosplhate-terminated RNA or nucleoside triphosphate indicates inhibition of said parasite RNA 5' triphosphatase by said test compound. Preferably the divalent cation cofactor is magnesium, manganese or cobalt. Although detection of hydrolysis may be by any method readily known to those having ordinary skill in this art, preferable methods include radioisotope assay, calorimetric assay, spectrophotometric assay, and fluorescence assay. A representative parasite RNA triphosphatase is the RNA triphosphatase from Plasmodium falciparum disclosed herein.

In accordance with the present invention, there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, "Molecular Cloning: A Laboratory Manual (1982); "DNA Cloning: A Practical Approach," Volumes I and II (D. N. Glover ed. 1985); "Oligonucleotide Synthesis" (M. J. Gait ed. 1984); "Nucleic Acid Hybridization" [B. D. Hames & S. J. Higgins eds. (1985)]; "Transcription and Translation" [B. D. Hames & S. J. Higgins eds. (1984)]; "Animal Cell Culture" [R. I. Freshney, ed. (1986)]; "Immobilized Cells And Enzymes" [IRL Press, (1986)]; B. Perbal, "A Practical Guide To Molecular Cloning" (1984). Therefore, if appearing herein, the following terms shall have the definitions set out below.

A "DNA molecule" refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes.

In general, expression vectors containing promoter sequences which facilitate the efficient transcription and translation of the inserted DNA fragment are used in connection with the host. The expression vector typically contains an origin of replication, promoter(s), terminator(s), as well as specific genes which are capable of providing phenotypic selection in transformed cells. A coding sequence is "operably linked" and "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence. The transformed hosts can be fermented and cultured according to means known in the art to achieve optimal cell growth.

Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell. A "cis-element" is a nucleotide sequence, also termed a "consensus sequence" or "motif", that interacts with other proteins which can upregulate or downregulate expression of a specific gene locus. A "signal sequence" can also be included with the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates to the host cell and directs the polypeptide to the appropriate cellular location. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes.

A cell has been "transformed" or "transfected" with exogenous or heterologous DNA when such DNA has been introduced inside the cell. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a vector or plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. A "clone" is a population of cells derived from a single cell or ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations. An organism, such as a plant or animal, that has been transformed with exogenous DNA is termed "transgenic".

As used herein, the term "host organism" is meant to include not only prokaryotes but also eukaryotes such as yeast, plant, protozoan, and animal cells. A recombinant DNA molecule or gene can be used to transform a host using any of the techniques commonly known to those of ordinary skill in the art. Prokaryotic hosts may include E. coli, S. tymphimurium, Serratia marcescens and Bacillus subtilis. Eukaryotic hosts include yeasts such as Pichia pastoris, mammalian cells, insect cells, and plant cells, such as Arabidopsis thaliana and Tobaccum nicotiana.

Claim 1 of 7 Claims

What is claimed is:

1. A method of screening for a compound that inhibits the catalytic activity of Plasmodium guanylyltransferase, comprising the steps of:

contacting said Plasmodium guanylyltransferase of SEQ ID NO: 1 or an enzymatically active fragment thereof with a guanosine triphosphate substrate and a divalent cation cofactor and a diphosphate-terminated RNA in the presence or absence of said compound; and

detecting formation of a GMP-capped RNA, wherein a lack of formation of said GMP-capped RNA or a decrease in formation of said GMP-capped RNA indicates said compound inhibits the catalytic activity of said guanylyltransferase.


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