Pharmacological targeting of mRNA cap formation for...

Details Pharmacological targeting of mRNA cap formation for... Pharmacological targeting of mRNA cap formation for...

2000-12-29

2002-09-17

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S194000, C435S320100, C435S325000, C536S023200

Reexamination Certificate

active

06451583

ABSTRACT:

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 Pgt1 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 (FIG.
1
). 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 (
FIG. 1
) [3].
H{dot over (a)}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 Cegt1, the RNA guanylyltransferase of
Saccharomyces cerevisiae
. The guanylyltransferase activity of Cegt1p 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 (denoted by dots in
FIG. 1
) and structure-activity relationships at these positions were subsequently determined by conservative replacements [10]. Many of the essential Cegt1 side chains correspond to moieties which, in the Chlorella virus capping enzyme crystal structure, make direct contact with GTP as denoted by the arrowheads in FIG.
1
.
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
Pct1, 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 a

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