Pharmacological targeting of mRNA cap formation

Details Pharmacological targeting of mRNA cap formation Pharmacological targeting of mRNA cap formation

2000-11-22

2002-07-16

Jones, W. Gary (Department: 1655)

Chemistry: molecular biology and microbiology

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

Fungi

C435S254110, C435S006120, C435S029000, C435S091200, C536S022100, C536S024200

Reexamination Certificate

active

06420163

ABSTRACT:

BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates generally to the fields of gene biochemical pharmacology and drug discovery. More specifically, the present invention relates to method of screening for a compound that inhibits formation of an organism's 5′ mRNA cap structure.
Description of the Related Art
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 [1].
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 [2-12]. Genetic and biochemical experiments highlight roles for the cap in protecting mRNA from untimely degradation by cellular 5′ exonucleases [13] and in recruiting the mRNA to the ribosome during translation initiation [14].
The physical and functional organizations of the capping apparatus differ in significant respects in fungi, metazoans, protozoa, and viruses. Hence, the cap-forming enzymes are potential targets for antifungal, antiviral, and antiprotozoal drugs that would interfere with capping of pathogen mRNAs, but spare the mammalian host capping enzymes. A plausible strategy for drug discovery is to identify compounds that block cell growth contingent on pathogen-encoded capping activities without affecting the growth of otherwise identical cells bearing the capping enzymes of the host organism. For this approach to be feasible, the capping systems of interest must be interchangeable in vivo.
The architecture of the capping apparatus differs between metazoans, fungi, protozoa, and DNA viruses. Metazoan species encode a two-component capping system consisting of a bifunctional triphosphatase-guanylyltransferase polypeptide (named Mce1p in the mouse and Hce1p in humans) and a separate methyltransferase polypeptide (Hem1p in humans) [6, 9, 15-22]. The budding yeast
Saccharomyces cerevisiae
encodes a three component system consisting of separate triphosphatase (Cet1p). guanylyltransferase (Ceg1p), and methyltransferase (Abd1p) gene products [7, 10, 11, 23]. In yeast, the triphosphatase (Cet1p) and guanylyltransferase (Ceg1p) polypeptides interact to form a heteromeric complex [11], whereas in mammals, autonomous triphosphatase and guanylyltransferase domains are linked in cis within a single polypeptide (Mce1p) [18].
Vaccinia
virus capping enzyme is a multifunctional protein that catalyzes all three reactions. The triphosphatase, guanylyltransferase, and methyltransferase active sites are arranged in a modular fashion within a single polypeptide—the
Vaccinia
D1 protein [24-30]. Other DNA viruses encode a subset of the capping activities; e.g., baculoviruses encode a bifunctional triphosphatase-guanylyltransferase (LEF-4) and
Chlorella
virus PBCV-1 encodes a monofunctional guanylyltransferase [31-33]. The guanylyltransferase and methyltransferase domains are conserved between DNA viruses, fungi, and metazoans. In contrast, the triphosphatase components are structurally and mechanistically divergent.
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 [34]. Both steps require a divalent cation cofactor, either magenesium or manganese.
E+pppG E
-
pG+PPi
(i)
E
-
pG+ppRNA GpppRNA+E
(ii)
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, 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
) [35].
Hakansson et al. [36] have determined the crystal structure of the
Chlorella
virus capping enzyme 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, 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. When the crystal is soaked in maganese, guanylyltransferase reaction chemistry occurs in
crystallo
and the covalent enzyme-GMP intermediate is formed. However, only the enzyme in the closed conformation is reactive.
Identification of essential enzymic functional groups has been accomplished by site-directed mutagenesis of Ceg1p, the RNA guanylyltransferase of
Saccharomyces cerevisiae
. The guanylyltransferase activity of Ceg1p is essential for cell viability. Hence, mutational effects on Ceg1p 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 [2, 5, 6] have been examined. Sixteen residues were defined as essential (denoted by asterisks in
FIG. 1
) and structure-activity relationships at these positions were subsequently determined by conservative replacements. Nine of the essential Ceg1p side chains correspond to moieties which, in the
Chlorella
virus capping enzyme crystal structure, make direct contact with GTP (arrowheads in FIG.
2
). These include: the motif I lysine nucleophile which contacts the alpha-phosphate of GTP; the motif I arginine and motif III glutamate, which contact the 3′ and 2′ ribose hydroxyls, respectively: the motif III phenylalanine, which stacks on the guanine base; the two motif V lysines, which contact the alpha-phosphate; the motif V aspartate, which interacts with the beta-phosphate: the motif VI arginine that interacts with the beta-phosphate; and the motif VI lysine, which contacts the gamma-phosphate of GTP [6, 36].
On the basis of sequence conservation outside motif I, III, IIIa, IV, V, and VI, the capping enzymes of fungi (
S. cerevisiae. S. pombe. C. albicans,
), metazoans (C. elegans and mammals) and
Chlorella
virus can be grouped into a discrete subfamily [6, 37]. The sequence alignment in
FIG. 2
highlights two motifs that are present in these capping enzymes, but not in the poxvirus enzymes, which can be designated motif P and motif Vc. In the
Chlorella
virus capping enzyme, motif P forms one wall of the guanosine binding pocket of domain 1 [36]. Motif Vc—(K/R)I(I/V)EC—is situated between motifs V and VI in domain 2. The glutamate residue of motif Vc is essential for the activity of the fungal guanylyltransferase Ceg1p [37].
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 yeast Cet1p, baculovirus LEF-4 and vaccinia D1), which require three conserved collinear motifs (A, B, and C) for activity [12, 28, 31, 32], and (ii) the divalent cation-independent RNA triphosphatases, e.g., the metazoan cellular enzymes and the baculovirus enzyme BVP [15, 17, 38-4

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