Cloning, sequence determination, and regulation of the ribonucleotide reductase subunits from Plasmodium falciparum: a target for antimalarial therapy.

Abstract

Malaria remains a leading cause of morbidity and mortality worldwide, accounting for more than one million deaths annually. We have focused on the reduction of ribonucleotides to 2'-deoxyribonucleotides, catalyzed by ribonucleotide reductase, which represents the rate-determining step in DNA replication as a target for antimalarial agents. We report the full-length DNA sequence corresponding to the large (PfRl) and small (PfR2) subunits of Plasmodium falkiparum ribonucleotide reductase. The small subunit (PfR2) contains the majior catalytic motif consisting of a tyrosyl radical and a dinuclear Fe site. Whereas PfR2 shares 59% amino acid identity with human R2, a striking sequence divergence between human R2 and PfR2 at the C terminus may provide a selective target for inhibition of the malarial enzyme. A synthetic oligopeptide corresponding to the C-terminal 7 residues of PfR2 inhibits mammalian ribonucleotide reductase at concentrations "10-fold higher than that predicted to inhibit malarial R2. The gene encoding the large subunit (PfRl) contains a single intron. The cysteines thought to be involved in the reduction mechanism are conserved. In contrast to mammalian ribonucleotide reductase, the genes for PfRl and PfR2 are located on the same chromosome and the accumulation of mRNAs for the two subunits follow different temporal patterns during the cell cycle.The spread of chloroquine resistance and the emergence of resistance to newer antimalarial agents is a serious problem in the therapy of malaria caused by Plasmodiumfalciparum, and clearly, additional agents are needed. Inhibition of ribonucleotide reductase (RR) encoded by pathogenic organisms is now recognized as a target for the design of antimicrobial agents. This approach has been most extensively investigated in the design of therapies for the herpes viruses (1-4 complex between the large subunit (Rl) dimer and the small subunit (R2) dimer. The association constant for this interaction is 0.5-1 x 107 M-1 (6). Kinetic studies of the association of E. coli Rl with full-length E. coli R2 compared to the association of Rl with a C-terminal truncated R2 strongly indicate that the interaction between Rl and R2 is entirely accounted for by C-terminal sequences of R2 (7). Synthetic peptides identical to the C terminus of R2 effectively inhibit RR activity by competing with R2 for association with Rl. This phenomenon was first demonstrated for herpes simplex virus RR where the acetylated nonapeptide, AcYAGAV-VNDL, had an IC50 value of 10-20 uM (2, 3, 8, 9). Further work showed that even modest changes in the sequence of the parent peptide can lead to large decreases in inhibitory potency (10, 11). We demonstrated that AcFTLDADF, corresponding to the C terminus of mouse R2, inhibits mammalian RR with an IC50 value of 20 uM (12). It was later shown that C-terminal peptides derived from E. coli, viral, or mammalian R2 sequences inhibited the RRs from which they were derived but did not cross-inhibit RRs from other species (13). In principle, therefore, a ...

Baculoviruses contain a gene for the large subunit of ribonucleotide reductase.

“…3. Alignment of the predicted amino acid sequences of the RR1 proteins of the baculoviruses SeMNPV and SpliMNPV with those of Homo sapiens (Hs ; Parker et al, 1991), Caenorhabditis elegans (Ce ; Sulston et al, 1992), Plasmodium falciparum (Pf ; Rubin et al, 1993), Schizosaccharomyces pombe (Sp ; Fernandez-Sarabia et al, 1993), vaccinia virus (VV ; Tengelsen et al, 1988), African swine fever virus (ASFV ; Boursnell et al, 1991), herpes simplex virus type 1 (HSV1 ; Nikas et al, 1986), herpesvirus saimiri (HVS ; Nicholas et al, 1992), bacteriophage T4 (Tseng et al, 1988) and E. coli (Ec ; Nillson et al, 1988). Gaps introduced to optimize alignment are indicated by dots.…”

Cloning and characterization of the R1 and R2 subunits of ribonucleotide reductase from Trypanosoma brucei

“…The only eukaryotic R1 proteins known to differ in this amino acid sequence are those from T. brucei (Fig. 2) and Plasmodium falciparum (the malaria parasite) (63), which instead contain the sequence DQGGGKRKG. This sequence identity in these two otherwise unrelated protozoans is interesting from an evolutionary standpoint.…”

Cloning, sequencing and expression of ribonucleotide reductase R2 from Trypanosoma brucei

“…Ribonucleotide reductase is an attractive target molecule for anticancer and antimicrobial drug development [21,22]. The enzyme can be inhibited by nucleotide analogs, by reduction of the essential tyrosyl radical and by preventing subunit interaction [7,20,21].…”

“…Developmental regulation of T. brucei ribonucleotide reductase may occur at the post-transcriptional level as it has been suggested for other proteins of this parasite [25]. Alternatively the levels of the R1 and R2 transcripts are not linked as in Plasmodium falciparum [22] and R1 may be regulated.…”

“…Interaction between the R1 and R2 proteins is entirely accounted for by the carboxy terminus of the small subunit [15,20]. Synthetic peptides containing the seven C-terminal residues of R2 are selective inhibitors of mammalian [20], viral [21] and probably plasmodial [22] ribonucleotide reductases.…”