Inhibition of Plasmodium falciparum Proliferationin Vitro by Ribozymes

Abstract

Catalytic RNA (ribozymes) suppressed the growth of the human malarial parasite Plasmodium falciparum in vitro. The phosphorothioated hammerhead ribozymes targeted unique regions of the P. falciparum carbamoylphosphate synthetase II gene. The P. falciparum carbamoyl-phosphate synthetase II gene encodes the first and limiting enzyme in the pathway, and its mRNA transcript contains two large insert regions absent in other carbamoyl-phosphate synthetases, including that from humans. These inserts are ideal targets for nucleic acid therapy. Exogenous delivery of ribozymes to cultures reduced malarial viability up to 55% at 0.5 M ribozyme concentrations, which is significantly greater than control levels (5-15% reduction), suggesting a sequence-specific inhibition. This inhibition was shown to be stagespecific, with optimal inhibitions being detected after 24 h, coincident with maximal production of the carbamoyl-phosphate synthetase enzyme in the course of the life cycle of the parasite. A decrease in total carbamoyl-phosphate synthetase activity was observed only in cultures treated with the ribozymes. The task of developing alternative therapeutic agents against malaria is urgent due to the evolution of drug-resistant strains of P. falciparum, the most virulent of all human malarial parasites. Another critical issue to be addressed is the possibility of eliminating or reducing any systemic toxicity to the host, which can potentially be provided by nucleic acid therapy. This work is the first reported assessment of the ability of ribozymes as antimalarials. Ribozyme inhibition assays can also aid in identifying important antimalarial loci for chemotherapy. The malarial parasite can, in turn, be a useful in vivo host to study the catalysis and function of new ribozyme designs.

Hammerhead ribozyme design and application

“…Other reported viral applications of hammerhead ribozymes include the inhibition of viral multiplication by targeting the polymerase gene of mouse hepatitis virus [218,219], suppression of MMLV replication in infected NIH3T3 cells after targeting of the packaging region [124] and inhibition (70-80%) of influenza A virus superinfection in stably ribozyme-expressing COS cells [166]. Reduction of the viability of the human malaria parasite Plasmodium falciparum in cultures after targeting of unique regions in the carbamoylphosphate synthetase II gene is a nonviral example [220]. Adenovirus-delivered ribozymes have been targeted to the nuclear antigen 1 of Epstein-Barr virus (EBV), which is essential for the proliferation of the virus [164].…”

Ribozyme cleavage of Plasmodium falciparum gyrase A gene transcript affects the parasite growth

“…While there is no alternative to the fatty acid type II biosynthesis pathway, the inhibition of LytB proved more effective in parasite growth inhibition. Both these ribozymes (mRz 49GyrA and mRz 876LytB ) were inhibiting the parasite growth at a much higher concentration than the ribozyme against the carbamoyl phosphate synthetase II mRNA described by Flores et al (1997). This discrepancy may have arisen due to the differences in the synthesis and purification protocols as suggested by Vinayak and Sharma (2007) or may be due to the differences in the folding of target RNA structure in vivo, in vitro, and in silico and thus differences in the accessibility of the target sites.…”

“…In the present study, we have designed and used the hammerhead ribozyme RZ 491GyrA to inhibit the expression of P. falciparum gyrase A so that it can be developed as a therapeutic agent. Ribozymes have been successfully used in controlling gene expression and inhibiting the growth of various parasites (Flores et al 1997;Dan et al 2000). They are attractive potential therapeutic agents due to their catalytic nature and lack of immunogenecity.…”

“…There was a gradual decrease in the amount of transcript and an increase in the product with increasing time. The cleavage rates (t 1/2 ) of the ribozymes were calculated as time at which 50% of the transcript was cleaved (Flores et al 1997 Cleavage of the target transcript by chemically modified synthetic ribozyme RZ 491GyrA in a cell-free system…”

Identification and Validation of Novel Drug Targets for the Treatment of Plasmodium falciparum Malaria: New Insights

“…PfCPSII also differs from its mammalian homologue by the presence of two inserted sequences, located between junctions of the glutamine aminotransferase and synthetase domains [161]. Although the absence of structural information and activity inhibitors, the druggable potential of this enzyme has already been demonstrated by the potent growth inhibitory effect of a synthetic ribozyme with specificity for the PfCPSII gene over P. falciparum cultures [162]. The same synthetic ribozyme has shown no toxicity to mammalian cells.…”