Targeting protein translation, RNA splicing, and degradation by morpholino-based conjugates in Plasmodium falciparum.
Identification and genetic validation of new targets from available genome sequences are critical steps toward the development of new potent and selective antimalarials. However, no methods are currently available for large-scale functional analysis of the Plasmodium falciparum genome. Here we present evidence for successful use of morpholino oligomers (MO) to mediate degradation of target mRNAs or to inhibit RNA splicing or translation of several genes of P. falciparum involved in chloroquine transport, apicoplast biogenesis, and phospholipid biosynthesis. Consistent with their role in the parasite life cycle, down-regulation of these essential genes resulted in inhibition of parasite development. We show that a MO conjugate that targets the chloroquine-resistant transporter PfCRT is effective against chloroquine-sensitive and -resistant parasites, causes enlarged digestive vacuoles, and renders chloroquine-resistant strains more sensitive to chloroquine. Similarly, we show that a MO conjugate that targets the PfDXR involved in apicoplast biogenesis inhibits parasite growth and that this defect can be rescued by addition of isopentenyl pyrophosphate. MO-based gene regulation is a viable alternative approach to functional analysis of the P. falciparum genome.
Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520;
Aprajita
A
Garg
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520;
Donna
D
Wesolowski
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520;
Dulce
D
Alonso
Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, NY 10065.
Kirk W
KW
Deitsch
Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520;
Choukri
C
Ben Mamoun
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520; sidney.altman@yale.edu.
Sidney
S
Altman
D000818:Animals; D000962:Antimalarials / Q000494:pharmacology; D037621:Artemisinins / Q000494:pharmacology; D002738:Chloroquine / Q000494:pharmacology; D015536:Down-Regulation / Q000187:drug effects; D004351:Drug Resistance / Q000187:drug effects; D005434:Flow Cytometry; D017930:Genes, Reporter; D045782:Hemiterpenes / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D060172:Morpholinos / Q000494:pharmacology*; D009943:Organophosphorus Compounds / Q000378:metabolism; D010271:Parasites / Q000187:drug effects / Q000235:genetics / Q000254:growth & development; D010455:Peptides / Q000494:pharmacology; D010963:Plasmodium falciparum / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D014176:Protein Biosynthesis / Q000187:drug effects*; D059748:Proteolysis / Q000187:drug effects*; D012326:RNA Splicing / Q000187:drug effects*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA
D000962:Antimalarials; D037621:Artemisinins; D045782:Hemiterpenes; D060172:Morpholinos; D009943:Organophosphorus Compounds; D010455:Peptides; D012333:RNA, Messenger; C004809:isopentenyl pyrophosphate; D002738:Chloroquine; C031327:artemisinin; D008156:Luciferases
gene expression; intraerythrocytic development; malaria; peptide conjugated morpholino oligomer; vivo morpholino oligomer