Plasmodium falciparum CTP:phosphocholine cytidylyltransferase possesses two functional catalytic domains and is inhibited by a CDP-choline analog selected from a virtual screening

• Malaria parasites require phosphatidylcholine to build membranes and to multiply.
• PfCCT, the rate-limiting enzyme of phosphatidylcholine biosynthesis, is a potential antimalarial target.
• PfCCT is atypical in having duplicated catalytic domains which are both active.
• Virtual screening identified one competitive inhibitor (C12) of PfCCT.
• C12 had an antimalarial effect and impaired PC biosynthesis in the malaria parasite.

Phosphatidylcholine is the major lipid component of the malaria parasite membranes and is required for parasite multiplication in human erythrocytes. Plasmodium falciparum CTP:phosphocholine cytidylyltransferase (PfCCT) is the rate-limiting enzyme of the phosphatidylcholine biosynthesis pathway and thus considered as a potential antimalarial target. In contrast to its mammalian orthologs, PfCCT contains a duplicated catalytic domain. Here, we show that both domains are catalytically active with similar kinetic parameters. A virtual screening strategy allowed the identification of a drug-size molecule competitively inhibiting the enzyme. This compound also prevented phosphatidylcholine biosynthesis in parasites and exerted an antimalarial effect. This study constitutes the first step towards a rationalized design of future new antimalarial agents targeting PfCCT.