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Phytochemical investigation of the figs of Ficus mucuso led to the isolation of three new isoflavone dimer derivatives, mucusisoflavones A-C (1-3), together with 16 known compounds. Some of the isolates were tested in vitro for their inhibitory properties toward β-glucuronidase and Plasmodium falciparum enoyl-ACP reductase (PfENR) enzymes. Compound 1 (IC₅₀) 0.68 μM) showed inhibitory activity on β-glucuronidase enzyme, while 3 (IC₅₀) 7.69 μM) exhibited a weak inhibitory activity against P. falciparum enoyl-ACP reductase (PfENR).

The importance of fatty acids to the human malaria parasite, Plasmodium falciparum, and differences due to a type I fatty acid synthesis (FAS) pathway in the parasite, make it an attractive drug target. In the present study, we developed and a utilized a pharmacophore to select compounds for testing against PfKASIII, the initiating enzyme of FAS. This effort identified several PfKASIII inhibitors that grouped into various chemical classes of sulfides, sulfonamides, and sulfonyls. Approximately 60% of the submicromolar inhibitors of PfKASIII inhibited in vitro growth of the malaria parasite. These compounds inhibited both drug sensitive and resistant parasites and testing against a mammalian cell line revealed an encouraging in vitro therapeutic index for the most active compounds. Docking studies into the active site of PfKASIII suggest a potential binding mode that exploits amino acid residues at the mouth of the substrate tunnel.

Cyclin dependent protein kinases (CDKs) have become attractive drug targets in an effort to identify effective inhibitors of the parasite Plasmodium falciparum, the causative agent of the most severe form of human malaria. We tested known CDK inhibitors for their ability to inhibit two malarial CDKs: Pfmrk and PfPK5. Many broad spectrum CDK inhibitors failed to inhibit Pfmrk suggesting that the active site differs from other CDKs in important ways. By screening compounds in the Walter Reed chemical database, we identified oxindole-based compounds as effective inhibitors of Pfmrk (IC(50) = 1.5 microM). These compounds have low cross-reactivity against PfPK5 and human CDK1 demonstrating selectivity for Pfmrk. Amino acid comparison of the active sites of Pfmrk and PfPK5 identified unique amino acid differences that may explain this selectivity and be exploited for further drug development efforts.

Malaria, a disease caused by protozoan parasites of the genus Plasmodium, is one of the most dangerous infectious diseases, claiming millions of lives and infecting hundreds of millions of people annually. The pressing need for new antimalarials has been answered by the discovery of new drug targets from the malaria genome project. One of the early findings was the discovery of two genes encoding Type II fatty acid biosynthesis proteins: ACP (acyl carrier protein) and KASIII (beta-ketoacyl-ACP synthase III). The initiating steps of a Type II system require a third protein: malonyl-coenzyme A:ACP transacylase (MCAT). Here we report the identification of a single gene from P. falciparum encoding pfMCAT and the functional characterization of this enzyme. Pure recombinant pfMCAT catalyzes malonyl transfer from malonyl-coenzyme A (malonyl-CoA) to pfACP. In contrast, pfACP(trans), a construct of pfACP containing an amino-terminal apicoplast transit peptide, was not a substrate for pfMCAT. The product of the pfMCAT reaction, malonyl-pfACP, is a substrate for pfKASIII, which catalyzes the decarboxylative condensation of malonyl-pfACP and various acyl-CoAs. Consistent with a role in de novo fatty acid biosynthesis, pfKASIII exhibited typical KAS (beta-ketoacyl ACP synthase) activity using acetyl-CoA as substrate (k(cat) 230 min(-1), K(M) 17.9 +/- 3.4 microM). The pfKASIII can also catalyze the condensation of malonyl-pfACP and butyryl-CoA (k(cat) 200 min(-1), K(M) 35.7 +/- 4.4 microM) with similar efficiency, whereas isobutyryl-CoA is a poor substrate and displayed 13-fold less activity than that observed for acetyl-CoA. The pfKASIII has little preference for malonyl-pfACP (k(cat)/K(M) 64.9 min(-1)microM(-1)) over E. coli malonyl-ACP (k(cat)/K(M) 44.8 min(-1)microM(-1)). The pfKASIII also catalyzes the acyl-CoA:ACP transacylase (ACAT) reaction typically exhibited by KASIII enzymes, but does so almost 700-fold slower than the KAS reaction. Thiolactomycin did not inhbit pfKASIII (IC(50)330 microM), but three structurally similar substituted 1,2-dithiole-3-one compounds did inhibit pfKASIII with IC(50) values between 0.53 microM and 10.4 microM. These compounds also inhibited the growth of P. falciparum in culture.