A mevalonate-independent pathway of isoprenoid biosynthesis present in Plasmodium falciparum was shown to represent an effective target for chemotherapy of malaria. This pathway includes 1-deoxy-D-xylulose 5-phosphate (DOXP) as a key metabolite. The presence of two genes encoding the enzymes DOXP synthase and DOXP reductoisomerase suggests that isoprenoid biosynthesis in P. falciparum depends on the DOXP pathway. This pathway is probably located in the apicoplast. The recombinant P. falciparum DOXP reductoisomerase was inhibited by fosmidomycin and its derivative, FR-900098. Both drugs suppressed the in vitro growth of multidrug-resistant P. falciparum strains. After therapy with these drugs, mice infected with the rodent malaria parasite P. vinckei were cured.

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The gcpE and lytB gene products control the terminal steps of isoprenoid biosynthesis via the 2-C-methyl-D-erythritol 4-phosphate pathway in Escherichia coli. In lytB-deficient mutants, a highly immunogenic compound accumulates significantly, compared to wild-type E. coli, but is apparently absent in gcpE-deficient mutants. Here, this compound was purified from E. coli v vlytB mutants by preparative anion exchange chromatography, and identified by mass spectrometry, 1 H, 13 C and 31 P NMR spectroscopy, and NOESY analysis as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP). HMB-PP is 10 4 times more potent in activating human VQ Q9/VN N2 T cells than isopentenyl pyrophosphate. ß

Cell surface molecules that can act as virus receptors may exert an important selective pressure on RNA viral quasispecies. Large population passages of foot-and-mouth disease virus (FMDV) in cell culture select for mutant viruses that render dispensable a highly conserved Arg-Gly-Asp (RGD) motif responsible for integrin receptor recognition. Here, we provide evidence that viability of recombinant FMDVs including a Asp-1433Gly change at the RGD motif was conditioned by a number of capsid substitutions selected upon FMDV evolution in cell culture. Multiply passaged FMDVs acquired the ability to infect human K-562 cells, which do not express integrin ␣ v ␤ 3 . In contrast to previously described cell culture-adapted FMDVs, the RGD-independent infection did not require binding to the surface glycosaminoglycan heparan sulfate (HS). Viruses which do not bind HS and lack the RGD integrin-binding motif replicate efficiently in BHK-21 cells. Interestingly, FMDV mutants selected from the quasispecies for the inability to bind heparin regained sensitivity to inhibition by a synthetic peptide that represents the G-H loop of VP1. Thus, a single amino acid replacement leading to loss of HS recognition can shift preferential receptor usage of FMDV from HS to integrin. These results indicate at least three different mechanisms for cell recognition by FMDV and suggest a potential for this virus to use multiple, alternative receptors for entry even into the same cell type. RNA viruses mutate at rates of 10Ϫ3 to 10 Ϫ5 misincorporations per nucleotide copied; as a consequence, they evolve as complex mutant distributions termed viral quasispecies (17,19,34,35,51,52,54). Evolution of RNA viral quasispecies does not occur by the steady accumulation of mutations as replication proceeds but rather proceeds as the outcome of population disequilibrium in response to population size variations and environmental modifications. This is reflected in frequent fitness variations of RNA viruses as they replicate in cell culture or in vivo (3,12,18,27,29,33,42,64; reviewed in reference 16). Perturbation of equilibrium may lead to the rapid dominance of subsets of variants which were previously present at low frequency in the mutant spectrum. Expression at the cell surface of particular molecules which can act as receptors or coreceptors for the virus may have a major influence on the mutant distributions in viral quasispecies.Foot-and-mouth disease virus (FMDV) has been used in our laboratory as a model system to study viral quasispecies evolution, including the molecular basis of fitness variations (21, 22) and changes in host cell tropism (3,20). FMDV is an important animal pathogen that belongs to the aphthovirus genus of the Picornaviridae family (5, 55) and infects cattle and other cloven-hooved animals (artiodactyls) (2, 9). Integrin ␣ v ␤ 3 was the first molecule identified as a primary receptor for FMDV (4,6,24,38). Recent evidence suggests that integrin ␣ v ␤ 3 is the functional receptor for FMDV infections of cattle (50). The integrin ...

Recombinant LytB protein from the thermophilic eubacterium Aquifex aeolicus produced in Escherichia coli was puri¢ed to apparent homogeneity. The puri¢ed LytB protein catalyzed the reduction of (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) in a de¢ned in vitro system. The reaction products were identi¢ed as isopentenyl diphosphate and dimethylallyl diphosphate. A spectrophotometric assay was established to determine the steady-state kinetic parameters of LytB protein. The maximal speci¢c activity of 6.6 þ 0.3 W Wmol min 31 mg 31 protein was determined at pH 7.5 and 60 ‡C. The k cat value of the LytB protein was 3.7 þ 0.2 s 31 and the K m value for HMBPP was 590 þ 60 W WM.

A continuous 7802 nucleotide sequence spanning the 94% of foot and mouth disease virus RNA between the 5'-proximal poly(C) tract and the 3'-terminal poly(A) was obtained from cloned cDNA, and the total size of the RNA genome was corrected to 8450 nucleotides. A long open reading frame was identified within this sequence starting about 1300 bases from the 5' end of the RNA genome and extending to a termination codon 92 bases from its polyadenylated 3' end. The protein sequence of 2332 amino acids deduced from this coding sequence was correlated with the 260 K FMDV polyprotein. Its processing sites and twelve mature viral proteins were inferred from protein data, available for some proteins, a predicted cleavage specificity of an FMDV encoded protease for Glu/Gly(Thr, Ser) linkages, and homologies to related proteins from poliovirus. In addition, a short unlinked reading frame of 92 codons has been identified by sequence homology to the polyprotein initiation signal and by in vitro translation studies.

Various bacterial and plastidic plant terpenoids are synthesized via the non-mevalonate1-deoxy-ᴅ-xylulose-5-phosphate (DOXP) pathway. The antibiotic and herbicidal compound fosmidomycin is known to inhibit growth of several bacteria and plants, but so far its mode of action was unknown. Here we present data which demonstrate that the DOXP pathway of isoprenoid biosynthesis is efficiently blocked by fosmidomycin. The results point to the DOXP reductoisom erase as the probable target enzyme of fosmidomycin.

Fosmidomycin acts through inhibition of 1-deoxy-D-xylulose 5-phosphate (DOXP) reductoisomerase, a key enzyme of the nonmevalonate pathway of isoprenoid biosynthesis. It possesses potent antimalarial activity in vitro and in murine malaria. In a recent clinical study, fosmidomycin was effective and well tolerated in the treatment of patients with acute uncomplicated Plasmodium falciparum malaria but resulted in an unacceptably high rate of recrudescence. In order to identify a potential combination partner, the interaction of fosmidomycin with a number of antimalarial drugs in current use was investigated in a series of in vitro experiments. Synergy was observed between fosmidomycin and the lincosamides, lincomycin and clindamycin. The efficacy of a combination of fosmidomycin and clindamycin was subsequently demonstrated in the Plasmodium vinckei mouse model.In humans, isoprenoids are synthesized via the mevalonate pathway. In contrast, they are synthesized by the nonmevalonate pathway (the 1-deoxy-D-xylulose 5-phosphate [DOXP] pathway, also called the MEP pathway) in a number of bacterial species and inside the plastides of algae and higher plants (22). Similarly, the enzymes of the nonmevalonate pathway are located inside the plastide-like organelle (apicoplast) of malaria parasites (7,27). The antibiotic fosmidomycin, originally isolated from Streptomyces lavendulae, represents a potent inhibitor of DOXP reductoisomerase, a key enzyme of the nonmevalonate pathway (15,28). Recently, it was demonstrated that fosmidomycin possesses potent antimalarial activity in vitro and in murine malaria (7). FR900098, a fosmidomycin derivative, was found to be twice as effective, while the prodrug derivatives had increased oral bioavailability in the mouse model (21).In a recent clinical study conducted in Gabon and Thailand, 20 patients with acute uncomplicated Plasmodium falciparum malaria were treated with fosmidomycin administered orally (B. Lell, R. Ruangweerayut, J. Wiesner, M. Missinou, A. Schindler, T. Baranek, M. Hintz, D. Hutchinson, H. Jomaa, and P. Kremsner, unpublished data). The treatment was well tolerated and resulted in rapid parasite and fever clearance times, comparable to those obtained with conventional quinoline antimalarial agents. All patients were clinically and parasitologically cured by day 7. By day 28, however, 9 out of 18 evaluable patients experienced recrudescence. A similarly high rate of recrudescence had been observed previously when the hydroxynaphthoquinone antimalarial agent atovaquone was evaluated as a single entity (17). Subsequently, proguanil was identified as a partner for atovaquone on the basis of in vitro synergistic activity, resulting in a highly effective and welltolerated fixed drug combination, approved and marketed as Malarone (3). Using a similar approach, we have investigated the interaction of fosmidomycin with most antimalarial agents in clinical use. MATERIALS AND METHODSMaterials. Blood components were provided by the local Institute of Clinical Immunology and Tran...