Naturally derived chemical compounds are the foundation of much of our pharmacopeia, especially in antiproliferative and anti-infective drug classes. Here, we report that a naturally derived molecule called carmaphycin B is a potent inhibitor against both the asexual and sexual blood stages of malaria infection. Using a combination of in silico molecular docking and in vitro directed evolution in a well-characterized drug-sensitive yeast model, we determined that these compounds target the β5 subunit of the proteasome. These studies were validated using in vitro inhibition assays with proteasomes isolated from Plasmodium falciparum. As carmaphycin B is toxic to mammalian cells, we synthesized a series of chemical analogs that reduce host cell toxicity while maintaining blood-stage and gametocytocidal antimalarial activity and proteasome inhibition. This study describes a promising new class of antimalarial compound based on the carmaphycin B scaffold, as well as several chemical structural features that serve to enhance antimalarial specificity.

Abstract. Clinical trials documented alarming post-treatment Plasmodium vivax recurrence rates caused by recrudescence of surviving asexual blood stages, relapse from hypnozoites, or new infections. Here we describe high rates of P. vivax recurrence (26-40% 180 days after treatment) in two cohorts of rural Amazonians exposed to low levels of malaria transmission after a vivax malaria episode treated with chloroquine-primaquine. Microsatellite analysis of 28 paired acute infection and recurrence parasites showed only two pairs of identical haplotypes (consistent with recrudescences or reactivation of homologous hypnozoites) and four pairs of related haplotypes (sharing alleles at 11-13 of 14 microsatellites analyzed). Local isolates of P. vivax were extraordinarily diverse and rarely shared the same haplotype, indicating that frequent recurrences did not favor the persistence or reappearance of clonal lineages of parasites in the population. This fast haplotype replacement rate may represent the typical population dynamics of neutral polymorphisms in parasites from low-endemicity areas.

Previous microsatellite analyses of sympatric populations of Plasmodium vivax and P. falciparum in Brazil revealed higher diversity in the former species. However, it remains unclear whether regional species-specific differences in prevalence and transmission levels might account for these findings. Here, we examine sympatric populations of P. vivax (n = 87) and P. falciparum (n = 164) parasites from Pursat province, western Cambodia, where both species are similarly prevalent. Using 10 genome-wide microsatellites for P. falciparum and 13 for P. vivax, we found that the P. vivax population was more diverse than the sympatric P. falciparum population (average virtual heterozygosity [HE], 0.87 vs. 0.66, P = 0.003), with more multiple-clone infections (89.6% vs. 47.6%) and larger mean number of alleles per marker (16.2 vs. 11.1, P = 0.07). Both populations showed significant multi-locus linkage disequilibrium suggestive of a predominantly clonal mode of parasite reproduction. The higher microsatellite diversity found in P. vivax isolates, compared to sympatric P. falciparum isolates, does not necessarily result from local differences in transmission level and may reflect differences in population history between species or increased mutation rates in P. vivax.

Plasmodium vivax parasites with chloroquine resistance (CQR) are already circulating in the BrazilianAmazon. Complete single-nucleotide polymorphism (SNP) analyses of coding and noncoding sequences of the pvmdr1 and pvcrt-o genes revealed no associations with CQR, even if some mutations had not been randomly selected. In addition, striking differences in the topologies and numbers of SNPs in these transporter genes between P. vivax and P. falciparum reinforce the idea that mechanisms other than mutations may explain this virulent phenotype in P. vivax.Plasmodium vivax is the most widely distributed human malaria parasite, causing approximately 80 to 300 million clinical cases of malaria each year (17). Numerous factors indicate that this burden will increase due to the emergence and spread of chloroquine-resistant parasites (3, 17).More than 50% of the malaria cases in Latin America occur in Brazil, and P. vivax predominates as the causative agent (16,21). Notably, failures of chloroquine treatment of P. vivax malaria in the Brazilian Amazon city of Manaus have been reported recently (1). The local confirmation of the presence of active P. vivax parasites resisting chloroquine at the proposed minimal effective concentration in plasma for sensitive strains is a public health concern deserving attention.Point mutations in two digestive-vacuole membrane proteins of P. falciparum, the P. falciparum chloroquine resistance transporter (PfCRT) and multidrug resistance 1 protein (PfMDR1), have been associated with chloroquine resistance (CQR), albeit to different extents (2, 10). Orthologues of these proteins in P. vivax (P. vivax CRT-O [PvCRT-O] and PvMDR1) have been identified previously (6, 15, 18), and recently, pvmdr1 mutant alleles were suggested to be associated with both in vitro and in vivo CQR in Southeast Asia (6,20).Here, we report a single-nucleotide polymorphism (SNP) analysis of pvmdr1 and pvcrt-o genes in P. vivax isolates from chloroquine-treated patients with and without recrudescent disease in the Brazilian Amazon region. In addition to complete coding sequences, we analyzed sequences from 5Ј flanking regions and introns.Field isolates were collected during a 28-day in vivo chloroquine efficacy study conducted in the city of Manaus, Brazil (8). Plasmatic chloroquine levels in all volunteers were measured by high-performance liquid chromatography on day 3 to confirm adequate dosing and good absorption of the oral chloroquine intake (three doses of 10, 7.5, and 7.5 mg/kg of body weight in 150-mg tablet form at 24-h intervals). Clinical treatment failure was defined as the occurrence of a positive blood smear result (confirmed by PCR diagnostic analysis) on day 14, 21, or 28 and the presence of parasites in peripheral blood (collected on the same day as the positive blood smear) containing Ͼ10 ng/ml of chloroquine as determined by high-performance liquid chromatography (7). Measurements of chloroquine and its active metabolite desethylchloroquine in whole blood were not obtained, as plasma samples were c...