(1994). Purification of mature-stage Plasmodium falciparum by gelatine flotation. Annals of Tropical Medicine & Parasitology: Vol. 88, No. 2, pp. 209-211.
ABSTRACT The antimalarial activity and pharmacology of a series of phenylthiazolyl-bearing hydroxamate-based histone deacetylase inhibitors (HDACIs) was evaluated. In in vitro growth inhibition assays approximately 50 analogs were evaluated against four drug resistant strains of Plasmodium falciparum . The range of 50% inhibitory concentrations (IC 50 s) was 0.0005 to >1 μM. Five analogs exhibited IC 50 s of <3 nM, and three of these exhibited selectivity indices of >600. The most potent compound, WR301801 (YC-2-88) was shown to cause hyperacetylation of P. falciparum histones, which is a marker for HDAC inhibition in eukaryotic cells. The compound also inhibited malarial and mammalian HDAC activity in functional assays at low nanomolar concentrations. WR301801 did not exhibit cures in P. berghei -infected mice at oral doses as high as 640 mg/kg/day for 3 days or in P. falciparum -infected Aotus lemurinus lemurinus monkeys at oral doses of 32 mg/kg/day for 3 days, despite high relative bioavailability. The failure of monotherapy in mice may be due to a short half-life, since the compound was rapidly hydrolyzed to an inactive acid metabolite by loss of its hydroxamate group in vitro (half-life of 11 min in mouse microsomes) and in vivo (half-life in mice of 3.5 h after a single oral dose of 50 mg/kg). However, WR301801 exhibited cures in P. berghei -infected mice when combined at doses of 52 mg/kg/day orally with subcurative doses of chloroquine. Next-generation HDACIs with greater metabolic stability than WR301801 may be useful as antimalarials if combined appropriately with conventional antimalarial drugs.
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.
Adequate clinical and parasitologic cure by artemisinin combination therapies relies on the artemisinin component and the partner drug. Polymorphisms in the Plasmodium falciparum chloroquine resistance transporter ( pfcrt ) and P. falciparum multidrug resistance 1 ( pfmdr1 ) genes are associated with decreased sensitivity to amodiaquine and lumefantrine, but effects of these polymorphisms on therapeutic responses to artesunate-amodiaquine (ASAQ) and artemether-lumefantrine (AL) have not been clearly defined. Individual patient data from 31 clinical trials were harmonized and pooled by using standardized methods from the WorldWide Antimalarial Resistance Network. Data for more than 7,000 patients were analyzed to assess relationships between parasite polymorphisms in pfcrt and pfmdr1 and clinically relevant outcomes after treatment with AL or ASAQ. Presence of the pfmdr1 gene N86 (adjusted hazards ratio = 4.74, 95% confidence interval = 2.29 – 9.78, P < 0.001) and increased pfmdr1 copy number (adjusted hazards ratio = 6.52, 95% confidence interval = 2.36–17.97, P < 0.001 ) were significant independent risk factors for recrudescence in patients treated with AL. AL and ASAQ exerted opposing selective effects on single-nucleotide polymorphisms in pfcrt and pfmdr1. Monitoring selection and responding to emerging signs of drug resistance are critical tools for preserving efficacy of artemisinin combination therapies; determination of the prevalence of at least pfcrt K76T and pfmdr1 N86Y should now be routine.
Single Nucleotide Polymorphisms (SNPs) in the Pfmdr1, and Pfcrt, genes of Plasmodium falciparum may confer resistance to a number of anti-malaria drugs. Pfmdr1 86Y and haplotypes at Pfcrt 72-76 have been linked to chloroquine (CQ) as well as amodiaquine (AQ) resistance. mefloquine (MQ) and lumefantrine (LU) sensitivities are linked to Pfmdr1 86Y. Additionally, Pfcrt K76 allele carrying parasites have shown tolerance to LU. We investigated the association between Pfmdr1 86/Pfcrt 72-76 and P. falciparum resistance to CQ, AQ, MQ and LU using field samples collected during 2008–2011 from malaria endemic sites in western Kenya. Genomic DNA from these samples was genotyped to examine SNPs and haplotypes in Pfmdr1 and Pfcrt respectively. Additionally, immediate ex vivo and in vitro drug sensitivity profiles were assessed using the malaria SYBR Green I fluorescence-based assay. We observed a rapid but steady percent increase in wild-type parasites with regard to both Pfmdr1 and Pfcrt between 2008 and 2011 (p<0.0001). Equally, a significant reciprocate decrease in AQ and CQ median IC50 values occurred (p<0.0001) during the same period. Thus, the data in this study point to a significantly rapid change in parasite response to AQ and CQ in the study period. This may be due to releasing of drug pressure on the parasite from reduced use of AQ in the face of increased Artemisinin (ART) Combination Therapy (ACT) administration following the intervention of the Global Fund in 2008. LU has been shown to select for 76K genotypes, thus the observed increase in 76K genotypes coupled with significant cross resistance between LU and MQ, may herald emergence of tolerance against both drugs in future.
Abstract : High-throughput in vitro anti-malarial drug screens have traditionally incorporated the use of radioactive substrates to measure the effect test compounds have on parasitic growth. Several alternative growth inhibition screening assays using fluorescent nucleic acid intercalating dyes have been recently published. In this study we evaluated the malaria SYBR Green I-based fluorescence (MSF) assay, described by Smilkstein et al., for its use in laboratory research and in support of the U.S. Army malaria drug resistance program and the Global Emerging Infection Surveillance and Response System (GEIS) objectives. We expanded upon Smilkstein's initial characterization and validation of the MSF assay to fit our program-specific drug screening needs by testing various culture conditions commonly used for resistance screening. Plasmodium falciparum strains D6 and W2 were treated with a panel of known anti-malarial drugs and their respective IC50s were determined using the MSF assay. The results were then compared to our IC50 data generated using our standard [3H]hypoxanthine incorporation assay. Assay conditions that could potentially affect MSF assay readout, including assay length, starting parasite density and hematocrit levels, microtiter plate selection, and different culture medium components, were also examined. The IC50 values from the MSF assay showed the expected pattern of drug resistance for both parasitic strains tested when compared to the values from the [3H]hypoxanthine incorporation assay. One possible limitation of the MSF assay for some drug resistance applications is due to a significant edge effect observed, which could influence IC50 calculation. The MSF assay was easily amended for use with our robotic plate and handling equipment. Compared to our gold standard radioactive assay, the MSF assay is more cost-effective, simple, and less hazardous, while still allowing for accurate high throughput, automated drug screening.
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