Lucia Gerena

Assessment and Continued Validation of the Malaria SYBR Green I-Based Fluorescence Assay for Use in Malaria Drug Screening

ABSTRACT Several new fluorescence malaria in vitro drug susceptibility microtiter plate assays that detect the presence of malarial DNA in infected erythrocytes have recently been reported, in contrast to traditional isotopic screens that involve radioactive substrate incorporation to measure in vitro malaria growth inhibition. We have assessed and further characterized the malaria SYBR Green I-based fluorescence (MSF) assay for its ability to monitor drug resistance. In order to use the MSF assay as a drug screen, all assay conditions must be thoroughly examined. In this study we expanded upon the capabilities of this assay by including antibiotics and antifolates in the drug panel and testing folic acid-free growth conditions. To do this, we evaluated a more expansive panel of antimalarials in combination with various drug assay culture conditions commonly used in drug sensitivity screening for their activity against Plasmodium falciparum strains D6 and W2. The detection and quantitation limits of the MSF assay were 0.04 to 0.08% and ∼0.5% parasitemia, respectively. The MSF assay quality was significantly robust, displaying a Z′ range of 0.73 to 0.95. The 50% inhibitory concentrations for each drug and culture condition combination were determined by using the MSF assay. Compared to the standard [3H]hypoxanthine assay, the MSF assay displayed the expected parasite drug resistance patterns with a high degree of global and phenotypic correlation (r2 ≥ 0.9238), regardless of which culture condition combination was used. In conclusion, the MSF assay allows for reliable one-plate high-throughput, automated malaria in vitro susceptibility testing without the expense, time consumption, and hazard of other screening assays.

Role of pfmdr1 amplification and expression in induction of resistance to artemisinin derivatives in Plasmodium falciparum.

ABSTRACT Artemisinin and its derivatives are the most rapidly acting and efficacious antimalarial drugs currently available. Although resistance to these drugs has not been documented, there is growing concern about the potential for resistance to develop. In this paper we report the selection of parasite resistance to artelinic acid (AL) and artemisinin (QHS) in vitro and the molecular changes that occurred during the selection. Exposure of three Plasmodium falciparum lines (W2, D6, and TM91C235) to AL resulted in decreases in parasite susceptibilities to AL and QHS, as well as to mefloquine, quinine, halofantrine, and lumefantrine. The changes in parasite susceptibility were accompanied by increases in the copy number, mRNA expression, and protein expression of the pfmdr1 gene in the resistant progenies of W2 and TM91C235 parasites but not in those of D6 parasites. No changes were detected in the coding sequences of the pfmdr1, pfcrt, pfatp6, pftctp, and pfubcth genes or in the expression levels of pfatp6 and pftctp. Our data demonstrate that P. falciparum lines have the capacity to develop resistance to artemisinin derivatives in vitro and that this resistance is achieved by multiple mechanisms, to include amplification and increased expression of pfmdr1, a mechanism that also confers resistance to mefloquine. This observation is of practical importance, because artemisinin drugs are often used in combination with mefloquine for the treatment of malaria.

Synthesis and biological evaluation of the first pentafluorosulfanyl analogs of mefloquine.

Two novel SF5 analogs of the antimalarial agent mefloquine were synthesized in 5 steps and 10-23% overall yields and found to have improved activity and selectivity against malaria parasites. This work also represents the first report of SF5-substituted quinolines.

New class of small nonpeptidyl compounds blocks Plasmodium falciparum development in vitro by inhibiting plasmepsins.

ABSTRACT Malarial parasites rely on aspartic proteases called plasmepsins to digest hemoglobin during the intraerythrocytic stage. Plasmepsins fromPlasmodium falciparum and Plasmodium vivax have been cloned and expressed for a variety of structural and enzymatic studies. Recombinant plasmepsins possess kinetic similarity to the native enzymes, indicating their suitability for target-based antimalarial drug development. We developed an automated assay of P. falciparum plasmepsin II andP. vivax plasmepsin to quickly screen compounds in the Walter Reed chemical database. A low-molecular-mass (346 Da) diphenylurea derivative (WR268961) was found to inhibit plasmepsins with a Ki of 1 to 6 μM. This compound appears to be selective for plasmepsin, since it is a poor inhibitor of the human aspartic protease cathepsin D (Ki greater than 280 μM). WR268961 inhibited the growth of P. falciparum strains W2 and D6, with 50% inhibitory concentrations ranging from 0.03 to 0.16 μg/ml, but was much less toxic to mammalian cells. The Walter Reed chemical database contains over 1,500 compounds with a diphenylurea core structure, 9 of which inhibit the plasmepsins, withKi values ranging from 0.05 to 0.68 μM. These nine compounds show specificity for the plasmepsins over human cathepsin D, but they are poor inhibitors of P. falciparum growth in vitro. Computational docking experiments indicate how diphenylurea compounds bind to the plasmepsin active site and inhibit the enzyme.

Development and validation of flow cytometric measurement for parasitemia in cultures of P. falciparum vitally stained with YOYO-1†‡

The need for improved malaria diagnostics has long been recognized.

Point mutations in the pfcrt and pfmdr-1 genes of Plasmodium falciparum and clinical response to chloroquine, among malaria patients from Nigeria

Abstract Chloroquine (CQ) resistance in Plasmodium falciparum has been associated with specific point mutations in the pfcrt and pfmdr-1 genes. In the present study, 30 children aged 1–12 years, who were all suffering from acute, uncomplicated, P. falciparum malaria in Ibadan, Nigeria, were evaluated to assess the association between these mutations and clinical outcome following treatment with CQ. The parasites, in blood samples collected pre-treatment and, in those who failed treatment, on the day symptoms re-occurred post-treatment, were genotyped using the polymorphic MSP1, MSP2 and GLURP loci and PCR-RFLP. The results showed that, pre-treatment, all 30 patients had polyclonal infections, the mean numbers of P. falciparum clones detected per infection being 2.6 with MSP1, 4.2 with MSP2 and 2.8 with GLURP. The T76 allele of pfcrt and the Y86 allele of pfmdr-1 were found in 53% and 40%, respectively, of the pre-treatment samples from the 15 patients who failed CQ treatment, but the Y1246 mutation in pfmdr-1 was never detected. Although the parasites from the two patients with high-grade (RIII) resistance to CQ had both of these point mutations, the presence of the T76 allele of pfcrt or the Y86 allele of pfmdr-1 (considered individually) could not be used to predict treatment outcome. However, a high frequency of clonal multiplicity may confound attempts to associate the point mutations in pfcrt or pfmdr-1 with clinical response to CQ. It remains unclear whether the present results represent the characteristics of the predominant parasite populations in the study area. Further studies are needed before the strength of the association between the point mutations identified as markers of drug resistance and clinical outcome can be accurately evaluated, in this and other regions of intense transmission.

Reversal of mefloquine resistance with penfluridol in isolates of Plasmodium falciparum from south-west Nigeria

The susceptibilities of isolates of Plasmodium falciparum from Nigeria and two reference cloned strains (D6 and W2) to mefloquine or chloroquine alone and in combination with either penfluridol, a piperidine analogue, or verapamil were determined using a modification of the semiautomated microdilution technique. Six of the isolates showed reduced susceptibility to mefloquine in vitro. The response of the 6 isolates was similar to that of the mefloquine resistant reference clone D6, with 50% inhibitory concentration (IC50) values = 3.29-9.72 ng/ml. Only 2 of the Nigerian isolates were sensitive to mefloquine (IC50 = 1.16 ng/ml and 2.62 ng/ml) and were similar to the reference mefloquine sensitive clone W2 (IC50 = 1.78 ng/ml). All the isolates tested were sensitive to chloroquine, with IC50 values = 1.5-3.04 ng/ml. Simultaneous incubation of the parasites with a constant sub-inhibitory concentration of penfluridol (5.0 x 10(-7)M) and mefloquine increased the susceptibility of the resistant parasites to mefloquine. Addition of the neuroleptic drug penfluridol did not alter the response of sensitive parasites to mefloquine or chloroquine. Similarly, addition of 1.0 x 10(-6)M verapamil did not affect the activity of mefloquine against the sensitive or resistant parasites.

Antimalarial Activity of Phenylthiazolyl-Bearing Hydroxamate-Based Histone Deacetylase Inhibitors

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 (IC50s) was 0.0005 to >1 μM. Five analogs exhibited IC50s 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.

Stereochemical evaluation of the relative activities of the cinchona alkaloids against Plasmodium falciparum.

Quinine and quinidine were over 100 times more active than 9-epiquinine and 9-epiquinidine against chloroquine-sensitive Plasmodium falciparum and over 10 times more active against chloroquine-resistant P. falciparum. Since the only structural difference between quinine, quinidine, 9-epiquinine, and 9-epiquinidine is their three-dimensional configuration, the three-dimensional structures of these four alkaloids were examined in order to explain the large difference in relative activities between the 9-epi alkaloids and quinine and quinidine. The crystal structure of 9-epiquinidine hydrochloride monohydrate was determined by X-ray diffraction and was compared with the crystal structures of quinine, quinidine sulfate dihydrate, and 9-epiquinine hydrochloride dihydrate. The crystallographic parameters for 9-epiquinidine hydrochloride monohydrate were as follows: chemical formula, C20H25N2O2+.Cl-.H2O; M(r), 378.9; symmetry of unit cell, orthorhombic; space group, P2(1)2(1)2(1); parameters of unit cell, a was 7.042 +/- 0.001 A (1 A = 0.1 nm), b was 9.082 +/- 0.001 A, c was 31.007 +/- 0.005 A; the volume of unit cell was 1,983.1 +/- 0.6 A3; number of molecules per unit cell was 4; the calculated density was 1.27 g cm-3; the source of radiation was Cu K alpha (lambda = 1.54178 A); mu (absorption coefficient) was 18.82 cm-1; F(000) (sum of atomic scattering factors at zero scattering angle) was 808; room temperature was used; final R (residual index) was 5.72% for 1,501 reflections with magnitude of F(o) greater than 3 sigma (F). The intramolecular distance from N-1 to O-12 in 9-epiquinidine and 9-epiquinine, although shorter than the corresponding distance in quinine and quinidine, was similar to those of other active amino alcohol antimalarial agents. In all four alkaloids, both the hydroxyl and amine groups formed intermolecular hydrogen bonds, showing the potential for forming hydrogen bonds with cellular constituents. However, the positioning of the N+-1--H-N1 and O-12--H-O12 groups relative to each other was quite different in the 9-epi alkaloids versus quinidine. This difference in positioning may determine the relative strengths, of the formation of hydrogen bonds with cellular constituents important to antimalarial activity and, therefore, may determine the relative strength of antimalarial activity.

Antimalarial Activities of New Pyrrolo[3,2-f]Quinazoline-1,3-Diamine Derivatives

ABSTRACT WR227825 is an antimalarial pyrroloquinazolinediamine derivative with a high potency but a low therapeutic index. A series of carbamate, carboxamide, succinimide, and alkylamine derivatives of WR227825 were prepared to search for compounds with an improved therapeutic index. The new acetamides and imide showed potent cell growth inhibition against four clones of Plasmodium falciparum (D-6, RCS, W-2, and TM91C235), with a 50% inhibitory concentration of ∼0.01 ng/ml, and were highly active against Plasmodium berghei, with 100% cure at doses from <0.1 mg/kg of body weight to 220 mg/kg. The carbamates and alkyl derivatives, however, showed weak activity against Plasmodium falciparum cell growth but were highly efficacious in tests against P. berghei by the Thompson test. The best compounds, bis-ethylcarbamate (compound 2a) and tetra-acetamide (3a) derivatives, further demonstrated high potency against the sporozoite Plasmodium yoelii in mice and P. falciparum and Plasmodium vivax in aotus monkeys. Against the AMRU-1 strain of P. vivax, which has four dihydrofolate reductase mutations and is highly resistant to antifolates, tetra-acetamide 3a cured the monkeys at doses of 1 and 3 mg/kg. Compound 2a cured only one out of two monkeys at 3 mg/kg. The results indicated that the new derivatives 2a and 3a not only have retained/improved the antimalarial efficacy of the parent compound WR227825 but also were less toxic to the animals used in the tests.