Selective inhibitors of human lactate dehydrogenases and lactate dehydrogenase from the malarial parasite Plasmodium falciparum.

Derivatives of the sesquiterpene 8-deoxyhemigossylic acid (2, 3-dihydroxy-6-methyl-4-(1-methylethyl)-1-naphthoic acid) were synthesized that contained altered alkyl groups in the 4-position and contained alkyl or aralkyl groups in the 7-position. These substituted dihydroxynaphthoic acids are selective inhibitors of human lactate dehydrogenase-H (LDH-H) and LDH-M and of lactate dehydrogenase from the malarial parasite Plasmodium falciparum (pLDH). All inhibitors are competitive with the binding of NADH. Selectivity for LDH-H, LDH-M, or pLDH is strongly dependent upon the groups that are in the 4- and 7-positions of the dihydroxynaphthoic acid backbone. Dissociation constants as low as 50 nM were observed, with selectivity as high as 400-fold.

A review of practical techniques for the diagnosis of malaria.

Malaria is a global health problem, responsible for nearly 3 million deaths each year, and on the increase worldwide. Improvements in malaria diagnostics should facilitate the identification of individuals infected with the malarial parasites and the treatment of such cases with appropriate drugs. Both traditional and contemporary methods for malaria diagnosis are the subjects of the present review. Traditional diagnosis, based on the examination of Giemsa-stained thick and thin blood smears under a microscope, is inappropriate for many areas because there are insufficient microscopes and/or trained microscopists to read and interpret the slides. Such traditional methods are discussed in the context of parasite quantification. Newer, more advanced malaria diagnostics are now available and the relative merits of methods based on fluorescent microscopy or the detection of nucleic acid (including PCR) are described, including comparisons of costs. Fluorescent microscopy and nucleic-acid techniques both require skills and equipment which are not universally available in many malaria-endemic countries. Recently introduced diagnostic tests based on immuno-assays solve this problem since they are easy to run and interpret, and do not require complex equipment or technical support. They are also rapid (< 10 min/test), cost-effective and at least as sensitive as traditional microscopy.

Lactate dehydrogenase and the diagnosis of malaria.

Over the past five years, several methods have been developed that exploit the differences between Plasmodium lactate dehydrogenase (pLDH) and the human LDH isoforms for the purposes of measuring pLDH in blood and in in vitro cultures. These methods have been incorporated into an easy screening method for the identification and quantitation of parasite growth in in vitro cultures using a Malstattrade mark reagent. In addition, another quantitative microplate method, the immunocapture pLDH (IcpLDH) assay, has been developed that utilizes monoclonal antibodies (mAbs) to capture the pLDH and then to measure the captured enzyme by its ability to reduce 3 acetyl pyridine adenine dinucleotide (APAD). In addition, a rapid immunochromatographic method, the OptiMAL® assay, has been formatted to capture pLDH as an antigen, and then to signal the presence of this captured antigen (enzyme) with a colloid conjugated antibody. The microplate IcpLDH assay, and the dipstick OptiMAL® assays, are both being used for the diagnosis and monitoring of malaria infections, as described here by Michael Makler, Rob Piper and Wil Milhous.

Plasmodium falciparum: evaluation of lactate dehydrogenase in monitoring therapeutic responses to standard antimalarial drugs in Nigeria.

The correlation of P. falciparum lactate dehydrogenase (pLDH) activities and patent infections was evaluated for monitoring therapeutic responses and drug resistance in 70 patients with microscopically confirmed P. falciparum malaria in Nigeria. Each patient was treated with standard dosages of artemether (53 patients), chloroquine (7 patients), sulfadoxine-pyrimethamine (6 patients), or halofantrine (4 patients). Response of infection to treatment was monitored by microscopic examination of thick and thin blood smears, clinical symptoms, and levels of pLDH activities in blood products. pLDH activity was determined using an antibody capture technique and 3-acetyl pyridine adenine dinucleotide developed to enhance sensitivity of the enzyme detection. All patients treated with artemether were cured while 5 patients treated with chloroquine, 1 treated with sulfadoxine-pyrimethamine, and 2 treated with halofantrine suffered recrudescent infections after treatment. pLDH activity was detected in blood products obtained from patients with patent or recrudescent infections determined by microscopy and clinical symptoms. Levels of pLDH activities in whole blood and packed cells from the patients correlated with qualitative detection of parasites in blood smears and in patients with high gametocyte counts. Gametocyte counts in the patients after treatment ranged from 40 gametocytes/microliter of blood to 4923 gametocytes/microliter of blood. There is a consistent relationship between patent infection and pLDH activities that could easily be determined in whole blood and packed cells from the patients. Further development of the procedure will enhance its valuable application in clinical management of drug-resistant malaria in the endemic areas.

Substrate and cofactor specificity and selective inhibition of lactate dehydrogenase from the malarial parasite P. falciparum.

Lactate dehydrogenase from the malarial parasite Plasmodium falciparum has many amino acid residues that are unique compared to any other known lactate dehydrogenase. This includes residues that define the substrate and cofactor binding sites. Nevertheless, parasite lactate dehydrogenase exhibits high specificity for pyruvic acid, even more restricted than the specificity of human lactate dehydrogenases M4 and H4. Parasite lactate dehydrogenase exhibits high catalytic efficiency in the reduction of pyruvate, kcat/Km = 9.0 x 10(8) min(-1) M(-1). Parasite lactate dehydrogenase also exhibits similar cofactor specificity to the human isoforms in the oxidation of L-lactate with NAD+ and with a series of NAD+ analogs, suggesting a similar cofactor binding environment in spite of the numerous amino acid differences. Parasite lactate dehydrogenase exhibits an enhanced kcat with the analog 3-acetylpyridine adenine dinucleotide (APAD+) whereas the human isoforms exhibit a lower kcat. This differential response to APAD+ provides the kinetic basis for the enzyme-based detection of malarial parasites. A series of inhibitors structurally related to the natural product gossypol were shown to be competitive inhibitors of the binding of NADH. Slight changes in structure produced marked changes in selectivity of inhibition of lactate dehydrogenase. 7-p-Trifluoromethylbenzyl-8-deoxyhemigossylic acid inhibited parasite lactate dehydrogenase, Ki = 0.2 microM, which was 65- and 400-fold tighter binding compared to the M4 and H4 isoforms of human lactate dehydrogenase. The results suggest that the cofactor site of parasite lactate dehydrogenase may be a potential target for structure-based drug design.

Steroidal geminal dihydroperoxides and 1,2,4,5-tetraoxanes: structure determination and their antimalarial activity.

Cholestane-derived gem-dihydroperoxides and tetraoxanes were synthesized starting from 5 alpha- and 5 beta-cholestan-3-ones by acid-catalyzed addition of hydrogen peroxide to the ketone. They were characterized by IR, NMR, and mass spectroscopy analysis aided by molecular mechanics calculations, and, in the instance of 5 beta-cholestane-3 alpha,3 beta-dihydroperoxide (6), by x-ray analysis. The synthesized compounds were tested in vitro against Plasmodium falciparum Sierra Leone (D6) and Indochina (W2) malaria clones. All compounds were inactive to both clones, with the exception of tetraoxane 7a, which exhibited modest activity toward D6 clone with IC50 = 155 nM.

Antimalarial dyes revisited: xanthenes, azines, oxazines, and thiazines.

In 1891 Guttmann and Ehrlich (P. Guttmann and P. Ehrlich, Berlin Klin. Wochenschr. 28:953-956, 1891) were the first to report the antimalarial properties of a synthetic, rather than a natural, material when they described the clinical cure of two patients after oral administration of a thiazine dye, methylene blue. Since that time, sporadic reports of the antimalarial properties of several xanthene and azine dyes related to methylene blue have been noted. We report here the results from a reexamination of the antimalarial properties of methylene blue. Janus green B, and three rhodamine dyes and disclose new antimalarial data for 16 commercially available structural analogs of these dyes. The 50% inhibitory concentrations for the chloroquine-susceptible D6 clone and SN isolate and the chloroquine-resistant W2 clone of Plasmodium falciparum were determined by the recently described parasite lactate dehydrogenase enzyme assay. No cross-resistance to chloroquine was observed for any of the dyes. For the 21 dyes tested, no correlation was observed between antimalarial activity and cytotoxicity against KB cells. No correlation between log P (where P is the octanol/water partition coefficient) or relative catalyst efficiency for glucose oxidation and antimalarial activity or cytotoxicity was observed for the dyes as a whole or for the thiazine dyes. The thiazine dyes were the most uniformly potent structural class tested, and among the dyes in this class, methylene blue was notable for both its high antimalarial potency and selectivity.

Parasite lactate dehydrogenase as an assay for Plasmodium falciparum drug sensitivity.

This report compares the use of the lactate dehydrogenase (pLDH) assay with 3H-hypoxanthine incorporation and Giemsa microscopy for the evaluation of anti-malaria drug inhibition of the growth of P. falciparum in vitro. The inhibition profiles and IC50 determinations of the pLDH assay were directly comparable to those determined by the radioactive uptake and microscopic methods. Furthermore, the pLDH culture sensitivity assay is reproducible, easily interpreted, rapid and inexpensive to perform, suggesting field applicability.

Measurement of the lactate dehydrogenase activity of Plasmodium falciparum as an assessment of parasitemia.

This report describes an enzyme assay for the detection of Plasmodium falciparum. The assay is based on the observation that the lactate dehydrogenase (LDH) enzyme of P. falciparum has the ability to rapidly use 3-acetyl pyridine NAD (APAD) as a coenzyme in the reaction leading to the formation of pyruvate from lactate. Human red blood cell LDH carries out this reaction at a very slow rate in the presence of APAD. We measured the development of APADH and found that the formation of this product could establish the basis of an assay that detected the presence of P. falciparum from in vitro cultures at parasitemia levels of 0.02%. We also had occasion to use this assay with clinical samples. We found a correlation between levels of parasitemia and the activity of parasite LDH. Parasite LDH (pLDH) activity could be measured in blood hemolysates and in plasma and serum from patients with malaria. We used the serum assay for pLDH and followed the level of pLDH in a patient with cerebral malaria prior to antimalarial treatment and during the recovery period. From these initial studies, it is evident that the measurement of pLDH has a correlation with parasitemia and may offer a method that can be developed into a simple test for the detection of Plasmodium parasitemia.

EGF is incomplete mitogen in porcine aortic smooth muscle cells: DNA synthesis without cell division.

To characterize growth effects of epidermal growth factor (EGF) in subconfluent quiescent porcine aortic vascular smooth muscle cells (VSMC), we measured DNA and protein synthesis by [3H]thymidine (Thd) and [35S]methionine (Met) incorporation, respectively, and cell proliferation rates over 0-6 days in Dulbecco's modified Eagle's-Ham's F-12 media containing 0.4% fetal calf serum (FCS) and insulin. EGF induced dose-dependent [3H]Thd uptake (P less than 0.001); after 10(-9) M EGF, DNA synthesis rate peaked at 24 h, averaging 77% of the response to 10% FCS, and then declined steeply with nadir at 48-60 h. Unexpectedly, EGF failed to induce cell proliferation in the first 4 days, leaving this initial burst of DNA synthesis (12-60 h) uncoupled from cell division. A second lesser but sustained phase of increased DNA synthesis, apparent by day 3-4, was associated with a small increase in cell number on day 6 (P less than 0.05). The early unsustained burst of DNA synthesis reflects EGF's potent mitogenic efficacy for DNA synthesis (G1- to S-phase traversal), probably acting on a subset of cells partially synchronized initially at an EGF-responsive G0/G1 locus; the minimal cell division despite brisk DNA synthesis documents EGF's limited efficacy for (or inhibition of) late cell-cycle events required for completion of mitosis. Late cell-cycle processes are thus rate limiting. EGF also increased protein synthetic rate over control (P less than 0.03) but to a lesser degree (P less than 0.01) than 10% FCS. Indomethacin (10(-6) M) did not alter DNA or proliferative responses to 10(-9) M EGF but transiently augmented EGF-induced protein synthesis (P less than 0.025) at 24 h only.(ABSTRACT TRUNCATED AT 250 WORDS)

Laboratory diagnosis of malaria.

Diagnostic procedures for the detection of malaria differ considerably depending on the aims of evaluation. The current requirements of any laboratory procedure for general application to the detection and diagnosis of malaria include: sensitivity, specificity, simplicity in application, unambiguous interpretation, and rapid turn-around time. Presently the differential stained thick and thin blood smear, examined under the microscope, remains the most reliable and definitive test for diagnosis of malaria.

Detection of Plasmodium falciparum infection with the fluorescent dye, benzothiocarboxypurine.

The fluorescent dye benzothiocarboxypurine (BCP) intensely stains nucleic acids. The dye does not penetrate viable white blood cells but does stain these cells following fixation. It has also been found that the dye stains the nucleic acid of viable Plasmodium falciparum. We have subsequently evaluated the staining of P. falciparum by benzothiocarboxypurine within red blood cells and have found that the red blood cell membrane is freely permeable to this dye and consequently P. falciparum is stained within the red blood cell. This finding prompted an in-depth analysis of the dye in the laboratory and in a field study as an alternative to Giemsa-stained blood smears and as a means of enhancing the microscopic diagnosis of malarial infection. In a field study the BCP dye allowed detection of malaria in fresh blood at a level equivalent to the Giemsa method (parasitemia ranged from 0.01% to 30%). The BCP staining procedure could also be used with fixed specimens although the differential staining characteristics were lost following specimen preparation. Of 111 blinded samples obtained in the field 22 were negative by Giemsa-stained thin smear, 16 were negative on thick smear and the same 16 were negative by BCP analysis. We have found that the BCP dye offers many advantages compared with the microscopic diagnosis of P. falciparum infection with standard Giemsa stains. These advantages are especially evident in conditions of low parasitemia, in the speed of staining and evaluation, and the relatively low level of training required to provide consistent results.

An enzyme immunoassay for the flow cytometer (FEIA).

This report describes the use of a fluorescent activated cell sorter (FACS) in combination with an enzyme immunosorbent assay (EIA). This combination of techniques expands the versatility of the flow cytometer. It introduces a new set of fluorescent enzyme products for antigen detection. These highly substantive fluorescent compounds permit the flow cytometer to quantify cell-EIA reactions and also to delineate subpopulations of cells with different quantities of surface antigens. Because the FEIA product is colored, as well as fluorescent, a simple light microscope may also be used to define the distribution of the label on the cell surface. These techniques have been applied to the examination of antigens on human erythrocytes, human T cell lymphoma cells (H9), and to surface markers on the tissue culture cell line K562.

Thiazole orange: a new dye for Plasmodium species analysis.

A rapid sensitive method for the determination of Plasmodium falciparum in in vitro culture is presented. The technique employs a fluorescent flow cytometer equipped with a 15-mwatt argon laser that emits light at 488 nm and a membrane-permeable fluorochrome thiazole orange (TO) that stains RNA. Parasitized red cells are stained by suspending them in 1 ml of phosphate-buffered saline (PBS) containing 10(-5) M of TO and incubating this mixture for 15 min in the dark at room temperature. The stained cells may be analyzed fresh or after fixation with 1% paraformaldehyde/PBS or 0.25% glutaraldehyde/PBS. Alternatively the cells may be fixed first and then stained. There is excellent correspondence between the number of fluorescent-labeled parasitized red cells and Giemsa-stained cells.

P. falciparum invasion of human red cells and cytoadherence to endothelial cells is dependent upon a parasite produced glycosidase.

This report describes biochemical and immunological evidence that P. falciparum produces an neuraminidase-like activity on invasion into the human red cell. The enzyme-like activity modifies the surface of glycophorin A on the infected red cell membrane to prevent further invasion of other merozoites, as well as to enhance the binding capabilities of the red cell to the host's endothelial cells (EC).