WO1993013197A1

WO1993013197A1 - Heme polymerase and method for treating malaria

Info

Publication number: WO1993013197A1
Application number: PCT/US1992/011279
Authority: WO
Inventors: Andrew F. G. Slater; Anthony Cerami
Original assignee: Picower Institute For Medical Research
Priority date: 1991-12-31
Filing date: 1992-12-29
Publication date: 1993-07-08
Also published as: EP0621894A1; AU3424793A

Abstract

A heme polymerase is described. The polymerase is critical in the biology of malaria. By using this novel polymerase, assays useful in developing anti-malaria drugs are made possible. Beyond providing a key for future drug studies and biochemical characterizations, treatments for malaria including quinoline-resistant malarias are made possible. Further, by employing polymerase-inhibiting techniques, such as polymerase inactivation or by offering an alternative substrate, significant breakthrough malaria treatment, prophylaxis and research are made possible.

Description

"Heme Polymerase and Method for Treating Malaria"

The invention described herein was made in the course of work under Grant No. Al 30660-01 from the National Institute of Health. The U.S. Government may have certain rights in this invention.

Background of the Invention

Throughout this application various additional publications are referenced and citations are provided in parentheses for them. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

Malaria is caused by a protozoan parasite of the genus Plasmodium. of which four species are known to infect humans: P. falciparum. P. vivax. . ovale and P. malariae. Infection is transmitted by female mosquitos of the genus Anopheles when, during a blood-meal, parasites are inoculated by the insect into a susceptible human host. Malaria transmission is indigenous in approximately 102 countries of the world and about 2.7 billion people (i.e., greater than 50% of the world's population) live in areas endemic for the disease. Although accurate evaluation of the global frequency of clinical malaria is made difficult by under-detection and under-reporting, the total incidence may be in the order of 100 million cases a year (statistics from WHO annual report, WHO Technical Report Series, 805, "Practical Chemotherapy of Malaria," WHO Geneva (1990) and New York Times, "Outwitted by Malaria, Desperate Doctors Seek New Remedies" Cl (February 12, 1991)). Malaria remains, despite considerable research and control efforts, the most prevalent and devastating parasitic disease of the tropics with global incidence of human malaria increasing during the past 20 years to an estimated 270

-i- million infected people and a staggering 2 million deaths per year.

Problems of controlling malaria are appreciated by private, national, and international health organizations, and presently, two main approaches are employed to combat the disease: the first based on empirical drug screening and the second based on vaccines. As is widely conceded, neither approach will make a significant impact on the control of malaria before the disease expands its current proportions. Moreover, there is a consensus among workers in the field that new chemotherapeutic approaches will only emerge following fundamental studies on the biochemistry of the parasite. With limited funding available and the lack of a substantial biochemical and molecular biological base, it is dubious that current efforts will make a serious dent in the malaria problem. Moreover, even when new drugs are developed, such as mefloquine, the cost is often prohibitive and prevents distribution to the general public.

The biology and life cycle of the malarial parasite are complex, and although known in broad outline, the biochemical and molecular details remain largely unknown. In the infected host, malarial parasites invade erythrocytes where they grow, mature and divide. Typically, this division is characterized by a synchronized 48 hour incubation period which causes an afflicted subject to suffer a series of high fevers. In milder cases, disruption of red blood cells leads to anemia; in more severe cases, red blood cells are trapped within small vessels of the brain and cerebral malaria ensues, which in turn results in stroke and ischemia, followed by death.

After an erythrocyte is invaded, hemoglobin is utilized by the parasite as a nutrient source. Hemoglobin comprises about 89% of the total cell protein and greater than 99% of the cytoplas ic protein. Since the malarial parasite has a limited capacity to synthesize amino acids de novo or to incorporate them exogenously, the abundant host cell hemoglobin is broken down to

-2- provide amino acids essential for parasite growth and maturation. It is estimated that 25-75% of an infected erythrocyte 's total hemoglobin is degraded by the parasite.

Concomitant with parasite maturation and hemoglobin breakdown, hemozoin (the black pigment associated with malaria) forms and accumulates within the food vacuole (large lysosomes that serve as digestive vacuoles in the proteolysis of ingested erythrocyte hemoglobin) . At the end of the erythrocytic cycle (schizogony) , the host red cell ruptures, merozoite-form parasites emerge, and the pigment-filled vacuole (known at this stage as the "residual body") is fully excised from the differentiated parasite. In vivo, the residual body is eventually scavenged by resident tissue macrophages and the pigment is accumulated in the liver, spleen, and brain of an infected patient. Hemozoin can remain in these organs indefinitely, even after the infection has been cleared. Although the process of hemoglobin degradation and hemozoin formation is of central importance to the parasite, it has not yet been successfully characterized at the molecular level.

To date, malaria has been fought primarily with quinoline- containing drugs, such as chloroquine and quinine. Quinoline- containing drugs accumulate within the acid food vacuoles of the intraerythrocytic stage malaria parasite. It has been suggested that quinolines exert their anti-malarial activity within this vacuole by raising Ph and thereby blocking hemoglobin breakdown, but it is in dispute whether this really occurs at relevant drug concentrations. Moreover, this theory fails to explain the relative inactivity of various steroisomers and congeners of chloroquine and quinine which also accumulate in the food vacuole. It has also been suggested that chloroquine forms a toxic.complex with heme inside the food vacuole; however it has not been shown that sufficient free heme is available for this to occur.

-3- The hypothesis that quinolines disrupt hemoglobin digestion or malaria pigment formation was first stated about twenty years ago. However, in spite of considerable effort, the antimalarial action of quinolines and their possible relationship to hemoglobin catabolism remained enigmatic on the molecular level. Previous attempts to study hemoglobin catabolism have been hampered by difficulties in purifying plasmodial vacuoles and their constituent enzymes. Additionally, malarial organisms resistant to quinoline-containing antimalarials have been discovered and play an increasingly important role in the epidemiology of malaria. Although initially noticed in Thailand and Colombia, quinoline- resistant malaria has been spreading throughout the world. The molecular mechanism of quinoline resistance is also unknown, but quinoline-resistance has contributed importantly to the increased incidence of human malaria over the last two decades.

Despite an appreciation that quinoline-containing antimalarial compounds accumulate in the acid food vacuoles of the parasite, the toxic mechanism of these drugs against the parasite, and more particularly the molecular basis for the specific vulnerability of growing intraerythrocytic malarial parasites to quinoline-containing drugs, has remained unascertained. As reviewed above, the primary function of the food vacuole is the proteolysis of ingested red cell hemoglobin to provide the growing parasite with essential amino acids. This breakdown of hemoglobin in the food vacuole releases heme which, were it left to accumulate in a soluble form could damage biological membranes and inhibit a variety of parasite enzymes. Rather than degrading or excreting this potentially toxic metabolite, the malarial parasite has evolved a novel pathway for its detoxification; heme is incorporated into the insoluble crystalline material called hemozoin (historically also known as "malaria pigment") . Hemozoin crystals form in the food vacuoles of the growing intraerythrocytic parasites concomitant with hemoglobin degradation, and remain there until the infected red cell bursts during schizogony. The structure of hemozoin, long

-4- a puzzle to scientists, has now been shown to comprise a polymer of heme subunits linked between the central ferric iron of one heme and a carboxylate side-group oxygen of another. This structure does not form spontaneously from either free heme or hemoglobin under physiological conditions, and the biochemistry of its formation has hitherto gone unexplained. See, A.F.G. Slater, Proc. Natl . Acad . Sci . , USA, 88: 325-329 (1991).

The present invention includes the discovery of a previously unknown heme polymerase activity. This polymerase has been identified and characterized from extracts of Plasmodium falciparum trophozoites and found to be inhibited by quinoline- containing drugs, such as chloroquine and quinine. The present invention provides an explanation of the quinolines' highly stage-specific antimalarial properties.

The subject invention further provides means for discovering and testing new anti-malarial agents, especially for use in treating quinoline-resistant malaria. By elucidating the mechanism through which quinoline-containing antimalarial drugs, either directly or indirectly, exert their specific toxicity within the food vacuole, and by clarifying the process in which malaria parasites detoxify heme, new insights into malaria treatment are realized. Further, the purified and isolated heme polymerase may be incorporated into assays to permit screening of antimalarial agents.

-5- Summary of the invention

It is an object of the subject invention to provide a heme polymerase, preferably in an isolated or purified form, suitable for use in developing treatments for malaria.

Another object of the subject invention is to provide a method for treating malaria by inhibiting the action of heme polymerase to minimize the damage caused by malarial parasites. Preferably, damage is minimized by reducing or eliminating infection through the hampering of heme polymerizing activity, effectively causing the parasites to die.

It is yet a further object of the subject invention to provide a method of screening drugs for use in treating malaria.

The subject invention provides a purified or isolated heme polymerizing enzyme characterized in that it is inhibited by quinolines, such as quinine, quinidine and chloroquine, and is further characterized in that enzymatic activity decreases at a Ph greater than about 6.5.

An agent for selectively binding with the heme polymerase is provided. This agent may be an alternate substrate or may directly act on the enzyme and may be employed in a method of inhibiting the polymerization of heme. By contacting the agent and polymerase in vivo, a prophylaxis or treatment of malaria may be accomplished. Also taught is a method of treating malaria which comprises contacting a heme polymerizing enzyme with a substrate other than heme. This method can be adapted for treating quinoline-resistant malaria and comprises administering to a subject afflicted with quinoline-resistant malaria an inhibitor of a heme polymerizing enzyme.

A method of testing a compound for its effectiveness in treating malaria is also provided. This method comprises contacting the compound to be tested, a heme polymerizing enzyme.

-6- and a substrate, and measuring the change in the amount of substrate or product. In one embodiment, the measuring comprises assaying the amount of product produced.

Lastly, the subject invention provides nucleic acids for probing and disabling the formation of the heme polymerase enzyme.

-7- Brief Description of the Figures

Figure 1A - A bar graph showing hemozoin formation (measured as nmoles heme) formed from trophozoite extracts (0.5 mg protein) incubated overnight at 37°C in 500 Mm sodium acetate pH 4.8 in the presence or absence of 400 μM hematin. Results (mean +/-sem) from 11 separate experiments are shown.

Figure IB - A bar graph showing hemozoin formation (measured as CPM from ¹C heme) formed from a trophozoite extract (65 μg protein) or a red cell extract (1.2 mg protein) incubated for 9 hr as above (see Figure 1A) in the presence or absence of 140 μM ^lC-hematin. Incorporation or radiolabel into hemozoin was determined in triplicate (mean +/-sem) .

Figure 2 - A Fourier-transform infrared spectrum of the hemozoin product after an enzyme assay described in Experiment 2. T is transmittance.

Figure 3A - A graph of hemozoin formation (pmoles heme) vs. time (mins) for an initial characterization of heme polymerase under conditions described in Experiment 3. Each data point shows mean +/- sem for triplicate assays.

Figure 3B - A graph of the rate of hemozoin formation (pmoles heme/hr) vs. amount of protein (ng) in the enzyme extract for a characterization of heme polymerase under conditions described in Experiment 3. Each data point shows mean +/- sem for triplicate assays.

Figure 3C - A graph of the rate of hemozoin formation (pmoles heme/hr) vs. pH for characterization of the heme polymerase under conditions described in Experiment 3. Sodium acetate (o) , citrate phosphate (•) or sodium phosphate (Δ) (200 mM) were used as buffers [control incubations in the absence of enzyme are also shown;

-8- acetate ( ) and citrate (*-*-)]. Each data point shows mean +/- sem for triplicate assays.

Figure 4A - A graph showing the % inhibition of heme polymerase vs. chloroquine concentration (mM) when assayed with ^Sc¬ heme as a substrate under conditions described in Experiment 4.

Figure 4B - A graph showing the % inhibition of heme polymerase vs. chloroquine concentration (mM) when assayed with a hemoglobin substrate under conditions described in Experiment 4.

Figure 4C - A graph showing the % inhibition of heme polymerase vs. drug concentration (mM) in a ¹⁴C-heme assay under conditions describe in Experiment 4. The drugs used were quinine (A), quinidine (•) and 9-epiquinine (■).

Figure 5 - A graph showing chloroquine % inhibition of purified, soluble heme polymerase. Enzyme activity is inhibited 50% at 30 mM chloroquine, which compares with 70 mM chloroquine when enzyme activity in the crude membrane fraction of a parasite extract is assayed.

-9- Detailed Description of the Invention

The inventors have discovered that malaria parasites detoxify heme in a unique way; i.e., heme is polymerized by a previously unknown polymerase to form hemozoin. The inventors have also successfully isolated an enzyme from Plasmodium falciparum having heme-polymerase activity.

The inventors propose that several other important medical and veterinary parasitic organisms have evolved a similar heme polymerizing activity to that actually identified in malaria. This is based on two lines of evidence. First, human parasites in the genus Schistosoma CS. mansoni. S. iaponicum) are known to accumulate a pigment with similar properties to that of Plasmodium hemozoin while infecting man (Homewood, Jewsbury & Chance, Comp. Biochem. Physiol. , 43B: 517-523 (1972)). It is therefore likely that heme polymerase activity is present in these organisms, and that it would be inhibited by similar compounds to those inhibiting the malaria polymerase. Second, parasites in the genus Eimeria (E. tenella) live most of their life in the intestines of their host, but under certain conditions invade the host's internal organs. While doing so they destroy and feed on red blood cells, and are known to be susceptible to chloroquine and other quinoline antimalarials (Chemotherapy of Parasitic Diseases (1986) eds. Campbell & Rew) . By analogy to malaria, it is possible that these

-10- parasites exhibit a heme polymerizing activity at such time, and that this activity is inhibited by the quinoline drug.

Isolated, purified and partially purified heme polymerizing enzymes may be incorporated into a number of biochemical assays. These assays include, but are not limited to, assays useful in screening anti-malarial agents. The assays may be performed in vivo or in vitro. By using an in vitro assay, a practical malaria drug screening system will finally be possible.

Development of the above assays are within the scope of those skilled in the art after reading the subject disclosure. Accordingly, these assays are only described briefly and not in detail. In an assay for screening drugs useful in treating malaria, the heme polymerizing enzyme of the present invention would be contacted with the drug in question in the presence of a substrate under suitable conditions. (An illustrative incubation being at 37°C, in a buffer such as 200 mM citrate phosphate at pH 5.5). The effectiveness of the drug at inhibiting the enzyme would then be evaluated by measuring the amount of decrease in product formation. Methods of detecting the product are described hereinbelow. Drugs that effectively inhibit the heme polymerizing enzyme are likely candidates for treating malaria.

Agents which selectively inhibit the newly discovered heme polymerase are also envisioned. Such agents may function by inhibiting the enzyme directly or may compete with or disable the substrate which may be heme or some other suitable substrate. An inhibiting agent may also be an alternate substrate which would likely contain a porphin macrocycle with either modified side chains or a different metal chelate. One or more inhibiting agents may be used to inhibit polymerization of heme jLn vivo or in vitro. If the contacting of the heme polymerase with an inhibiting agent occurs in vivo, it may serve as a prophylaxis or treatment for malaria. Preferably, the agent is introduced in a physiologically/ pharmaceutically acceptable carrier. The

-11- choice of physiologically acceptable carrier is readily determinable by one skilled in the art. Such carriers may be either liquid or solid and may include, but are not limited to, saline, sterile water, dextrose solutions, talcs, stearic acid, clay, sugars, salts, etc. Moreover, the mode of administering the agent for treating malaria may include, but is not limited to, injection (intravenous, intramuscular, subcutaneous, etc.), oral administration, transdermal application, inhalant, or suppository.

The invention may also be useful for treating quinoline- resistant malaria by aiding in identifying non-quinoline inhibitors and alternate or blocking substrates of heme polymerase. Typically, the agent for interfering with heme polymerase activity will be introduced in a physiologically acceptable carrier.

-12- Other agents may be produced which specifically bind with heme polymerase, such as antibodies or other molecules (e.g., substrates) . Specific binding agents may be used in assays, morphological studies and to treat malaria and related conditions.

Nucleic acids corresponding to the heme polymerase may also be identified and produced which could then be used as genetic probes or in the manufacture of anti-sense genetic material. Further, identification of the genetic material that encodes for all or segments of the active site of the heme polymerizing enzyme may be used in designing new drugs. It is to be understood that the nucleic acids may be DNA or RNA depending on the conditions of use.

For convenience and to maximize quality control, any of the enzymes, polymers and agents described in the present application may be packaged as a kit. For example, a drug screening kit might contain a labeled substrate, a heme polymerase and a buffer.

Polymerase Experimental Detail

When trophozoite extracts and radiolabeled hematin were incubated in sodium acetate at pH 4.8, a fraction of the heme was converted into a product having hemozoin-like properties (see Figure 1A) . This product was resistant to hydrolysis in weakly basic solvents and was insensitive to protease digestion. A Fourier-transform infrared spectrum of the isolated product confirmed its identify with hemozoin (Figure 2) . Hemozoin specific-absorbances at 1665 cms¹ and 1211 cms¹ were enhanced following incubation, while the spectrum did not contain features characteristic of free hematin (for example, no broad peak centered at 1226 cms^"1.) These results suggest that a component(s) of trophozoites can promote the polymerization of heme to form hemozoin. To overcome the problem of native hemozoin in the extract, experiments were repeated with ¹⁴C-heme

-13- as the substrate, such that liquid scintillation counting detected only de-novo synthesized hemozoin. The trophozoite extract catalyzed the conversion of heme into hemozoin (Figure IB) , while in similar experiments, extracts of either uninfected red blood cells or macrophages did not. Solutions of either denatured hemoglobin or albumin (both having a high non-specific affinity for heme) were also negative for heme polymerase activity in this assay. The malaria trophozoite extract is also capable of utilizing hemoglobin as the source of heme for hemozoin formation (see Figure 4B) . This is significant as it is not known whether the physiological substrate in the food vacuole is hemoglobin bound-heme, free heme, or heme associated with another vacuolar heme-binding protein.

If trophozoite extract is separated by centrifugation into a soluble protein-rich fraction and a pellet containing predominantly membranes and hemozoin, heme polymerase activity is only found in the pellet. The same result is obtained when trophozoites are extracted in the presence of either 0.5% CHAPS or 1% TRITON X-100, although the activity was destroyed following extraction in 1% SDS. Activity declines rapidly during storage of the pellet at -20°C, although pretreatment of the pellet fraction with either protease E or protease K (1 mg. ml"¹ in 50 mM Tris»HCl, pH 7.4) did not affect enzyme activity. If the hemozoin/membrane pellet is replaced in the assay with a ten-fold excess of either synthetic hemozoin or P. falciparum hemozoin purified of any protein or lipid contaminants, activity is not present. Accordingly, a component of the freshly prepared trophozoite pellet distinct from the hemozoin itself is responsible for heme polymerase activity.

The pellet fraction obtained from homogenized trophozoites was used to further characterize heme polymerase activity. Product formation was linear with time (Figure 3A) and amount of protein added (Figure 3B) . Activity increased slightly as the ionic strength of the buffer was raised, although considerable product formation occurred in all samples (50 mM - l.o M sodium

-14- acetate, pH 5.0). Optimum enzyme activity was found between pH 5.0 and 6.0 (Figure 3C) , which covers the estimated pH of malaria food vacuoles (i.e., pH 5.0 to 5.4). When pH is raised above 6.0, activity falls dramatically, possibly due to ionization of the carboxylate side-chains of the heme substrate. The linear time and protein dependence of hemozoin formation indicate the presence of a novel protease-resistant heme polymerase enzyme in malaria trophozoites, which tightly associates with the hemozoin product during isolation.

Chloroquine inhibits heme polymerase activity present in an extract of P. falciparum trophozoites (IC₅₀ ~ 120 μM, Figure 4A) . This inhibition occurs at a pH and drug concentration similar to that estimated to be achieved in the malaria food vacuole in treated patients. In similar experiments, 3-methyl chloroquine (an antimalarially active congener of chloroquine) gives 60% inhibition of heme polymerase at 1 mM, while 8-chloroquine (inactive as an antimalarial) gives no detectable inhibition at 5 mM. Chloroquine also inhibits heme polymerase when a hemoglobin-rich red cell lysate was used as the source of heme for the enzyme (Figure 4B) . The quinoline antimalarial amodiaquin, which unlike chloroquine does not form a complex with free heme, was also found to inhibit heme polymerase (IC₅₀- 250 μM) . This suggests that enzyme inhibition is not caused by a substrate-drug interaction.

Although the weak base properties of quinine and its iso ers quinidine and epiquinine are identical, their antimalarial activities vary several hundred fold. These drugs also vary in their capacity to inhibit malaria heme polymerase (Figure 4C) : quinidine (IC₅₀ - 90 μM) is the best inhibitor, followed by quinine (IC₅₀ - 300 μM) and epiquinine (IC₅₀ > 5mM) . This is the same rank order as their antimalarial activity and is consistent with heme polymerase being the physiological target for these drugs.

-15- During chemotherapy, chloroquine may reach millimolar concentrations in the food vacuole of a susceptible malaria trophozoite. This probably inhibits heme polymerase, thereby disrupting the ordered conversion of hemoglobin-bound heme into hemozoin. Heme is known to be highly toxic for proteases, and its presence would therefore be expected to rapidly block further hemoglobin degradation by malarial proteases. This may explain the specific vulnerability of growing intraerythrocytic malaria parasites. to quinoline-containing drugs. A detailed understanding of the mechanisms of action of heme polymerase opens the possibility of designing new classes of antimalarial agents, both as direct inhibitors of the polymerase and as alternative substrates to preoccupy the polymerase.

Experiment 1

The results of Experiment 1 are graphically depicted in Figures 1A-C. Experiment 1 determined hemozoin formation in an extract of malaria trophozoites. (a) Trophozoite extracts (0.5 mg protein) were incubated overnight at 37°C in 500 mM sodium acetate pH 4.8 in the presence or absence of 400 μM hematin. Results (mean +/- sem) from 11 separate experiments are shown in Figure 1. (b) A trophozoite extract (65 μg protein) or a red cell extract (1.2 mg protein) was incubated for 9 hr as above in the presence or absence of 140 μM ¹⁴C-hematin. Incorporation of radiolabel into hemozoin was determined in triplicate (mean +/- sem) .

P. falciparum clone HB-3 was cultured in A+ human erythrocytes. Synchrony was maintained by sorbitol treatment. Trophozoites were harvested by saponin lysis, washed twice in phosphate buffered saline (PBS) pH 7.0 and stored at -70°C. Trophozoite pellets were resuspended in PBS pH 7.0 with 1.5 mM MgCl_2Λ 1 mM PMSF, 1 mM 1,10 phenanthroline, 0.1 mM leupeptin and 50 μM pepstatin, and mechanically disrupted with a Dounce homogenizer. A red cell extract was prepared by homogenizing 100 μl washed, packed A+ human red cells in 10 ml PBS as above.

-16- Stock solutions of 2 mM hematin in 0.01 N NaOH were prepared fresh; 3 ml of this stock was added to 1 μCi ¹⁴C-hematin (e.g., from Leeds Radioporphyrin, UK) for the experiments in (b) . At the end of each experiment free heme was removed by extracting the sample in 1 ml of 2% SDS in 0.1 M sodium bicarbonate, pH 9.1. Hemozoin was pelleted by centrifugation, washed in bicarbonate buffer, and incubated overnight in 1 mg Protease E (Sigma) in 50 mM TrisΗCl pH 7.5. Hemozoin was again recovered by centrifugation, and the amount of heme incorporated determined either by the pyridine hemochrome method (a) or by scintillation counting (b) .

Experiment 2

The results of Experiment 2 are graphically depicted in Figure 2. Experiment 2 determined the Fourier-transform infrared spectrum of the hemozoin product after an enzyme assay.

Heme polymerase in a trophozoite extract (2.3 mg protein, 47 moles hemozoin-associated heme) was assayed with heme as described in Experiment 1(a). The product remaining after SDS- bicarbonate and protease treatment was washed three times in deionized water (dH₂0) and lyophilized. KBr pellets were prepared, and the spectrum acquired for 30 cycles in a Fourier- transform infrared spectrometer (Perkin Elmer model 1980) . A control spectrum obtained from a trophozoite extract alone was very similar to that shown, except that the peaks were less intense.

Experiment 3

The results of Experiment 3 are graphically depicted in Figure 3A-C. Experiment 3 was an initial characterization of heme polymerase determining (a) time dependent enzyme activity, (b) concentration dependent enzyme activity, and (c) pH dependent enzyme activity.

-17- Trophozoite extracts were prepared as described in Experiment 1, and centrifuged at 25,000 x g for 30 min at 4°C. The supernatant was discarded, and the membrane/hemozoin pellet resuspended in PBS pH 7.0. In (a,b) heme polymerase activity was assayed at 500 mM sodium acetate pH 5.0, while for the pH curve in (c) 200 mM sodium acetate (o) , citrate phosphate (•) or sodium phosphate (Δ) were used as buffers [control incubations in the absence of enzyme are also shown; acetate (A) and citrate (o)]. The enzyme was incubated with ¹⁴C-heme for 0-12 hr (a) or 9 hrs (b,c) . Each data point shows mean +/- sem for triplicate assays.

Experiment 4

The results of Experiment 4 are depicted graphically in Figure 4. Experiment 4 evaluated the inhibition of heme polymerase by quinoline-containing antimalarial drugs. The following were determined: (a) effect of chloroquine on heme polymerase activity assayed with ¹⁴C-heme as substrate, (b) effect of chloroquine on heme polymerase assayed with a hemoglobin substrate, and (c) comparison of quinine, quinidine and 9- epiquinine as inhibitors of heme polymerase in the ¹⁴C-heme assay.

Trophozoite extracts and ¹⁴C-heme were prepared as described in Experiment l; leupeptin and pepstatin were omitted from the trophozoite extraction buffer for the experiments shown in (b) .

Washed A+ human red cells were lysed in three volumes of dH₂0, and membranes removed by centrifugation at 25,000 x g for 30 min.

In (a) and (c) , 20 μg trophozoite protein was incubated at 30°C in 500 mM sodium acetate pH 5.0 for 10 hrs in the presence of 140 μM ^I4C-heme and various concentrations of either chloroquine diphosphate (a) or quinine hydrochloride dihydrate, quinidine sulfate dihydrate or 9-epiquinine hydrochloride dihydrate (c) .

In (b) 350 μg trophozoite protein was incubated as above in the presence of a red cell lysate (0.33 μmoles hemoglobin) and various concentrations of chloroquine diphosphate. The samples were processed as described in Experiment l, and the amount of heme incorporated determined by scintillation counting (a,c) or

-18- the pyridine hemochrome method (b) . Results are expressed as % inhibition relative to hemozoin formation in a drug-free control, and show either mean +/- sem for triplicates (a,c) or single assays (b) .

Experiment 5

Heme polymerase was purified as follows:

P. falciparum trophozoites were homogenized in 20 mM bis Tris propane pH 7.3 ("buffer A") in the presence of protease inhibitors and centrifuged at 25,000 x g for 40 min at 4°c. The supernatant was removed, and the pellet resuspended by brief sonication in buffer A containing 1 M NaCl and 10% acetonitrile. The sonicate was diluted in three volumes acetonitrile and shaken for 3 hours at 4°C, and then centrifuged as above. The upper phase of the supernatant was collected, and the pellet reextracted under identical conditions. The soluble upper phases were pooled, diluted in buffer A to 10% acetonitrile, and loaded onto a Mono Q 5/5 fplc column (Pharmacia) . Bound proteins were eluted with a linear gradient from 40 mM to 1 M NaCl. Fractions containing heme polymerase activity were pooled, diluted in one volume buffer A to 5% acetonitrile, and loaded onto a phenyl superose 5/5 fplc column (Pharmacia) . Bound proteins were eluted with a linear gradient from 5% to 90% acetonitrile. Fractions containing heme polymerase activity were pooled, and constitute the material referred to as purified enzyme. Purifications and activities are expressed in Table 1. Enzyme activity is expressed as cpm of ¹⁴C heme incorporated into pigment during the assay.

-19- Table 1

Specific Sample Protein Activity Activity Fold

(μg/ml) (cpm/μl) (cpm/μg protein) Purity

Parasite membrane sonicate 840 194 231 1

Acetonitrile upper soluble phase 44 71 1614 7

MonoQ filtered

Active fractions 2 44 22000 95

Phenyl superose active fractions (l* 47 >47000 )203

* Protein concentration was too low to be detected by conventional assay. Protease sensitivity of the purified heme polymerase activity indicates that protein is present.

Experiment 6

Soluble heme polymerase was purified as described in Experiment 5. Enzyme was eluted from a phenyl superose fplc column, and assayed in the presence of increasing concentrations of chloroquine. Heme polymerase enzyme activity is graphically expressed in Figure 5 as % inhibition relative to activity recorded in the absence of chloroquine.

Experiment 7

Sensitivity of purified, soluble heme polymerase to boiling and protease treatment was determined as follows:

Soluble heme polymerase was purified as described in Experiment 5. Enzyme eluted from a phenyl superose fplc column was assayed either before or after boiling at 100°c for 15 mins (see Table 2) . Enzyme activity is expressed as both cp of ^I4C heme incorporated into pigment during the assay and as % inhibition of activity relative to that of untreated heme polymerase enzyme. The results of this experiment are expressed in Table 2.

-20- Table 2

Treatment activity (cmp) % inhibition

control 750 boiled 29 96.1

Uponreading the subject application, various alternative embodiments will become obvious to those skilled in the art. These variations are to be considered within the scope and spirit of the subject invention and the preferred examples discussed herein are not to be construed as limiting. The subject invention is only to be limited by the claims which follow and their equivalents. Moreover, although various mechanisms are suggested throughout the application to yield a better understanding of subject invention, they are not to be considered as limiting the invention, nor are they to be held binding against the inventors.

-21-

Claims

What is claimed is:

1. An isolated heme polymerizing enzyme.

2. A purified heme polymerizing enzyme.

3. An enzyme of claim 1 or 2 that is inhibited by a quinoline.

4. An enzyme of claim 3 that is inhibited by quinine.

5. An enzyme of claim 3 that is inhibited by quinidine.

6. An enzyme of claim 3 that is inhibited by chloroquine.

7. An enzyme of claim 1 or 2 characterized in that enzymatic activity decreases at a pH greater than about 6.5.

8. An enzyme of claim 1 or 2 characterized in that it is not degraded by protease E.

9. An enzyme of claim l or 2 isolated from the genus Plasmodium.

10. An enzyme of claim 9 isolated from P. falciparum. P. vivax, P. ovale or P. malariae.

11. An enzyme of claim 10 isolated from P. falciparum.

12. An agent which selectively inhibits the enzyme of claim l or 2.

13. An agent of claim 12, wherein the agent is a substrate for a heme polymerizing enzyme.

-22-

14. An agent of claim 12, wherein the agent is a competitive inhibitor of a heme polymerizing enzyme.

15. An agent of claim 12, wherein the agent comprises a porphin ring.

16. An agent of claim 15, wherein the porphin ring is a porphyrin.

17. An agent of claim 16, wherein the porphyrin is a proto-porphyrin.

18. An agent of claim 16, wherein the porphyrin is a metallo-porphyrin.

19. An agent which selectively binds to the enzyme of claim 1 or 2.

20. An agent of claim 19, wherein the agent comprises an antibody.

21. A method of inhibiting the polymerization of heme which comprises contacting a heme polymerizing enzyme with an agent which selectively inhibits heme polymerizing activity.

22. A method of claim 21, wherein the contacting is performed j-n vitro.

23. A method of claim 21, wherein the contacting is performed .in vivo.

24. A method of claim 23, wherein the in, vivo contacting is within a subject afflicted with malaria.

25. A method of claim 24, wherein the agent is administered together with a pharmaceutically acceptable carrier.

-23-

26. A method of claim 25, wherein the subject is afflicted with a drug-resistant malaria.

27. A method of claim 26, wherein the subject is afflicted with quinoline-resistant malaria.

28. A method of claim 23, wherein the in vivo contacting is within a subject not having malaria.

29. A method of claim 28, wherein a prophylaxis for malaria results.

30. A method of testing a compound for its effectiveness in treating malaria which comprises contacting the compound to be tested, a heme polymerizing enzyme, and a substrate, and measuring the change in the amount of substrate.

31. A method of claim 30, wherein the substrate is heme.

32. A method of claim 30, wherein the enzyme is an isolated heme polymerizing enzyme.

33. A method of claim 30, wherein the measuring comprises assaying the amount of product formed from the enzyme's action on the substrate.

34. A pharmaceutical composition comprising a heme polymerizing enzyme inhibitor and a pharmaceutically acceptable carrier.

35. Apharmaceutical composition comprising an inhibitor to the heme polymerizing enzyme of claim 1 or 2 and a pharmaceutically acceptable carrier.

36. An assay kit comprising the enzyme of claim 1 or 2.

37. An assay kit comprising the enzyme of claim 11.

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38. A nucleic acid probe useful for identifying a nucleic acid encoding for a heme polymerizing enzyme.

39. A probe of claim 38 comprising DNA.

40. A probe of claim 38 comprising RNA.

41. A nucleic acid which binds with a nucleic acid encoding for a heme polymerizing enzyme.

42. A nucleic acid of claim 41 which comprises an anti- sense nucleic acid.

43. A nucleic acid of claim 42, wherein the anti-sense nucleic acid comprises DNA.

44. A nucleic acid of claim 42, wherein the anti-sense nucleic acid comprises RNA.

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