WO2011060271A1 - Screening for inhibitors of p. falciparum using luciferase based high throughput screening assay - Google Patents

Abstract

The present invention includes a novel, high-throughput, luciferase based assay for identifying anti-malarial compounds. The present invention further includes novel anti-malarial compounds useful in the prevention and treatment of malaria that have been identified using the methods of the invention.

Description

TITLE OF THE INVENTION

Screening for Inhibitors of P. falciparum Using Luciferase Based High Throughput Screening Assay

BACKGROUND OF THE INVENTION

Approximately, 3.3 billion people are at risk for malaria and about 150 million of them contract the disease in any given year. Of those, malaria is responsible for almost 1 million deaths worldwide every year (Breman et al., 2001, Am. Trop. Med. Hyg. 10 64, 1-1 1),

P. falciparum, the causative agent of malaria, enters the host bloodstream via the bite of an infected mosquito. The parasite migrates to and multiplies in the liver before returning to the blood stream where it invades erythrocytes. The parasite continues to multiply within the erythrocytes until they burst, releasing large numbers of parasites into the blood stream.

Anti-malaria chemotherapy includes quinine, an alkaloid isolated from the trunk bark of the cinchona tree has been used to treat malaria since the 1600s. Chloroquine, a quinine derivative developed by Bayer during World War II, and artemisinin isolated from Artemisa annua, are also effective drugs used to treat malaria. More recently, anti-malarial compounds for use world-wide comprise an artemisinin-base combination therapy ((ACT) Coartem® a combination of artemether and lumefantrine)).

Drugs currently available for the treatment of malaria have a number of serious short-comings. In particular, current anti-malarial dmgs are accompanied by significant side effects, including rash, vomiting, diarrhea, fever and headache (atovaquone and mefloquine); cardiovascular and CNS effects (chloroquine); and blood dyscrasias (pyrimethamine and primaquine). Pharmacokinetics are typically sub-optimal with long half-life values (e.g., atovaquone, 1.5-3 days; chloroquine, days to weeks; pyrimethamine, 80-95 hours; mefloquine, 20 days), excessive protein binding (e.g., 99% with atovaquone; .about 98% with mefloquine), double peaking (e.g., atovaquone), massive volumes of distribution (chloroquine over 100 Vkg mefloquine, several times the volume of body water), and erratic and incomplete absorption of oral doses. In addition, both chloroquine-resistant and nrulti-drug resistant strains of, e.g., P. falciparum, have emerged. Drug-resistant malaria has become one of the most important problems in malaria control in recent years. Resistance //; vivo has been reported in all anti-malarial drugs, except artemisinin and its derivatives. Drug resistance necessitates the use of drugs which are more expensive and may have dangerous side effects. However there is little ongoing drug-development for the prophylaxis and treatment of malaria, and it is likely that malaria will become unbeatable.

Because of significant side-effects, cost, and the emergence of various P. falciparum strains that are resistant to virtually all commonly used anti-malarial dnigs, there is an urgent need in the art for new compounds for the treatment and prevention of malaria. The present invention meets this need.

BRIEF SUMMARY OF THE INVENTION

The invention includes a method of identifying an anti-malarial compound. The method comprises the step of introducing a detectable gene marker comprising a luciferase into a P. falciparum. The method further comprises the step of culturing the P. falciparum comprising the luciferase in the presence of red blood cells (RBCs) to generate infected RBCs. The method further comprises the step of contacting the infected RBCs with either a test or a control compound. The method further comprises the step of adding a luciferase substrate to the infected RBCs so contacted. The method further comprises the step of measuring the level of luminescence emitted from the infected RBCs so contacted, wherein the compound is identified as an antimalarial compound when the level of luminescence in the infected RBCs contacted with the test compound is inhibited >85% when compared with the level of luminescence in the infected RBCs contacted with the control compound.

In one embodiment, the compound is selected from the group consisting of an acridinedione, a cyclopentylideneamino-oxy-carbonyi, a pyrimidine, a tetrahydropirido indole, a carbazole, a dimethoxy aniline, a 1,4-naphtoquinone, a urea, a 4-piperidinopiperidine, a idenebenzene amine, a hydrazone, a piperazine, a 1- amino piperidine, a 4-amino piperidine, and a phenol. In another embodiment, the compound is selected from the group consisting of compounds 1-24, S(-)-UH-301 hydrochloride, amperozide hydrochloride, hexahydro-sila-difenidol hydrochloride pfluoro analog, BW 284c51 and SKF 95282 dimalate (Zolantidine), ion channel blockers 3',4' dichlorobenzamil, benzamil hydrochloride, 5-(N-methyl-N-isobutyl) amiloride and 5-(N,N-dimethyl) amiloride hydrochloride. In yet another embodiment, the phenol is selected from the group consisting of compounds 1-12. In yet another embodiment, the 4-amino-piperidine is selected from the group consisting of compounds 13-24. In yet another embodiment, the naphthoquinone is selected from the group consisting of compounds PC-0012225, PC-0983253, PC-1018916,

PC-1018917, PC-1018918, PC-1018919, PC-1018920, and PC-1018921.

The invention also includes a method of preventing P. falciparum infection in a mammal in need thereof. The method comprises the step of

administering a therapeutically effective amount of an anti-malarial compound to the mammal, wherein the mammal has a reduced risk of infection with P. falciparum when the anti-malarial compound is administered to the mammal. In one

embodiment, the anti-malarial compound is selected from the group consisting of an acridinedione, a cyclopentylideneamino-oxy-carbonyl, a pyrimidine, a

tetrahydropirido indole, a carbazole, a dimetoxy aniline, a 1,4-naphthoquinone, a urea, a 4-piperidinopiperidine, a idenebenzene amine, a hydrazone, a piperazine, a 1-amino piperidine, a 4-amino piperidine, and a phenol. In another embodiment, the antimalarial compound is selected from the group consisting of S(-)-UH-301

hydrochloride, amperozide hydrochloride, hexahydro-sila-difenidol hydrochloride pfluoro analog, BW 284c51 and SKF 95282 dimalate (Zolantidine), ion channel blockers 3',4' dichlorobenzamil, benzamil hydrochloride, 5-(N-methyl-N-isobutyl) amiloride and 5-(N,N-dimethyl) amiloride hydrochloride. In yet another embodiment, the phenol is selected from the group consisting of compounds 1 -12. In yet another embodiment, the 4-amino-piperidine is selected from the group consisting of compounds 13-24. In yet another embodiment, the naphthoquinone is selected from the group consisting of compounds PC-0012225, PC-0983253, PC-1018916,

PC-1018917, PC-1018918, PC-1018919, PC-1018920, and PC-1018921. In yet another embodiment, the mammal is a human. In yet another embodiment, the antimalarial compound is administered to the mammal in combination with another therapeutic agent.

The invention also includes a method of treating a mammal in need thereof infected with P. falciparum. The method comprises the step of administering to the mammal a therapeutically effective amount of an anti-malarial compound, wherein, when the anti-malarial compound is administered to the mammal, the mammal experiences either a reduction or cessation in the symptoms associated with infection with the P. falciparum , or the P. falciparum can no longer be detected in a body sample from the mammal.

In one embodiment, the anti-malarial compound is selected from the group consisting of an acridmedione, a cyclopentylideneamino-oxy-carbonyl, a pyrimidine, a tetrahydropirido indole, a carbazole, a dimetoxy aniline, a 1,4- naphtoquinone, a urea, a 4-piperidinopiperidine, a idenebenzene amine, a hydrazone, a piperazine, a 1 -amino piperidine, a 4-amino piperidine, and a phenol. In another embodiment, the anti-malarial compound is selected from the group consisting of S(-)-UH-301 hydrochloride, amperozide hydrochloride, hexahydro-sila-difenidol hydrochloride pfluoro analog, BW 284c51 and SKF 95282 dimalate (Zolantidine), ion channel blockers 3',4' dichlorobenzamil, benzamil hydrochloride, 5-(N-methyl-N- isobutyl) amiloride and 5 -(N,N- dimethyl) amiloride hydrochloride. In yet another embodiment, the phenol is selected from the group consisting of compounds 1-12. In yet another embodiment, the 4-amino-piperidine is selected from the group consisting of compounds 13-24. In yet another embodiment, the naphthoquinone is selected from the group consisting of compound PC-0012225, PC-0983253, PC-1018916, PC-1018917; PC-1018918; PC-1018919; PC-1018920, and PC-1018921. In yet another embodiment, the mammal is a human. In yet another embodiment, the anti- malarial is administered to the mammal in combination with another therapeutic agent.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are depicted in the drawings certain embodiments of the invention. However, the invention is not limited to the precise arrangements and instrumentalities of the embodiments depicted in the drawings.

Figure 1, comprising Figures 1A-1C, is a series of images depicting optimization of the P. falciparum luciferase assay. Figure 1 A is an image depicting the high throughout screen (HTS) method as described, herein. Figure IB is a series of graphs depicting (i) hematocrit concentration determination, (ii) optimal % parasitemia, (iii) luminescence reading stability over time, and (iv) optimal histidine rich protein (HRP) 2 promoter activity assay. Figure 1C, reproduced as Table 4, depicts the results of the HTS assay for known anti-malarial compounds evaluated in this assay.

Figure 2, comprising Figures 2A-2B, is a series of images depicting P. falciparum Luciferase HTS 384 well plate validation. Figure 2A depicts HTS statistics for the LOP AC, NINDS, and ChemBridge libraries. Figure 2B, reproduced as Table 5, is a series of graphs which depict luminescence measured as a function of well number for a sample (i) LOP AC assay plate, (ii) NINDS assay plate, and (iii) ChemBridge assay plate.

Figure 3, comprising Figures 3A-3B, is a series of images depicting the structures and distribution of (a) ChemBridge library HTS hits scaffolds distribution (b) number of hits for each scaffold.

Figure 4 is a table summarizing the cytotoxicity (IC₅₀) of selected hits from HTS against HA C and HepG2 cells. Data shown are means from 3 replicates ± standard errors of the means. Doxorubicin was used as the positive control.

Figure 5 is a table illustrating IC₅₀ values of amperozide derivatives.

Figure 6 is a table illustrating IC₅₀ values of H2 antagonists.

Figure 7, comprising Figures 7A-7B, is a table illustrating the

ChemBridge hits in terms of scaffold distribution, number of hits and IC₅₀ activity range.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is related to compounds, compositions and methods useful in the treatment and prevention of malaria. Definitions:

As used herein, each of the following terms has the meaning associated with it in this section.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

The term "about" will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used.

As used herein, the term " pharmaceutically acceptable derivative", means any pharmaceutically acceptable salt, solvate, or prodrug e.g. ester or carbamate of a compound, which upon administration to the recipient is capable of providing (directly or indirectly) the compound, or an active metabolite or residue thereof. Such derivatives are recognizable to those skilled in the mi, without undue experimentation. Nevertheless, reference is made to the teaching of Burger's

Medicinal Chemistiy and Drug Discovery, 5th Edition, Vol 1: Principles and Practice, which is incorporated herein by reference to the extent of teaching such derivatives. In one aspect of the invention pharmaceutically acceptable derivatives are salts, solvates, esters and carbamates. In another aspect of the invention pharmaceutically acceptable derivatives are salts, solvates and esters. In a further aspect,

pharmaceutically acceptable derivatives are salts and solvates.

As used herein, the term "pharmaceutically acceptable salts" refers to salts that retain the desired biological activity of the subject compound and exhibit minimal undesired toxicological effects. For a review on suitable salts see Berge et al, J, Pharm. Sci., 1977, 66: 1-19. The term "pharmaceutically acceptable salts" includes both pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts. These pharmaceutically acceptable salts may be prepared in situ during the final isolation and purification of the compound, or by separately reacting the purified compound in its free acid or free base form with a suitable base or acid, respectively. The salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent.

As used herein, "treatment" means: (1) the amelioration or prevention of the condition being treated or one or more of the biological manifestations of the condition being treated, (2) the interference with (a) one or more points in the biological cascade that leads to or is responsible for the condition being treated or (b) one or more of the biological manifestations of the condition being treated, or (3) the alleviation of one or more of the symptoms or effects associated with the condition being treated. The skilled artisan will appreciate that "prevention" is not an absolute term. In medicine, "prevention" is understood to refer to the prophylactic

administration of a drug to substantially diminish the likelihood or severity of a condition or biological manifestation thereof, or to delay the onset of such condition or biological manifestation thereof.

As used herein, "safe and effective amount" means an amount of the compound sufficient to significantly induce a positive modification in the condition to be treated but low enough to avoid serious side effects (at a reasonable benefit/risk ratio) within the scope of sound medical judgment. A safe and effective amount of a compound of the invention will vary with the particular compound chosen (e.g. depending on the potency, efficacy, and half-life of the compound); the route of administration chosen; the nature of the infection and/or condition being treated; the severity of the infection and/or condition being treated; the age, size, weight, and physical condition of the patient being treated; the medical history of the patient to be treated; the duration of the treatment; the nature of concurrent therapy; the desired therapeutic effect; and like factors, but can nevertheless be routinely determined by the skilled artisan.

Description:

The present invention includes a novel, high-throughput, luciferase- based assay for identifying anti-malarial compounds,

The present invention further includes anti-malarial compounds useful in the prevention and treatment of malaria that have been identified using the methods of the invention. An anti-malarial compound of the instant invention is one which inhibits any of the red blood cell stages of P. falciparum growth or development, including the merozoite stage, ring stage, trophozoite stage and schizont stage.

The present invention also includes methods for the treatment and prevention of malaria in a mammal infected with, or at risk of becoming infected with the P. falciparum using the anti-malarial compounds disclosed herein.

I. Luciferase based Assay for Identifying compounds that inhibit P. falciparum

The invention encompasses a novel luciferase-based high throughput screening (HTS) assay for identifying compounds that inhibit P. falciparum development in red blood cells. The assay comprises a method for screening at least one test compound, in the presence of at least one control for efficacy of the test compound in inhibiting P. falciparum development in red blood cells.

The assay comprises the steps of;

(a) Transfecting P. falciparum with a detectable gene marker. In one embodiment, the marker is an enzyme that acts upon a substrate to produce a detectable signal. The substrate may be endogenous to the cell or supplied to the cell exogenousiy, for example in a culture medium. In another embodiment, the marker is a bioluminescent gene marker. In yet another embodiment, the biomarker is a luciferase. It will be understood by the skilled artisan that the instant invention is not limited to the markers recited herein, but encompasses any detectable marker, both known and unknown in the art, provided that the marker fulfills the requirements of the assay as described herein.

(b) Culturing the transfected P. falciparum in the presence of red blood cells under conditions whereby the red blood cells are infected with the transfected P. falciparum.

(c) Contacting the red blood cells infected with the transfected P. falciparum with a test compound, It will be readily understood by the skilled artisan that more than one test compound may be screened at a time in a given assay. In one embodiment, at least one test compound in screened in an assay. In another embodiment, between 1 and 10 test compounds are screened in an assay. In still another embodiment between 1 and 100 test compounds are screened in an assay. In yet another embodiment, between 1 and 1,000 test compounds are screened in an assay. In yet another embodiment, between 1 and 5,000 test compounds are screened in an assay. In still another embodiment between 1 and 10,000 test compounds are screened in an assay.

(d) Optionally, providing a substrate for the gene marker. In one embodiment, when the gene marker is luciferase, a luciferase substrate is added.

(e) Quantifying the gene marker in cells contacted with a test compound and comparing the amount of gene marker detected in the presence of a test compound to the amount of gene marker detected in a control. In one embodiment, the luminescence resulting from the reaction of a luciferase with its substrate is measured using techniques well known in the art. If a test compound inhibits the luminescence resulting from the reaction of a luciferase with its substrate by greater than or equal to 85% of the luminescence of a control, the test compound is identified as an anti-malarial compound.

At least one control must be run for each assay regardless of the number of test compounds being evaluated in a given assay. In one embodiment, a control used in the assay is a compound or composition known not to inhibit the growth or' development of P. falciparum. Accordingly, a control in the instant assay may comprise a vehicle, such as a buffer, solution, or medium, used for the preparation of a test compound where the vehicle does not further comprise a test drug. A control in the assay may also comprise a compound known not to be an anti- malarial compound. In another embodiment, a control comprises exposing uninfected red blood cells to a test compound. In still another embodiment, a control comprises infecting P. falciparum with a control vector that does not comprise a functional gene marker.

The control is measured for luminescence resulting from the reaction of luciferase and its substrate at the same time as the luminescence for test compounds is quantified. The luminescence measurements for a test compound and a control are compared and the percent inhibition is calculated for a test compound relative to a control.

In a preferred embodiment, the assay comprises the steps of tiansfecting P. falciparum 3D7 parasites with firefly luciferase under the control of histidine rich protein (HRP) 2 promoter. Ten microliters (10 μL) of culture media are dispensed into each well of a multi-well plate, In a subset of wells, one hundred and ten (110) nL of a test solution, wherein a test solution comprises a test compound, is transferred to individual wells. In another subset of wells, one hundred and ten (110) nL of a control solution is transferred to individual wells, wherein a control solution comprises either a vehicle without a test drug or a drug known not to be an anti-malarial compound, Thirty (30) μL of red blood cells infected with synchronized parasites at the ring stage are added to each well containing both test and control solutions. The plates are covered to prevent evaporation and maintained at 37°C for 48 hours. After this period, 40 μL of luciferase substrate is added to each well and luminescence signal detected in an Envision plate reader,

Test molecules for use in the method of identifying an anti-malarial compound may be obtained using any of the numerous approaches in combinatorial library methods known in the art, including biological libraries, spatially-addressable parallel solid phase or solution phase libraries, synthetic library methods requiring decon volution, the "one -bead one- compound" library method, and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, nonpeptide oligomer, or small molecule libraries of compounds (Lam, 1997,

Anticancer Drug Des. 12: 145),

Examples of methods for the synthesis of molecular libraries may be found in the art, for example, in: DeWitt et al., 1993, Proc, Natl. Acad. Sci. USA 90:6909-6913; Erb et al., 1994, Proc. Natl. Acad. Sci. USA 91 : 11422-1 1426;

Zuckermann et al., 1994, J. Med. Chem. 37:2678-2685; Cho et al., 1992, Science

261 : 1303-1305; Carell et al., 1994, Angew. Chem. Int. Ed. Engl. 33:2059-2061 ;

Carell et ali., 1994, Angew. Chem. Int. Ed. Engi. 33:2061-2064; and Gallop et al., 1994, J. Med. Chem.37: 1233-1251.

Libraries of compounds may be presented in solution (e.g., Houghten,

1992, Bio/Techniques 13:412-421), or on beads (Lam, 1991, Nature 354:82-84), chips

(Fodor, 1993, Nature 364:555-556), bacteria (U.S. Pat. No. 5,223,409), spores (U.S.

Pat. Nos. 5,571 ,698; 5,403,484; and 5,223,409), plasmids (Cull et al., 1992, Proc. Natl. Acad. Sci. USA 89: 1865-1869), or phage (Scott and Smith, 1990, Science

249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al., 1990, Proc. Natl.

Acad. Sci. USA 87:6378-6382; and Felici, 1991, J Mol. Biol. 222:301-310).

The resulting libraries of candidate molecules may be screened using the assay described herein for efficacy in inhibiting P. falciparum development in the red blood cells of a mammal.

II. Compositions

The anti-malarial compounds of the instant invention comprise compounds identified using the assay described herein. These compounds inhibit the progression of the invasion and rupture of erythrocytes by P. falciparum.

A compound useful in the present invention as a potential prophylactic agent for preventing malaria in a mammal may be a peptide, a nucleic acid, a small molecule, or other drug that inhibits P. falciparum development in the red blood cells of a mammal.

A compound useful in the present invention as a potential therapeutic agent for treating malaria in a mammal may be a peptide, a nucleic acid, a small molecule, or other drug that inhibits P. falciparum development in the red blood cells of a mammal. Accordingly, the compounds of the instant invention are not limited to those recited specifically herein.

Nucleic Acids

When the anti-malarial compound of the invention comprises a nucleic acid, any number of procedures may be used for the generation of an isolated nucleic acid encoding the compound as well as derivative or variant forms of the isolated nucleic acid, including using recombinant DNA methodology well known in the art (see Sambrook et al., 2001 , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York; Ausubel et al., 2001 , Current Protocols in Molecular Biology, Green & Wiley, New York) or direct synthesis of the nucleic acid. For recombinant nucleic acids encoding the compound and in vitro

transcription, DNA encoding RNA molecules can be obtained from known clones of the compound, by synthesizing a DNA molecule encoding an RNA molecule, or by cloning the gene encoding the RNA molecule. Techniques for in vitro transcription of RNA molecules and methods for cloning genes encoding known RNA molecules are described by, for example, Sambrook et al. 2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

As an example, a method for synthesizing nucleic acids de novo involves the organic synthesis of a nucleic acid from nucleoside derivatives, This synthesis may be performed in solution or on a solid support, One type of organic synthesis is the phosphotriester method, which has been used to prepare gene fragments or short genes. In the phosphotriester method, oligonucleotides are prepared which can then be joined together to form longer nucleic acids. For a description of this method, see Narang et al. (1979, Meth. Enzymol., 68:90) and U.S. Pat. No. 4,356,270. The phosphotriester method can be used in the present invention to synthesize an isolated anti-malarial nucleic acid.

In addition, the compositions of the present invention can be synthesized in whole or in part, or an isolated anti-malarial nucleic acid can be conjugated to another nucleic acid using organic synthesis such as the phosphodiester method. See Brown et al. (1979, Meth. Enzymol. 68: 109) for a description of this method. As in the phosphotriester method, the phosphodiester method involves synthesis of oligonucleotides which are subsequently joined together to form the desired nucleic acid.

A third method for synthesizing nucleic acids, described in U.S. Pat. No. 4,293,652, is a hybrid of the above-described organic synthesis and molecular cloning methods. In this process, the appropriate number of oligonucleotides to make up the desired nucleic acid sequence is organically synthesized and inserted sequentially into a vector which is amplified by growth prior to each succeeding insertion.

In addition, molecular biological methods, such as using a nucleic acid as a template for a PCR reaction, or cloning a nucleic acid into a vector and transforming a cell with the vector can be used to make large amounts of the nucleic acid of the present invention,

Anti-malarial compounds may include small synthetic nucleic acid compounds. Thus, oligonucleotide agents are incorporated herein and include otherwise unmodified RNA and DNA as well as RNA and DNA that have been modified, e.g., to improve efficacy, and polymers of nucleoside surrogates.

Unmodified RNA refers to a molecule in which the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are the same or essentially the same as that which occur in nature, preferably as occur naturally in the human body. The art has referred to rare or unusual, but naturally occurring, RNAs as modified RNAs, see, e.g., Limbach et al. (1994, Nucleic Acids Res, 22: 2183-2196), Such rare or unusual RNAs, often termed modified RNAs, are typically the result of a post- transcriptional modification and are within the term unmodified RNA as used herein. Modified RNA, as used herein, refers to a molecule in which one or more of the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are different from that which occur in nature, preferably different from that which occurs in the human body,

As nucleic acids are polymers of subunits or monomers, many of the modifications described below occur at a position which is repeated within a nucleic acid, e.g., a modification of a base, or a phosphate moiety, or a non-linking O of a phosphate moiety. In some cases the modification will occur at all of the subject positions in the nucleic acid but in many, and in fact in most cases it will not. By way of example, a modification may only occur at a 3' or 5' terminal position, in a terminal region, e.g., at a position on a terminal nucleotide, or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. A component can be attached at the 3' end, the 5' end, or at an internal position, or at a combination of these positions. For example, the component can be at the 3' end and the 5' end; at the 3' end and at one or more internal positions; at the 5' end and at one or more internal positions; or at the 3' end, the 5' end, and at one or more internal positions. For example, a phosphorothioate modification at a non-linking O position may only occur at one or both termini, or may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of the oligonucleotide. The 5' end can be phosphorylated.

For increased nuclease resistance and/or binding affinity to the target, an oligonucleotide agent, can include, for example, 2^,-modified ribose units and/or phosphorothioate linkages. E.g., the 2' hydroxyl group (OH) can be modified or replaced with a number of different "oxy" or "deoxy" substituents.

Examples of "oxy" -2' hydroxyl group modifications include alkoxy or aryloxy (OR, e.g., R = H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar);

polyethyleneglycols (PEG), 0(CH₂CH₂0)_nCH₂CH₂OR; "locked" nucleic acids (LNA) in which the 2' hydroxyl is connected, e.g., by a methylene bridge, to the 4' carbon of the same ribose sugar; amine, 0-AMINE and aminoalkoxy, 0(CH₂)_nAMINE, (e.g., AMINE = NH₂; alkylamino, dialkylamino, heterocyclyl amino, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino). It is noteworthy that oligonucleotides containing only the methoxyethyl group (MOE), (OCH₂CH₂OCH₃, a PEG derivative), exhibit nuclease stabilities comparable to those modified with the robust phosphorothioate modification.

Preferred substitutents include but are not limited to 2' -methoxyethyl, 2'-OCH₃, 2'-0-allyl, 2'-C- ally], and 2'-fluoro.

"Deoxy" modifications include hydrogen (i.e. deoxyribose sugars); halo (e.g., fluoro); amino (e.g. NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid);

NH(CH₂CH₂NH)_nCH₂CH₂ -AMINE (AMINE = NH₂; alkylamino, dialkylamino, heterocyclyl amino, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino), -NHC(0)R (R = alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., an amino functionality.

One way to increase resistance of a nucleic acid to nuclease activity, particularly RNA to RNase activity, is to identify cleavage sites and modify such sites to inhibit cleavage. For example, the dinucleotides 5'-UA-3', 5'-UG-3', 5'-CA-3', 5'- UU-3', or 5' -CC-3' can serve as cleavage sites. Enhanced nuclease resistance can therefore be achieved by modifying the 5' nucleotide, resulting, for example, in at least one 5'-uridine-adenine-3' (5'-UA-3') dinucleotide wherein the uridine is a 2'- modified nucleotide; at least one 5 '-uridine -guanine -3' (5'-UG-3') dinucleotide, wherein the 5'-uridine is a 2' -modified nucleotide; at least one 5'-cytidine-adenine-3' (5' -CA-3') dinucleotide, wherein the 5'-cytidine is a 2' -modified nucleotide; at least one 5' uridine-uridine -3' (5'-UU-3') dinucleotide, wherein the 5'-uridine is a 2'- modified nucleotide; or at least one 5' -cytidine-cytidine-3' (5¹ -CC-3') dinucleotide, wherein the 5'-cytidine is a 2'-modified nucleotide. In certain embodiments, all the pyrimidines of the miRNA inhibitor carry a 2'-modification, and the miRNA inhibitor therefore has enhanced resistance to endonucleases.

In addition, to increase nuclease resistance, the 2' modifications can be used in combination with one or more phosphate linker modifications (e.g., phosphorothioate). The so-called "chimeric" oligonucleotides are those that contain two or more different modifications.

With respect to phosphorothioate linkages that serve to increase protection against RNAse activity, the miRNA inhibitor can include a

phosphorothioate at least the first, second, or third internucleotide linkage at the 5' or 3' end of the nucleotide sequence. In one embodiment, the miRNA inhibitor includes a 2' -modified nucleotide, e.g., a 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-0-methyl, 2'-0- methoxyethyl (2'-0-MOE), 2'-0-aminopropyl (2'-0-AP), 2'-0-dimethylaminoethyl (2'-0-DMAOE)_} 2'-0-dimethylaminopropyl (2'-0-DMAP)_} 2*-0- dimethylaminoethyloxyethyl (2'-0-DMAEOE), or 2'-0-N-methylacetamido (2'-0- NMA). In a preferred embodiment, the miRNA inhibitor includes at least one 2'-0- methyl-modified nucleotide, and in some embodiments, all of the nucleotides of the miRNA inhibitor include a 2'-0-methyl modification.

The 5' -terminus can be blocked with an aminoalkyl group, e.g., a 5'-0- alkylamino substituent. Other 5' conjugates can inhibit 5'-3' exonucleoiytic cleavage. While not being bound by theory, a 5' conjugate, such as naproxen or ibuprofen, may inhibit exonucleoiytic cleavage by sterically blocking the exonuclease from binding to the 5' end of the oligonucleotide. Even small alkyl chains, aryl groups, or

heterocyclic conjugates or modified sugars (D-ribose, deoxyribose, glucose etc.) can block 3'-5'exonucleases.

The oligonucleotide can be constructed using chemical synthesis and/or enzymatic ligation reactions using procedures known in the art. For example, an oligonucleotide can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the oligonucleotide and target nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Other appropriate nucleic acid modifications are described herein. Alternatively, the oligonucleotide can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i. e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest (e.g., an mR A, pre-mRNA, or an miRNA).

Any polynucleotide of the invention may be further modified to increase its stability in vivo. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends; the use of phosphorothioate or 2' O-methyl rather than phosphodiester linkages in the backbone; and/or the inclusion of nontraditional bases such as inosine, queosine, and wybutosine and the like, as well as acetyl- methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine, and uridine.

Peptides

When the anti-malarial compound of the invention is a peptide, the peptide may be chemically synthesized by Merrifield-type solid phase peptide synthesis. This method may be routinely performed to yield peptides up to about 60- 70 residues in length, and may, in some cases, be utilized to make peptides up to about 100 amino acids long. Larger peptides may also be generated synthetically via fragment condensation or native chemical ligation (Dawson et al., 2000, Ann. Rev. Biochem. 69:923-960). An advantage to the utilization of a synthetic peptide route is the ability to produce large amounts of peptides, even those that rarely occur naturally, with relatively high purities, i.e., purities sufficient for research, diagnostic or therapeutic purposes.

Solid phase peptide synthesis is described by Stewart et al. in Solid Phase Peptide Synthesis, 2nd Edition, 1 84, Pierce Chemical Company, Rockford, Illinois; and Bodanszky and Bodanszky in The Practice of Peptide Synthesis, 1984, Springer- Verlag, New York. At the outset, a suitably protected amino acid residue is attached through its carboxyl group to a derivatized, insoluble polymeric support, such as cross-linked polystyrene or polyamide resin. "Suitably protected" refers to the presence of protecting groups on both the a. -amino group of the amino acid, and on any side chain functional groups. Side chain protecting groups are generally stable to the solvents, reagents and reaction conditions used throughout the synthesis, and are removable under conditions which will not affect the final peptide product.

Stepwise synthesis of the oligopeptide is carried out by the removal of the N- protecting group from the initial amino acid, and coupling thereto of the carboxyl end of the next amino acid in the sequence of the desired peptide. This amino acid is also suitably protected. The carboxyl of the incoming amino acid can be activated to react with the N-terminus of the support-bound amino acid by formation into a reactive group, such as formation into a carbodiimide, a symmetric acid anhydride, or an "active ester" group, such as hydroxybenzotriazole or pentafluorophenyl esters.

Examples of solid phase peptide synthesis methods include the BOC method, which utilizes tert-butyloxcarbonyl as the a-amino protecting group, and the FMOC method, which utilizes 9-fluorenylmethyloxcarbonyl to protect the a-amino of the amino acid residues. Both methods are well-known by those of skill in the art.

Incorporation of N- and/or C- blocking groups may also be achieved using protocols conventional to solid phase peptide synthesis methods. For incorporation of C-terminal blocking groups, for example, synthesis of the desired peptide is typically performed using, as solid phase, a supporting resin that has been chemically modified so that cleavage from the resin results in a peptide having the desired C-terminal blocking group. To provide peptides in which the C-terminus bears a primary amino blocking group, for instance, synthesis is performed using a p- methylbenzhydrylamine (MBHA) resin, so that, when peptide synthesis is completed, treatment with hydrofluoric acid releases the desired C-terminally amidated peptide. Similarly, incorporation of an N-methylamine blocking group at the C-terminus is achieved using N-methylaminoethyl-derivatized DVB (divinylbenzene) resin, which upon hydrofluoric acid (HF) treatment releases a peptide bearing an N- methyl amida ted C-terminus. Blockage of the C-terminus by esterification can also be achieved using conventional procedures. This entails use of resin/blocking group combination that permits release of side-chain peptide from the resin, to allow for subsequent reaction with the desired alcohol, to form the ester function. FMOC protecting group, in combination with DVB resin derivatized with

methoxyalkoxybenzyl alcohol or equivalent linker, can be used for this purpose, with cleavage from the support being affected by trifluoroacetic acid (TFA) in

dichloromethane. Esterification of the suitably activated carboxyl function, e.g. with dicyclohexylcatbodiimide (DCC), can then proceed by addition of the desired alcohol, followed by de-protection and isolation of the esterified peptide product.

Incorporation of N-terminal blocking groups may be achieved while the synthesized peptide is still attached to the resin, for instance by treatment with a suitable anhydride and nitrile. To incorporate an acetyl blocking group at the N- terminus, for instance, the resin-coupled peptide can be treated with 20% acetic anhydride in acetonitrile, The N-blocked peptide product may then be cleaved from the resin, deprotected and subsequently isolated.

Prior to its use as an anti-malarial compound in accordance with the invention, a peptide is purified to remove contaminants. Anyone of a number of a conventional purification procedures may be used to attain the required level of purity including, for example, reversed-phase high-pressure liquid chromatography (HPLC) using an alkylated silica column such as C₄ -,C₈- or CJS- silica. A gradient mobile phase of increasing organic content is generally used to achieve purification, for example, acetonitrile in an aqueous buffer, usually containing a small amount of trifluoroacetic acid. Ion-exchange chromatography can be also used to separate polypeptides based on their charge. Affinity chromatography is also useful in purification procedures.

Peptides may be modified using ordinaiy molecular biological techniques to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent, Analogs of such polypeptides include those containing residues other than naturally occurring

L-amino acids, e.g., D-amino acids or non-naturally occurring synthetic amino acids.

The polypeptides useful in the invention may further be conjugated to non-amino acid moieties that are useful in their application. In particular, moieties that improve the stability, biological half-life, water solubility, and immunologic characteristics of the peptide are useful, A non-limiting example of such a moiety is polyethylene glycol

(PEG).

Small Molecules

When the anti-malarial compound of the invention is a small molecule, the small molecule may be obtained using standard methods known to the skilled artisan. Such methods include chemical organic synthesis or biological means.

Biological means include purification from a biological source, recombinant synthesis and in vitro translation systems, using methods well known in the art.

Combinatorial libraries of molecularly diverse chemical compounds potentially useful in treating a variety of diseases and conditions are well known in the art as are methods of making such libraries. The method may take advantage of a variety of techniques well-known to the skilled artisan including solid phase synthesis, solution methods, parallel synthesis of single compounds, synthesis of chemical mixtures, rigid core structures, flexible linear sequences, deconvolution strategies, tagging techniques, and generating unbiased molecular landscapes for lead discovery vs. biased structures for lead development.

In a general method for small library synthesis, an activated core molecule is condensed with a number of building blocks, resulting in a combinatorial library of covalently linked, core-building block ensembles. The shape and rigidity of the core determines the orientation of the building blocks in shape space. The libraries can be biased by changing the core, linkage, or building blocks to target a characterized biological structure ("focused libraries") or synthesized with less structural bias using flexible cores.

In one embodiment, an anti-malarial compound of the instant invention is selected from the gr oup consisting of acrid inediones, cyclopentylideneamino-oxy- carbonyl, pyrimidines, tetrahydropirido indoles, carbazoles, dimetoxy anilines, 1,4- naphtoquinones, ureas, 4-piperidinopiperidine, idenebenzene amines, hydrazones, piperazine, 1- amino piperidines, 4-aminopiperidines, and phenols.

In another embodiment, an anti-malarial compound of the invention comprises a phenol based compound, including but not limited to compounds 1 -12 as 25 described below and in Table I.

Compound (1 ) is 2,4-dibromo-6-(N-butyl-N-methyl-amino)phenol, Compound (2) is 2-(N-benzyl-N-methyl-amino)-4,6-dibromophenol, Compound (3) is 2,4-dibromo-6-(phenethylamino)phenol. Compound (4) is 2-(N-benzyl-N- butylamino)phenol. Compound (5) is 4-methoxy-2-(piperidin-I-yl)phenol.

Compound (6) is 2,4-dichloro-6-(3-methylpiperidin-l-yl)phenol. Compound (6a) is 2-bromo-6-(3-methylpiperidin-l-yl)phenol. Compound (7) is 2-(octahydroquinolin-l (2H)-yl)phenol. Compound (8) is 2-(azocan-l -yl)-6-chIorophenol. Compound (8a) is 2-(azocan- l-yl)-6-bromophenol, Compound (9) is 2-bromo-6-((N-cyclopropylmethyl- N-propyl)amino)-4-methoxyphenol. Compound (9a) is 2,4-dichloro-6-((N- cyclopropylmethyl-N-propyl)amino)phenol. Compound (9b) is 2,4-dibromo-6-((N- cyclopropylmethyl-N-propyi)amino)phenol. Compound (10) is 2-chloro-6-(5-(4- fluorophenyl)-4H-pyrazol-3-yl)phenol. Compound (11) is 2-(5-(4-bromophenyl)-4H- pyrazol-3-yl)-6-methylphenol, Compound (12) is 2-chloro-6-(5-(4-chlorophenyl)-4H- pyrazol-3-yl)phenol.

In another embodiment, an anti-malarial compound of the invention comprises a 4-amino-piperidine based compound, including but not limited to compounds 13-24 as described below and in Table 2.

Compound (13) is N-benzhydryl-l-propylpiperidin-4-amine.

Compound (14) is N-(2,2-diphenylethyl)-l-propylpiperidin-4-amine, Compound (15) is l-benzyl-N(3-chlorobenzyl)piperidin-4-amine. Compound (16) is l-benzyl-N-(4- chlorobenzyl)-piperidin-4-amine. Compound (17) is I-benzyl-N-(4-methoxybenzyl)- piperidin-4-amine. Compound (18) is l-benzyl-N-^S-dimethoxybenzylJpiperidin^- amine. Compound (19) is N-cyclohexyl-l-phenethyipiperidin-4-amine. Compound (20) is N-(3-bromophenyl)-l-phenethyIpiperidin-4-aminc. Compound (21) is N-(3- chlorophenyl)-l-phenethylpiperidin-4-amine. Compound (22) is N-(3-ffuorophenyl)- l-phenethylpiperidin-4-amine. Compound (23) is N-(3,4-dimethoxyphenyl)- l- phenethylpiperidin-4-amine. Compound (24) is N-(3,5-dimethoxyphenyl)-l- phenethylpiperidin -4-amine.

In still another embodiment, an anti-malarial compound of the instant invention comprises S(-)-UH-301 hydrochloride, amperozide hydrochloride, hexahydrosila-difenidol hydrochloride pfluoro analog, BW 284c51 and SKF 95282 dimalate (Zolantidine), ion channel blockers 3 ',4' dichlorobenzamil, benzamil hydrochloride, 5-(N-methyl-N-isobutyl) amiloride and 5-(N,N-dimethyl) amiloride hydrochloride which are amiloride derivatives, chloroquine, artensunate. dihydro- artemisini, mefloquine, or atovaquone.

In another embodiment, an anti-malarial compound of the invention comprises a naphthoquinone, In still another embodiment, an anti-malarial compound of the invention comprises a naphthoquinone selected from the group consisting of PC-0012225, PC-0983253, PC- 1018916, PC-1018917, PC-1018918, PC-1018919, PC-1018920, and PC-1018921, as depicted in Table 3. ( )

F

III. Pharmaceutical Compositions

The compounds of the invention will norm lly, but not necessarily, be formulated into pharmaceutical compositions prior to administration to a patient. In one aspect, the invention is directed to pharmaceutical compositions comprising a compound of the invention. In another aspect the invention is directed to

pharmaceutical compositions comprising a compound of the invention and a pharmaceutically acceptable carrier and/or excipient. The carrier and/or excipient must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

The pharmaceutical compositions of the invention may be prepared and packaged in bulk form wherein a safe and effective amount of a compound of the invention can be extracted and then given to the patient such as with powders or syrups. Alternatively, the pharmaceutical compositions of the invention may be prepared and packaged in unit dosage form wherein each physically discrete unit contains a safe and effective amount of a compound of the invention. When prepared in unit dosage form, the pharmaceutical compositions of the invention typically contain from about 0.1 to 100 mg, in another aspect 0.1 mg to about 50 mg of a compound of the invention.

The pharmaceutical compositions of the invention typically contain one compound of the invention, However, in certain embodiments, the

pharmaceutical compositions of the invention contain more than one compound of the invention. For example, in certain embodiments the pharmaceutical compositions of the invention contain two compounds of the invention. In addition, the

pharmaceutical compositions of the invention may optionally further comprise one or more additional active therapeutic compounds. The pharmaceutical compositions of the invention typically contain more than one pharmaceutically acceptable excipient. However, in certain embodiments, the pharmaceutical compositions of the invention contain one pharmaceutically acceptable excipient.

As used herein, the term "pharmaceutically acceptable" means suitable for pharmaceutical use.

The compound of the invention and the pharmaceutically acceptable excipient or excipients will typically be fonnulated into a dosage form adapted for administration to the patient by the desired route of administration. For example, dosage forms include those adapted for (1) oral administration such as tablets, capsules, caplets, pills, troches, powders, syrups, elixirs, suspensions, solutions, emulsions, sachets, and cachets; (2) parenteral administration such as sterile solutions, suspensions, and powders for reconstitution; (3) transdermal administration such as transdermal patches; (4) rectal administration such as suppositories; (5) inhalation such as aerosols and solutions; and (6) topical administration such as creams, ointments, lotions, solutions, pastes, sprays, foams, and gels.

Suitable pharmaceutically acceptable excipients will vary depending upon the particular dosage form chosen. In addition, suitable pharmaceutically acceptable excipients may be chosen for a particular function that they may serve in the composition. For example, certain pharmaceutically acceptable excipients may be chosen for their ability to facilitate the production of uniform dosage forms. Certain pharmaceutically acceptable excipients may be chosen for their ability to facilitate the production of stable dosage forms. Certain pharmaceutically acceptable excipients may be chosen for their ability to facilitate the carriage or transport of the compound or compounds of the invention from one organ, or portion of the body, to another organ, or portion of the body, once administered to the patient. Certain

pharmaceutically acceptable excipients may be chosen for their ability to enhance patient compliance.

Suitable pharmaceutically acceptable excipients include the following types of excipients: binders, disintegrants, lubricants, glidants, granulating agents, coating agents, wetting agents, solvents, co-solvents, suspending agents, emuisifiers, sweeteners, flavoring agents, flavor masking agents, coloring agents, anticaking agents, humectants, chelating agents, plasticizers, viscosity increasing agents, antioxidants, preservatives, stabilizers, surfactants, and buffering agents, The skilled artisan will appreciate that certain pharmaceutically acceptable excipients may serve more than one function and may serve alternative functions depending on how much of the excipient is present in the formulation and what other ingredients are present in the formulation.

Skilled artisans possess the knowledge and skill in the mi to enable them to select suitable pharmaceutically acceptable excipients in appropriate amounts for use in the invention, In addition, there are a number of resources that are available to the skilled artisan which describe pharmaceutically acceptable excipients and may be useful in selecting suitable pharmaceutically acceptable excipients. Examples include Remington's Pharmaceutical Sciences (Mack Publishing Company), The Handbook of Pharmaceutical Additives (Gower Publishing Limited), and The Handbook of Pharmaceutical Excipients (the American Pharmaceutical Association and the Pharmaceutical Press).

The pharmaceutical compositions of the invention are prepared using techniques and methods known to those skilled in the art. Some of the methods commonly used in the art are described in Remington's Pharmaceutical Sciences (Mack Publishing Company).

In one aspect, the invention is directed to a solid or liquid oral dosage form such as a liquid, tablet, lozenge or a capsule, comprising a safe and effective amount of a compound of the invention and a carrier. The carrier may be in the form of a diluent or filler. Suitable diluents and fillers in general include lactose, sucrose, dextrose, mannitol, sorbitol, starch (e.g. corn starch, potato starch, and pre-gelatinized starch), cellulose and its derivatives (e.g. microcrystalline cellulose), calcium sulfate, and dibasic calcium phosphate. A liquid dosage form will generally consist of a suspension or solution of the compound or salt in a liquid carrier for example, ethanol, olive oil, glycerine, glucose (syrup) or water (e.g. with an added flavoring, suspending, or coloring agent). Where the composition is in the form of a tablet or lozenge, any pharmaceutical carrier routinely used for preparing solid formulations may be used. Examples of such carriers include magnesium stearate, terra alba, talc, gelatin, acacia, stearic acid, starch, lactose and sucrose. Where the composition is in the form of a capsule, any routine encapsulation is suitable, for example using the aforementioned carriers or a semi-solid e.g. mono di-glycerides of capric acid, Gelucire™. and Labrasol™, or a hard capsule shell e.g gelatin. Where the composition is in the form of a soft shell capsule e.g. gelatin, any pharmaceutical carrier routinely used for preparing dispersions or suspensions may be considered, for example aqueous gums or oils, and may be incorporated in a soft capsule shell.

An oral solid dosage form may further comprise an excipient in the form of a binder. Suitable binders include starch (e.g. com starch, potato starch, and pre-gelatinized starch), gelatin, acacia, sodium alginate, alginic acid, tragacanth, guar gum, povidone, and cellulose and its derivatives (e.g. microcrystalline cellulose). The oral solid dosage form may further comprise an excipient in the form of a

disintegrant. Suitable disintegrants include crospovidone, sodium starch glycolate, croscarmelose, alginic acid, and sodium carboxymethyl cellulose. The oral solid dosage form may further comprise an excipient in the form of a lubricant. Suitable lubricants include stearic acid, magnesium stearate, calcium stearate, and talc.

There is further provided by the present invention a process of preparing a pharmaceutical composition, which process comprises mixing at least one compound of Formula I or Formula A or a pharmaceutically acceptable derivative thereof, together with a pharmaceutically acceptable carrier and/or excipient.

Preparations for oral administration may be suitably formulated to give controlled/extended release of the active compound.

All publications, including but not limited to patents and patent applications cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference as though fully set forth.

IV, Methods

The instant invention provides methods for preventing malaria in a subject at risk of becoming infected with P. falciparum, The method comprises administering to the subject a therapeutically effective amount of an anti-malarial compound identified according to the method of the invention.

The instant invention also provides methods of treating a subject infected with P. falciparum. The method comprises administering to a mammal in need thereof, a therapeutically effective amount of an anti-malarial compound identified in the assay described herein.

In one embodiment, the present invention comprises a method of inhibiting development, growth, division or replication of P. falciparum in the red blood cells of a subject. In one embodiment, the present invention comprises a method of inhibiting P. falciparum transformation from merozoites to rings, the method comprising administering to a subject in need thereof, a therapeutically effective amount of a compound identified in the assay described herein as an anti-malarial.

In another embodiment, the present invention comprises a method of inhibiting P. falciparum transformation from rings to trophozoites, the method comprising administering to a subject in need thereof, a therapeutically effective amount of a compound identified in the assay described herein as an anti-malarial.

In another embodiment, the present invention comprises a method of inhibiting P. falciparum transformation from trophozoites to schizont, the method comprising administering to a subject in need thereof, a therapeutically effective amount of a compound identified in the assay described herein as an anti-malarial.

In one embodiment, the compound comprising an anti-malarial compound of the instant invention is selected from the group of chemical scaffolds consisting of acridinediones, cyclopentylideneamino-Oxy-Carbonyl, pyrimidines, tetrahydropirido indoles, carbazoles, dimethoxy anilines, 1 ,4 naphtoquinones, ureas,

4- piperidinopiperidine, idenebenzene amines, hydrazones, piperazine, 1-amino piperidines, 4 amino piperidines, and phenols.

In another embodiment, an anti-malarial compound of the invention comprises a phenol based compound, including but not limited to compounds 1-12 as described in Table 1.

In another embodiment, an anti-malarial compound of the invention comprises a 4 amino piperidine based compound, including but not limited to compounds 13-24 as described in Table 2.

In still another embodiment, a compound of the instant invention comprises S(-)-UH-301 hydrochloride, amperozide hydrochloride, hexahydro- siladifenidol hydrochloride pfluoro analog, BW 284c51 and S F 95282 dimalate (Zolantidine), ion channel blockers 3',4' dichlorobenzamil, benzamil hydrochloride,

5- (N-methyl-N-isobutyl) amiloride and 5- N,N-dimethyl) amiloride hydrochloride which are amilloride derivatives, chloroquine, artensunate, dihydro-artemisini, mefloquine, or atovaquone.

In still another embodiment, a compound of the instant invention comprises a naphthoquinone. In still another embodiment, an anti-malarial compound of the invention comprises a naphthoquinone selected from the group consisting of PC-0012225, PC-0983253, PC-1018916, PC-1018917, PC-1018918, PC-1018919, PC- 1018920, and PC- 1018921, as depicted in Table 3.

In one embodiment of the invention, the subject is a mammal. In another embodiment of the invention, the subject is a human. The subject may be diagnosed with malaria. In another embodiment, the subject is at risk of infection with P. falciparum.

The methods of the present invention can be used in combination with other treatment regimens, including other anti-malarial compounds, virostatic and virotoxic agents, antibiotic agents, antifungal agents, anti-inflammatory agents (steroidal and non-steroidal), antidepressants, anxiolytics, pain management agents, (acetaminophen, aspirin, ibuprofen, opiates (including morphine, hydrocodone, codeine, fentanyl, methadone), steroids (including prednisone and dexamethasone), and antidepressants (including gabapentin, amitriptyiine, imipramine, doxeprn) antihistamines, antitussives, muscle relaxants, bronchodilators, beta-agonists, anticholinergics, corticosteroids, mast cell stabilizers, leukotriene modifiers, methylxanthines, nucleic acid based therapeutic agents, as well as combination therapies, and the like. The compounds of the present invention may be administered before, during, after, or throughout administration of any other therapeutic agents used in the treatment of a subject.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified, Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

The materials and methods employed in the experiments disclosed herein are now described.

Parasite Culturing

The P. falciparum expressing 3D7 (drag sensitive) parasites were cultured in RPMI 1640 media containing L-glutamine (Invitrogen) supplemented with 50 mg/L Hypoxanthine (Sigma), 25 mM Hepes (Invitrogen/Gibco), 10 mg/L

Gentamicin (Invitrogen/Gibco), 25 mM sodium bicarbonate (Invitrogen), and 0.5% Albumax II (Invitrogen). Packed red blood cells (RBCs) were obtained from

Biological Specialty Corporation (Colmer, PA) and the blood type varied week to week. Parasites were cultured in 4% hematocrit at 37°C with gas composed of 5% 02, 5% C0₂, and balanced with N2 in a modular incubator chamber (Hot Box system, Billups-Rothenberg Inc.)

Compound Libraries

The University of Pennsylvania Center for Molecular discovery has a diverse set of compounds libraries, including commercially available drags, as well as small molecules with unknown pharmacological activity. The compounds libraries used in this screening were as follows:

(1) Library of Pharmacologically Active Compounds (XOPAC)

LOPAC consists of 1280 compounds from Sigma Aldrich with known activity in cell signaling (9%), phosphorylation (8%), cell stress (4%), lipids (4%), ion channels (6%), G-proteins (3%), ap opto sis/cell cycle (2%) gene regulation (3%), hormone related (3%), neuroscience related (58%). Compounds were distributed in four 384 well plates, holding 10 μL of compounds at 2 μΜ in DMSO.

(2 Library from the National Institute of Neurological Disorders and Stroke

(MENDS^')

MENDS consists of 1040 compounds from MicroSource Discovery Systems, Inc. (MDSI). Compounds were distributed in four 384 well plates, holding 10 μL of compounds at 2 μΜ in DMSO.

(3) The ChemBridee Compound Library

ChemBridge consists of 10,000 diverse drugs like small molecule compounds, covering the broadest part of biologically relevant pharmacophore diversity space. Compounds were distributed in thirty two 384 well plates, holding 10 L of compounds at 2 μΜ in DMSO.

Luciferase Assay

Parasites were synchronized at rings stage using 5% Sorbitol solution (Sigma) for at least two cycles prior to the assay. To obtain a more robust signal, the assay was set up at 1% parasitemia during the late-ring stage and 3% hematocrit. Using a Wellmate Microplate Dispenser (Matrix Technologies) 10 μΐ of complete culturing media was dispensed under sterile conditions into a 384 white flat bottom tissue culture treated polystyrene well plate (Greiner 781080). Afterwards, using the EP3 workstation (Perkin Elmer), 1 10 nl of compounds in DMSO at 2 mM were tratlsferred from the compound plate into the assay plate where columns 1, 2, 23, and 24 received only DMSO. Subsequently, 30 μΐ of infected culture were dispensed into the 384-weIl plate in all columns except 1 and 23. In columns 1 and 23, 30 μΐ of complete media with 3% uninfected RBCs were dispensed to serve as negative controls. These plates were then covered with a Breath-Easy seal (Diversified Biotech) and a Kalypsys metal lids (Kalypsys, Inc.) to reduce evaporation effects. The plates were incubated in a water jacketed C02 incubator BEPA class 100

(Thermo Electron Corporation) for approximately one life cycle (48 hours) under normal conditions as described above.

To assay the plates, the metal lid and seal were removed and 40ul of BrightGlo® Luciferase Substrate (Promega) was dispensed. Immediately following, the plates were centrifuged at 200 rpm and luminescence read by an EnVision muitilable reader 2102 (Perkin Elmer). The compounds identified according to these methods were run in a luciferase counter screen to evaluate for compounds targeting luciferase. The assay consisted of monitoring the inhibitory activity of those hits obtained in the primary HTS by detecting the luminescence signal generated by the ATP consumption by luciferase using CellTiter-Glo® (Promega). Ten (10) pL of 10 nM ATP in culture media and 10 μΐ. of CellTiter-Glo® was added to each well.

Resveratiol (Sigma), a known luciferase inhibitor was used as a control compound at 273 μΜ in columns 1 and 23 in a 384 well plates (Greiner 781080). The signal was read on the Envision to monitor the luminescence due to luciferase activity. Data Analysis

Data were analyzed in the IDBS ActivityBase. Each HTS plate contained compounds (5.5uM in 0.3% DMSO) in columns 3-22, controls (3% uninfected RBCs, no compounds) in columns 2 and 24, and blanks (1 % parasitemia during the late-ring stage and 3% hematocrit) in column 1 and 23. HTS percent inhibition was calculated for each compound from the signal in luminescence units and the mean of the plate controls and the mean of the plate blanks using the following equation:

% Inhibition = 100*(l-((signal mean - blank mean)/(control mean - blank mean))) A hit cut-off of 85% inhibition was selected. Based on this cutoff, a hit rate of 5.2 for LOPAC library, 8.4 for NTNDS library and 1.5 for ChemBridge library was observed: Hits (>85% inhibition): 183 compounds; Inactive (<85% inhibition): 12137 compounds. The results of the experiments presented in this Example ate now described.

Experimental Example 1 : Luciferase base high tliroughput screening (HTS) 384 well plate assay development

A high throughput- screening (HTS) assay was used to identify small molecules that prevent P. falciparum growth. The approach used took advantage of a novel whole organism luciferase based assay using P. falciparum. For this assay, firefly luciferase, under the control of the histidine rich protein (HRP) 2 promoter, was stably transfected into P. falciparum 3D7 parasites. Greiner 384 well plates were used. Ten (10) Ε of culture media were dispensed into each well of the plate, 1 10 nL of test solution comprising test compounds (columns 3-22) and control solution comprising DMSO (columns 1-2, 23-24) were transferred to individual wells using a pintool. Either thirty (30) \L of red blood cells infected with synchronized parasites at the ring stage (columns 2-22 and 24), or 30 μ!_, of uninfected red blood cells, were added (columns 1 and 23). Breath easy membranes and metal lids were used to cover the plates and control evaporation. Variation across the plate because of edge effects was prevented by wrapping the plate with wet paper towels and keeping the humidity at 96%, The plates were then transferred to a gas controlled modular incubator chamber in presence of a mix of 5% ⁽¾ 5% CO2, and balanced with N₂. These chambers were sealed and then transferred to an incubator and maintained at 37 °C during 48 hours. After this incubation period, the plates were removed from the incubator, the seal and lid removed. Forty (40) iL of Bright-Glo® Luciferase Substrate were added to each well of the plate and any resulting luminescence signal was detected in an envision plate reader (Figure 1A).

To determine the suitability of the erythrocytic P. falciparum luciferase base assay in a 384 well format, the hemotocrit, parasitemia and growing conditions were evaluated. Three per cent (3%) hematocrit (Figure lB-i) and 1 % parasitemia (Figure IB-ii) gave a satisfactory rate of invasion as well as luminescence signal. The reading of samples using Bright-Glo® Luciferase Substr ate gave a reliable detection 30 minutes after addition of the reagent (Figure ΙΒ-iii). A robust linear relationship was found between starting parasitemia levels and luminescence values, indicating that this assay can report parasite growth over a range of parasitemia levels. It was confirmed that the HHP 2 promoter is most active at the late ring stage of parasite development and therefore, it provides a good marker for the viability of recently invaded parasites. This allows for testing of parasite viability through an entire cycle of intracellular growth, rupture and reinvasion.

Table 4

In addition, an ECJQ dose response was established for those known antimalarial compounds listed in Table 4. EC₅o values were obtained that were similar to those reported for in the literature for those compounds. Further optimization of the assay in 384-well format demonstrates that the assay conditions yield signal parameters beneficial for high throughput screening. A 300X signal to background ratio, reproducible plate-to-plate results, an average coefficient of variation ratio less than 10%, and an average Z-factor of 0.7 were evident (Table 5).

Experimental Example 2: Compound Libraries High Throughput Screening (HTS

The main aim of this study was to identify and evaluate compounds capable of inhibiting the progression of the rupture and invasion of erythrocytes by P. falciparum. A HTS assay was performed using three different compound libraries, two comprising known and FDA approved dmgs including some anti-malarial agents (LOPAC library: 1,280 compounds and NTNDS library: 1 ,040 compounds), and one, the ChemBridge library, composed of 10,000 small molecules with unknown activity. After optimizing the luciferase assay, compounds were screened for the ability to inhibit the progression of the erythrocytic cycle of the parasite. A negative control plate was evaluated to determine the characteristic of the signal under 0.2% DMSO. A plate with 10 μΜ of artesunate was used as a positive control and as a standard for 50-60% inhibition of parasite invasion of RBCs.

The statistical parameters which determine the reliability and confidence of the data obtained for each library in this HTS campaign are described in Table 5 and include the signal to background (S/B) ratio, Z-factor, coefficient of variation (CV), and hits rate for each of the libraries. Each of the libraries where evaluated separately, but in the same week. An average S/B ratio of 305, Z-factor of 0.68, and CV of 11 % CV was obtained for each library using this cell base assay.

Figure 2B-i shows the effect of compounds from the LOPAC library that decreased the luciferase signal more than 85% compared to the control. Figures 2B-ii and 2B-iii present data for compounds from the NTNDS and ChemBridge libraries, respectively. Resveratrol was used as a control compound capable of inhibiting 50% of luciferase at 273 μΜ as is described in material and methods.

Experimental Example 3: LOP AC screening

LOPAC compound library consists of 1280 compounds from Sigma Aldrich with known activities in cell signaling (9%), phosphorylation (8%), cell stress (4%), lipids (4%), ion channels (6%), G-proteins (3%), apoptosis/cell cycle (2%), gene regulation (3%), endocrine related (3%), and neuroscience related (58%).

Of the 1,280 compound screened, 67 compounds reported luminescence inhibition higher than 85%, giving a 5.2% hit rate in this primary screen. IC₅₀ were evaluated for the identified inhibitors and seventeen (17) compounds ranging from 0.001 to 1.2 μΜ, giving a 1.3% hit rate in this confirmation screen. The identification of quinine derivatives as inhibitors or the parasite confirms the reliability of this assay. Other well-known bioactive compounds that have been proven to have anti-malarial activity were also found to inhibit Plasmodium development in this assay, including anticancer drugs like vincristine (0.001 μΜ IC₅o) and vinblastine (0.002 μΜ IC₅o), which target microtubule assembly, emetine (0.001 μΜ ICso) which inhibits RNA protein translation, and 5(N-ethyl N-isopropyl) amilloride (0.825 μΜ IC₅o), an ion channel blocker. The following compounds: S(-)-UH-301 hydrochloride (0.005 μΜ ICso), amperozide hydrochloride (0.035 μΜ IC₅₀), hexahydro-sila-difenidol hydrochloride pfluoro analog (0.004 μΜ IC₅₀), BW 284c51 (0.107 μΜ IC₅₀) and SKF 95282 dimalate (Zolantidine) (0.001 μΜ IC₅₀), ion channel blockers 3^,,4* dichlorobenzamil (0.143 μΜ IC₅₀), benzamil hydrochloride (0.217 μΜ IC₅₀), 5-(N- methyl-N-isobutyl) amiloride (0.549 μΜ IC₅o) and 5-(N,N-dimethyl) amiloride hydrochloride (1.1 μΜ IC₅₀) which are amilloride derivatives, are identified for the first time herein as having anti-malarial activity.

Experimental Example 4: NINDS screening

The library from the National Institute of Neurological Disorders and Stroke (NINDS) consist of 1,040 compounds limn MicroSource Discovery Systems, Inc. (MDSI). Out of the 1 ,040 compound screened, 87 compounds reported a percent inhibition higher than 85% for an 8.4 % hit rate in the primary screen, 16 of them were confirmed for a 1.5% hit rate after cherry picking and IC₅₀ evaluation, Table 6 describes the names, activities and the IC₅₀ value in this assay for the hits obtained from this library in this HTS. Because the main characteristic of compounds in this librmy is that they have neurological activity, only one known anti-malarial compound, cinchonine, a quinine derivative, was found to have a 0.005 μΜ IC₅₀. Four (4) additional compounds, namely tyrothicin, pentamidine isethionate, astemizole and hycanthon, have been reported to have anti-malarial activity in different studies. Sulfamerazine is an antibacterial compound which lyses the red blood cells in this assay. The other ten hits from this library are distributed as anti- infectant, anti-inflammatory, antifungal, emetic among other activities as is described in Table 6.

Experimental Example 5: ChemBridge screening

The ChemBridge library consists of 10,000 diverse compounds. Of those 10,000 compounds, 1.7% had greater than 85% inhibition in this HTS. One hundred thirty (130) compounds, equivalent to 1.3% of the total number of compounds from this library, exhibited confirmed inhibitory activity of luminescence. A more meticulous classification of those hits allowed the categorization of71 (60.8%) of them into 14 main scaffolds groups. The remaining 59 (39.2%») are compounds that were identified as hits in this screening having diverse structures which do not fit on the classification criteria (SAR) used in those 14 scaffolds. Figure 3a shows the structures and distribution of those scaffolds. Figure 3b describes the number of hits from each scaffolds found in this libraiy. The activity of those hits range from 0.027 μΜ to 20 μΜ. Experimental Example 6: ChemBridge Scaffolds

Phenolic compound are aromatic structures with a phenol group as core scaffold. Twelve per cent (12%) of the 130 hits confirmed to be active from this library are characterized as having a phenolic scaffold. These 16 compounds vary mainly in their substituent in position 2, 4 and 6 of the phenolic ring. Their activities range from 0.036 μΜ to 15.1 μΜ, and cyclopropy line thy! propyl amino methyl, 2- phenylethyl amino methyl, 1-azo cany lme thy 1 among others, can be found as substituent in position 6 in this phenolic scaffold. Halogen atoms, mainly bromide and chloride, are found as substituent in positions 2 and 4 (Table 1). There is no reported data about this compounds being evaluated against Plasmodium and this group of molecules do not have activity against luciferase based on their IC₅₀ values being higher than 68.2 μΜ (Table 1). This is the first time these compounds have been reported to have anti-malarial activity.

Four amino piperidines have been studied as anti-malarial compounds. Twelve (12) compounds or 9.2% of the hits evaluated from this library were classified in this group of molecules. Their activity ranges from 0.246 μΜ to 1.3 μΜ. These types of inhibitors have been found to be active against plasniepsin II. However, none of the structures found in this study have been reported as inhibitors of this enzyme. Compounds 13 and 14 have a propylpipendine-4-aniine as a scaffold. Compounds 15-18 have a benzyl piperidine-4-amine as their main scaffold. Compounds 19-24 have a phenylethyl-piperidine-4-amine as the scaffold. This group of molecules does not have activity against luciferase judging by their IC₅₀ values being higher than 68.2 μΜ (Table 2).

Piperazines are compounds with reported activity against choroquine resistant P. falciparum. Ten (10) piperazines, or 7.7% of the hits, were found to be active from this library in this HTS assay. The activity of the piperazines found in this study range from 0.105 μΜ to 5.4 μ . None of the piperazines found in this study have been previously reported as P. falciparum inhibitors.

Hydrazones, primarily aroy!hydrazones~ have been reportied to have antimalarial activity against chloroquine resistant and sensitive parasites by acting as iron chelators. Out of the 130 hits obtained for this ChemBridge library, 8 compounds, or 6% of the hits, exhibit inhibition of P. falciparum in this assay, ranging from 0.030 μΜ to 5.7 μΜ. None of the hydrazones found in this study has been reported to have anti-malarial activity.

Idenebenzene amines represent the 4.7 % of the total hits obtained from this library, This is equivalent to 6 compounds, and this is the first time they are reported as inhibiting the development of P. falciparum with an IC₅₀ ranging between 0.530 μΜ to 5.7 μΜ, These compounds do exhibit some activity against luciferase enzyme (13.8 μΜ), but this is insufficient to account for the inhibition of the P.

falciparum parasite.

Ureas were found to be active in inhibiting the growth of P.

falciparum. Four (4) compounds of this class, or 3.1 % of the total hits, were found to have inhibitory effects on the growth of P. falciparum in this assay, with a maximum activity of 0.526 μΜ and a minimum of 1.4 μΜ.

1,4-Naphthoquinone compounds are related to atovaquone, one of the most effective anti-malarial compounds, Using the assay of the invention, 4 inhibitors of P. falciparum were detected with the 1,4-Naphthoquinone scaffold. The IC₅₀ for these compounds ranged from 0.0053 μΜ to 5.4 μΜ.

Tetrahydropirido indoles have been found to be potent inhibitors o PfENR, one of the enzymes responsible for the fatty acid biosynthesis in the P.

falciparum. Two compounds, or 1 ,5% of the compounds found in this screen, are active inhibiting P. falciparum growth. IC₅₀ for these compounds ranged from IC₅₀ of 0.495 μΜ ιο 1.6 μΜ.

Five (5) compounds, or 3,8% of the hits from this library, with the 4- piperidinopiperidine scaffold were found to inhibit P. falciparum growth. The IC₅0 for these compounds ranged from 0.211 μΜ to 0.969 μΜ,

Acridinediones were among the most active compounds in this study.

Two (2) compounds with this scaffold, or 1.5% of the total hits reported, exhibited a maximum inhibitory activity of 0.092 μΜ and a minimum of 1.1 μΜ,

Two compounds with a pyrimidine scaffold, or 1.5% of the total number of compounds screened, inhibited P. falciparum growth in this assay. The activity of the pyrimidine compounds range from 0.099 μ to 0.368 μΜ.

Cyclopentylideneamino-oxy-carbonyl is a scaffold with no previously reported anti P falciparum activity. Two compounds with this scaffold were found to be active against P. falciparum, corresponding to 1.5% of the total hits found in this study. Their IC₅₀ ranged from 2.4 μΜ to 4 μΜ

Others compounds not characterized as belonging to any of the scaffolds described were 39% of the hits found, corresponding to 51 compounds, many of which had IC₅₀ higher than 5 μΜ

The luciferase enzymatic assay used in this screening also allowed the determination of which hits also had some direct inhibitory effects on the luciferase reporting system. Only 14 compounds, or 10.7%) of the total hits obtained in this HTS assay, exhibited luciferase inhibitory activity.

Experimental Example 6: 1.4-naphthoquinone derivatives as inhibitors of P.

falciparum

Naphthoquinones are known to have antibacterial, antitumor and antimalarial activity. Atovaquone is a naphthoquinone used in combination with proguanil hydrocloride in the treatment of malaria. Atovaquone is known to inhibit the mitochondrial electron transport vital in the parasite respiratory system, more exactly inhibiting the ubiquinone cytochrome c oxidoreductase (bcl complex). It is known that there are at least 5 mitochondrial dehydrogenases in the parasite:

Rotenone-insensitive NADH dehydrogenase, Glycerol 3-phosphate dehydrogenase, Dihydroorotate dehydrogenase (DHOD), Succinate dehydrogenase, Malate-quinone oxidoreductase. All of them generate coenzyme Q (CoQ) which is re-oxidized by Complex III to make CoQ available to continue the electron transport chain, which is essential for the survival of the parasite (Srivastava et al., 1996, J. Biol. Chem.

272:3961-3966; Painter et al., 2007, Nature 446:88-91 ; Biagini et al., 2008, Mol. Pharm. 73: 1347-1355).

Compound PC-00 12225 (Table 3) has demonstrated activity against different cell lines of the malaria parasite with an IC₅₀ value of 14nM in this assay and an approximated IC₅₀ value of 86nM when it was evaluated in a chloroquine sensitive cell line (3d7) and an IC₅₀ value of 96nM in a chloroquine resistant cell line (Dd2) as is described in Figure 1.

Compound PC-0012225 and its derivatives (Table 3) have not previously been studied as anti-malarial drugs. Compound PC-1 0 18918 is the most potent in this series (0.7 nM ICj₀). The subnanomolar activity of compound PC- 1018918 as well as its possible different mechanism of action compare to that described for atavaquone, makes this compound and the naphthoquinones found in this study a potential drugs to be used in the treatment of malaria. Experimental Example 7: Further Investigation of 1,4-Napthoquinones,

Since this series of 1,4-napthoquinones was potent, drug-like, and structurally related to atovaquone, investigation of their mechanism of action was further pursued. Atovaquone is thought to act by inhibiting the quinol oxidation site (Qo) of the bc_\ complex of the parasite mitochondria (Fry & Pudney, 1992, Biochem. Pharmacol. 43: 1545 53; Korsinczky et ai., 2000, Antimicrob. Agents Chemother. 44:2100-08; Srivastava et al., 1999, Mol, Microbiol. 33:704-1 1; Syafruddin et al„ 1999, Mol. Biochem. Parasitol, 104: 185-94). All potent 2,3-diamino-l,4- naphthoquinones were tested against both drug-sensitive (3D7) and

chloroquine/atovaquone-resistant (TM90C2B) parasite lines using the DAPI-based method (Table 7). All the newly identified test compounds demonstrated potent activity against the atovaquone-resistant parasite line, indicating that it is unlikely that they share a mechanism with atovaquone. This observation was confirmed, as compound PC-1018918 demonstrated no inhibitoty activity ai 14μΜ against both the parasite bc_\ complex and beef-heart bc_\. Furthermore, PC-1018918 did not show any inhibitory activity (at ^4 μΜ) against the other quinone-dependent respiratory enzyme, NADH biquinone oxidoreductase (Pf DH2). Taken together, these data indicate that the potent 2,3-diamino-l ,4-naphthoquinone series identified from this HTS is likely to possess a novel mechanism of action distinct from that of atovaquone, although it contains a similar core scaffold.

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without depatling from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMS What is claimed:

1. A method of identifying an anti-malarial compound, said method comprising:

(a) introducing a detectable gene marker comprising a luciferase into a P. falciparum;

(b) cultunng said P. falciparum comprising said luciferase in the presence of red blood cells (RBCs) to generate infected RBCs;

(c) contacting said infected RBCs with either a test or a control compound;

(d) adding a luciferase substrate to said infected RBCs so contacted;

(e) measuring the level of luminescence emitted from said infected RBCs so contacted, wherein said compound is identified as an antimalarial compound when the level of luminescence in said infected RBCs contacted with said test compound is inhibited≥85% when compared with the level of luminescence in said infected RBCs contacted with said control compound.

2, The anti-malarial compound identified according to the method of claim 1, wherein said compound is selected from the group consisting of an acridinedione, a cyclopentylideneamino-oxy-carbonyl, a pyrimidine, a

tetrahydropirido indole, a carbazole, a dimethoxy aniline, a 1,4-naphtoquinone, a urea, a 4-piperidinopiperidine, a idenebenzene amine, a hydrazone, a piperazine, a 1 -amino piperidine, a 4-amino piperidine, and a phenol.

3, The antimalarial compound identified according to the method of claim 1, wherein said compound is selected from the group consisting of compounds 1-24, S(-)-UH~301 hydrochloride, amperozide hydrochloride, hexahydro-sila- difenidol hydrochloride pfluoro analog, BW 284c51 and SKF 95282 dimalate (Zolantidine), ion channel blockers 3',4' dichlorobenzamil, benzamil hydrochloride, 5- (N-methyl-N-isobutyl) amiloride and 5-(N,N-dimethyl) amiloride hydrochloride.

4. The anti-malarial compound identified according to the method of claim 2, wherein said phenol is selected from the group consisting of compounds 1-12.

5. The anti-malarial compound identified according to the method of claim 2, wherein said 4-amino-piperidine is selected from the group consisting of compounds 13-24.

6. The anti-malarial compound identified according to the methods of claim 2, wherein said naphthoquinone is selected from the group consisting of compounds PC-0012225, PC-0983253, PC-1018916, PC-1018917, PC-1018918, PC- 1018919, PC- 1018920, and PC- 1018921.

7. A method of preventing P, falciparum infection in a mammal in need thereof, said method comprising administering a therapeutically effective amount of an anti-malarial compound to said mammal, wherein said mammal has a reduced risk of infection with P. falciparum when said anti-malarial compound is administered to said mammal.

8. The method of claim 7, wherein said anti-malarial compound is selected from the group consisting of an acridinedione, a cyclopentylideneamino-oxy- carbonyl, a pyrimidine, a tetrahydropirido indole, a carbazole, a dimetoxy aniline, a 1,4 -naphthoquinone, a urea, a 4-piperidinopiperidine, a idenebenzene amine, a hydrazone, a piperazine, a 1-amino piperidine, a 4-amino piperidine, and a phenol.

9. The method of claim 7, wherein said anti-malarial compound is selected from the group consisting of S(-)-UH-301 hydrochloride, amperozide hydrochloride, hexahydro-sila-difenidol hydrochloride pfluoro analog, BW 284c51 and SKF 95282 dimalate (Zolantidine), ion channel blockers 3',4' dichlorobenzamii, benzamil hydrochloride, 5-(N-methyl-N-isobutyl) amiloride and 5 -(N,N-di methyl) amiloride hydrochloride.

10. The method of claim 8, wherein said phenol is selected from the group consisting of compounds 1-12.

1 1. The method of claim 8, wherein said 4-amino-piperidine is selected from the group consisting of compounds 13-24.

12. The method of claim 8, wherein said naphthoquinone is selected from the group consisting of compounds PC-0012225, PC-0983253, PC-1018916, PC-1018917, PC-1018918, PC-10189I9, PC-1018920, and PC-1018921.

13. The method of claim 7, wherein said mammal is a human.

14. The method of claim 7, wherein said anti-malarial compound is administered to said mammal in combination with another therapeutic agent.

15. A method of treating a mammal in need thereof infected with P. falciparum, said method comprising administering to said mammal a therapeutically effective amount of an anti-malarial compound, wherein, when said anti-maiarial compound is administered to said mammal, said mammal experiences either a reduction or cessation in the symptoms associated with infection with said

P. falciparum, or said P. falciparum can no longer be detected in a body sample from said mammal,

16. The method of claim 15, wherein said anti-malarial compound is selected from the group consisting of an acrid inedione, a cyclopentylideneamino-oxy- carbonyl, a pyrimidine, a tetrahydropirido indole, a carbazole, a dimetoxy aniline, a 1,4-naphtoquinone, a urea, a 4-piperidinopiperidine, a idenebenzene amine, a hydrazone, a piperazine, a 1 -amino piperidine, a 4-amino piperidine, and a phenol.

17. The method of claim 15, wherein said anti-malarial compound is selected from the group consisting of SQ-UH-301 hydrochloride, amperozide hydrochloride, hexahydro-sila-difenidol hydrochloride pfluoro analog, BW 284c51 and SKF 95282 dimalate (Zolantidine), ion channel blockers 3'_}4' dichlorobenzamil, benzamil hydrochloride, 5-(N-methyl-N-isobutyl) amiloride and 5-(N,N-dimethyl) amiloride hydrochloride.

18. The method of claim 16, wherein said phenol is selected from the group consisting of compounds 1-12.

19. The method of claim 16, wherein said 4-amino-piperidine is selected from the group consisting of compounds 13-24.

20. The method of claim 16, wherein said naphthoquinone is selected from the group consisting of compound PC-0012225, PC-0983253, PC-1018916, PC- 1018917; PC-1018918; PC-1018919; PC-1018920, and PC-1018921.

21. The method of claim 15, wherein said mammal is a human.

22. The method of claim 15, wherein said anti-malarial is administered to said mammal in combination with another therapeutic agent.