Mitochondria-targeted drugs as Anti-parasite therapy

Abstract

The present disclosure provides a method of inhibiting proliferation of a parasite or treating a disease caused by a parasite in a subject by administration of a mitochondria-targeted drug. Nonlimiting examples of the parasites include Brugia malayi and Dirofilaria immitis. Advantageously, movement of both male and female microfilaria can be inhibited by the mitochondria-targeted drug.

Description

Attorney Docket No. 650053.01255MITOCHONDRIA-TARGETED DRUGS AS ANTI-PARASITE THERAPYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U. S. Provisional Patent Application No.63 / 719,395 filed November 12, 2024, the content of which is hereby incorporated by reference in its entirety.BACKGROUND

[0002] Brugia malayi and Wuchereria bancrofti are parasitic nematodes that cause lymphatic filariasis (elephantiasis), a neglected tropical disease affecting over 100 million people across Asia, Africa, and South America. Transmission occurs via bites from infected mosquitoes, and disease manifestations range from asymptomatic microfilaremia to severe lymphedema and disability once adult parasites colonize lymph nodes and trigger chronic inflammation. Current treatments rely on combinations of ivermectin, albendazole, and doxycycline. However, emerging resistance and incomplete macrofilaricidal activity underscore the continued need for new, targeted therapies with minimal host toxicity.

[0003] Other filarial nematodes also cause life-threatening infections in animals, including dogs and cats. Dirofilaria immitis (heartworm), a blood-borne parasite transmitted by mosquitoes, infects dogs, cats, and other mammals. Larvae migrate to pulmonary arteries and the heart, maturing into adults up to 30 cm long that can persist for years and produce millions of circulating microfilariae. In heavily infected dogs, hundreds of worms may accumulate, impairing cardiac output, inducing pulmonary vascular inflammation, and ultimately causing cardiopulmonary disease, congestive heart failure, or embolic death, if untreated.

[0004] Standard treatment for canine heartworm combines melarsomine, an arsenic-based adulticide that presumably inhibits phosphofructokinase (glycolysis), with macrocyclic lactones such as ivermectin, which paralyze microfilariae by activating glutamate-gated chloride channels. While effective, these drugs have notable drawbacks: host toxicity, contraindications in MDR1 -mutant dogs, and rising reports of macrocyclic lactone resistance. Moreover, no comprehensive studies have examined the effects of melarsomine or ivermectin on parasite mitochondrial bioenergetics.

[0005] Mitochondria are critical for energy production and survival for the parasites. Studies have shown that parasites also hijack the host cell’s mitochondria and obtain additional nutrients for energy to survive and replicate. Drugs targeting mitochondrial energy metabolism have been used to combat parasitic infections. The mitochondrial respiratory chain of parasitesAttorney Docket No. 650053.01255is identified as a suitable target for antiparasitic drug development, as mitochondrial respiratory chain of parasitic cells differs from those of host cells. Drugs that potently disrupt the mitochondrial function of parasites with minimal disruption to host cell mitochondrial function are being developed.

[0006] Mitochondria are central to parasite survival, growth, and reproduction through ATP production via oxidative phosphorylation (OXPHOS). Several anthelmintics, including emodepside, monepantel, and flutolanil, exploit this vulnerability by targeting electron transport chain (ETC) complexes II (succinate dehydrogenase) and IV (cytochrome c oxidase). However, host mitochondrial toxicity is a concern. Filarial worms also exhibit metabolic plasticity; early larvae rely predominantly on glycolysis, whereas adults shift toward OXPHOS. This dynamic reprogramming may underlie drug tolerance, emphasizing the need for therapies that disrupt both glycolysis and OXPHOS across life stages.

[0007] In cancer and chronic disease chemotherapy, it was discovered that many malignant / damaged eukaryotic cells shift their metabolism from oxidative phosphorylation to aerobic glycolysis to produce ATP. Because of that shift, the redox potential (charge) of mitochondrial membranes from diseased cells and normal cells can vary dramatically. Capitalizing on that difference, it was shown that lipophilic cationic antioxidants can easily penetrate mitochondrial lipid bilayers, accumulate in mitochondria of the abnormal cells and kill them. Potent antioxidants reduce damage due to oxygen free radicals and have many clinical applications in noncommunicable diseases.

[0008] Two approaches to delivery of antioxidants to mitochondria are (1) ‘'tagging” compounds with a moiety (TPP) that directs an attached antioxidant to the “abnormal” mitochondria, and (2) tagging compounds to enhance the activity of drugs that themselves block the transcription of mitochondrial genes. Capitalizing on that difference, it was shown that lipophilic cationic antioxidants accumulate in mitochondria of cancer cells and inhibit respiration and proliferation.

[0009] Mitochondria-targeted agents (MT As) represent a promising strategy to selectively impair parasite bioenergetics. Mitochondrial targeting involves attaching a triphenylphosphonium cation (TPP+) through an alkyl chain of varying length to a quinone, nitroxide or a phenolic moiety. The optimal side chain length has 10 carbons. By conjugating bioactive molecules to the lipophilic triphenylphosphonium (TPP+) cation, MTAs accumulate in mitochondria in response to their negative membrane potential. Initially developed for cancer therapy, MTAs can, in principle, be repurposed for parasitic diseases to achieve greater potencyAttorney Docket No. 650053.01255and specificity. Examples of two mitochondria-targeted drugs are Mito-CP (mitochondrially targeted carboxy proxyl nitroxide and Mito Q (mitochondrially targeted ubiquinone).

[0010] Respiration in mitochondria from parasitic helminths may be mediated by a number of different electron transport pathways. Aerobic respiratory pathways have been identified that appear similar in most respects to the mammalian electron transport chain in addition to alternative respiratory pathways.

[0011] Recent research has elucidated the mitochondrial genome of B. malayi microfilariae, and the critical role of mitochondrial respiration and metabolism and drugs targeting energy metabolism in filarial nematodes.

[0012] Atovaquone (ATO), ahydroxyl 1,4-napthaquinone analog of coenzyme Q10, targets mitochondrial complex 3 and inhibits mitochondrial respiration. ATO is a competitive inhibitor of ubiquinol. ATO (FIG. 1) is an FDA-approved prophylactic drug used to treat and prevent malaria, Pneumocystis pneumonia and Toxoplasmosis, and inhibits mitochondrial complex 3 and respiration in the Plasmodium falciparum parasite. ATO has an excellent safety profile and a long half-life and a single dose of atovaquone (Malarone) reportedly achieves 5-10 microM serum concentrations. Pharmacokinetic studies of atovaquone show that it has a long half-life (50-84 hours) and a single dose of oral Malarone achieves a concentration in plasma as high as 10 microM. While Atovaquone is used as a prophylactic drug against malaria, it works at much higher concentrations in male but not female larvae of these parasites.

[0013] Recently, the structure of ATO was modified by attaching an alkyl side chain containing the triphenylphosphonium (TPP+) group to generate a new class of mitochondria-targeted compounds known as the Mito-ATOs. Mito-ATO (FIG. 1) targets both mitochondrial complex 1 and complex 3 of the respiratory chain in cancer cells. Mito-ATO was significantly more potent than ATO in inhibiting tumor cell proliferation. Mitochondria targeted atovaquone exhibits 100-fold increased activity against cancer cells.

[0014] There remains a need for novel anti-parasite agents and therapies.SUMMARY OF THE INVENTION

[0015] In one aspect, the present disclosure provides a method of inhibiting proliferation of a parasite in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound of formula (I), (II), (III), (IV), (V), (VI), VII), or (VIII), or a pharmaceutically acceptable salt thereof,Attorney Docket No. 650053.01255(VIII) whereinR1is Mito;one of R2and R3is Mito, the other is H;each of R4, R5, R6, R7, and R8is Mito;Z is NH or O;Attorney Docket No. 650053.01255L is Ci-2oalkylene, C2-2oalkenylene, LI-RC-L2, or amino acid;Li and L2 are each independently absent or C1-C10 alkylene;Rcis - (CH2CH2O)q-, arylene, or cycloalkylene;q is 1-20;Y at each occurrence is independently -CF3, Me, Cl, OMe, C(O)CH3, NO2, N(Me)2, COOH, F, Br, I, or OH;m at each occurrence is independently 0, 1, 2, 3, 4, or 5; andX is a counterion.

[0016] In one aspect, the present disclosure provides method of treating a disease caused by a parasite in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound of formula (I), (II), (III), (IV), (V), (VI), (VII), or (VIII), or a pharmaceutically acceptable salt thereof, as described herein.

[0017] In some embodiments, the parasite comprises parasitic nematode, parasitic protozoa, parasitic trematode, parasitic cestode, or parasitic conoidasida. As nonlimiting examples, the parasite can comprise Brugia malayi, Dirofilaria immitis, Leishmania spp., Trypanosoma cruzi, Borrelia burgdorferi, Toxoplasma, Pneumocystis, Plasmodium sp, amoebae, Giardia, Paramecium Balantidium, Trypanosoma, Trichomonas, Cryptosporidium. Babesia, filariae, Brugia. Onchocerca. Wuchereria. Mansonella, Loaiasis. Necator, Ancyclostoma, Schistosoma, Fasciola, Clonorchis, Paragonimus, Dirofilariae. Dictocalus, hookworm, Strongyloides, Onchocerca, ascariasis, cestodes, Coccidia, or a combination thereof. In some embodiments, the parasite comprises Brugia malayi, Dirofilaria immitis, or a combination thereof.

[0018] In some embodiments, the parasite comprises both male and female microfilaria, and the compound as described herein, or a pharmaceutically acceptable salt thereof, inhibits movement of both the male and female microfilaria.Attorney Docket No. 650053.01255

[0019] In some embodiments, the parasite comprises both adult male and adult female parasites, and the compound as described herein, or a pharmaceutically acceptable salt thereof, inhibits movement of both the adult male and adult female parasites.

[0020] In some embodiments, the parasite comprises both male and female parasites, and the compound as described herein, or a pharmaceutically acceptable salt thereof, reduces the number of microfilariae released by the female parasite.

[0021] In some embodiments, the disease caused by a parasite in a subject is transmitted by mosquitoes infected by the parasite. As nonlimiting examples, the disease can include elephantiasis, dirofilariasis, leishmaniasis, chagas disease, Lyme disease, gastroenteritis, colitis, parasitemia, lymphatic filariasis, ascariasis, hookworm, strongyloidiasis, malaria, babesiosis, or trypanosomiasis. In some embodiments, the disease is elephantiasis or dirofilariasis.

[0022] The subject as described herein can be, for example, a human, a dog, a cat, a cow, a horse, a sheep, a pig, a goat, a donkey, or a mule.

[0023] The methods as described herein can further include administering a therapeutic agent to the subject. For example, the therapeutic agent can include melarsomine, 2-deoxy glucose, or a combination thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] For a more complete understanding of the embodiments and the advantages thereof, reference is now made to the following description, in conjunction with the accompanying figures briefly described as follows:

[0025] FIG. 1 shows the chemical structures of atovaquone (ATO), mitochondrial-tagged ATO, HNK, and Mito-HNK.

[0026] FIGS. 2A-2B show the effects of Mito- ATO on adult B. Malayi movements. FIG.2A: Adult B. Malayi movements (scored from 1 as normal movement to 0 as no movement).FIG. 2B: Both male and female B. Malayi were treated with Mito-ATO as indicated, and the movements were monitored and scored for five days. **, P<0.01 vs untreated control group at each time point. Data shown are the mean±SD, n=6.

[0027] FIGS. 3A-3F show the effects of Mito-ATO or ATO on adult female and male B. Malayi movements. Both male and female B. Malayi were treated with Mito-ATO (FIG. 3A) or ATO (FIG. 3D) as indicated and the movements were monitored and scored for five days. Effects of Mito-ATO (FIG. 3B) or ATO (FIG. 3E) in female B. Malayi movements. Effects of Mito-ATO (FIG. 3C) or ATO (FIG. 3F) in male B. Malayi movements. **, P<0.01 vsAttorney Docket No. 650053.01255untreated control group at each time point. Data shown are the mean±SD, n numbers are as indicated.

[0028] FIG. 4 shows the effects of Mito-ATO on microfilaria larvae releasing by female B. Malayi after 48 h treatment. Microfilaria larvae observed at 48 h.

[0029] FIG. 5 shows the effects of Mito-ATO on microfilaria larvae releasing by female B. Malayi after treatment for five days as compared to the untreated control. The female B. Malayi treated with Mito-ATO as indicated for five days. The microfilaria larvae released in the culture media were shown.

[0030] FIGS.6A-6B show the effects of Mito-ATO on female B. Malayi cellular structures at 1 Ox lens (FIG. 6A) and 4x lens (FIG. 6B). The female B. Malayi cellular structures were observed by fluorescence staining for active mitochondria (Mito-tracker deep red) and cell death (SYTOX Green Nucleic Acid Stain) as indicated. Mito-tracker deep red dye is a cellpermeant MitoTracker probe which stains active mitochondria in live cells for labeling and localization in fluorescent cell imaging. SYTOX Green allows quick determination of cell viability. It will not cross intact membranes but will easily penetrate compromised membranes characteristic of dead cells. Because it exhibits >500-fold fluorescence enhancement upon binding nucleic acids, the SYTOX Green method labels the nuclei of dead cells, yielding green fluorescence.

[0031] FIG. 7 shows the fluorescent labelled (Mitotracker Green™) Brugia malayi (left) and Mito CP-treated Brugia malayi (right). Mito CP-treated parasites reveal swollen, granular mitochondria associated with rapid parasite death.

[0032] FIG. 8 shows both Mito Q and Mito CP rapidly kill male and female Brugia malayi at concentrations less than one micromolar.

[0033] FIG. 9A shows that Male and female B. malayi were treated with Mito-HNK or HNK as indicated, and mobility was assessed for four days.

[0034] FIG. 9B shows that D. immitis microfilariae were treated with Mito-HNK as indicated, and motility was monitored for three days. Dog microfilaria (n=4). ** P<0.01 vs. control group at each time point. Data are presented as mean ± SD; n=3 for each sex.

[0035] FIG. 9C shows the effect of Mito-ATO on B. malayi microfilariae, showing decreased release from adult females into the culture medium.

[0036] FIG. 9D shows that adult B. malayi treated for 24 h were stained with MitoTracker Deep Red (left) to visualize mitochondria and SYTOX Green (right) to detect cell death, showing pronounced mortality after Mito-ATO treatment.Attorney Docket No. 650053.01255

[0037] FIGS. 10A-10D show in vivo measurement of mitochondrial respiration and mobility in B. malayi microfilaria: effect of Mito-HNK. FIG. 10A shows schematic representation of the Seahorse XF96 Analyzer used for real-time bioenergetic profiling of live B. malayi microfilariae. The design of the measurement is shown with the sensor cartridge in the resting (left) and lowered (right) positions. When lowered, a transient microchamber is formed above the microfilarial suspension, enabling continuous monitoring of oxygen and proton flux. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) are calculated from the slopes of oxygen and pH traces, respectively, providing quantitative indices of mitochondrial respiration and glycolytic activity. FIG. 10B shows longitudinal measurements of OCR and parasite density from Day 1 to Day 6 demonstrate the stability of microfilarial mitochondrial activity over several days, establishing the feasibility of extended, real-time metabolic monitoring in live parasites. FIG. 10C shows effect of Mito-HNK on mitochondrial bioenergetics, OCR (left) and ECAR (right) of B. malayi microfilariae. FIG.10D shows correlation between mitochondrial respiration (OCR) and microfilarial mobility following treatment with 0.5 pM Mito-HNK, indicating that mitochondrial function is closely linked to parasite motility and viability.

[0038] FIGS. 11A-11D shows dual targeting of mitochondrial function and mobility in B. malayi microfilariae. (FIG. 11A) Dose-dependent effects of 2-deoxyglucose (2-DG) on B. malayi mitochondrial oxygen consumption rate (OCR) when administered alone (left) or in combination with Mito-HNK (right). (FIG. 11B) Effects of 2-DG on extracellular acidification rate (ECAR) alone (left) and in combination with Mito-HNK (right), illustrating complementary inhibition of glycolytic and mitochondrial pathways. (FIG. 11C) Combined treatment with Mito-HNK and 2-DG on B. malayi microfilarial mobility. (FIG. 11D) Inhibitory effects of Mito-HNK and 2-DG, alone and in combination, on microfilarial mobility in D. immitis (canine heartworm).

[0039] FIGS. 12A-12C shows the effects of melarsomine on B. malayi microfilaria mitochondrial functions and mobility with or without glycolysis inhibitor, 2-DG. Effects of melarsomine on B. malayi microfilaria mitochondrial functions (oxygen consumption. OCR). B. malayi microfilaria were treated with melarsomine alone (FIG. 12A, left) or in combination with 10 mM of 2-DG (FIG 12A, right) as indicated. The microfilaria mitochondrial OCR were monitored for seven days. Effects of melarsomine on B. malayi microfilaria mitochondrial functions (glycolysis functions, ECAR). B. malayi microfilaria were treated with melarsomine alone (FIG 12B, left) or in combination with 10 mM of 2-DG (FIG 12B, right) as indicated.Attorney Docket No. 650053.01255The microfilaria mitochondrial ECAR were monitored for seven days. (FIG 12C) Effects of melarsomine in combination with 2-DG on B. malayi microfilaria movements.

[0040] FIG. 13 shows uptake of Mito-ATO and Mito-HNK into mitochondria driven by transmembrane potential difference.

[0041] FIG. 14 shows mitochondrial electron transport chain and energy production. Inhibition of complex I by Mito-HNK and complexes I and III by Mito-ATO.

[0042] FIG. 15 shows metabolism of glucose and glutamine to ATP.

[0043] FIG. 16 shows the chemical structures of 2-DG and melarsomine.

[0044] The drawings illustrate only example embodiments and are therefore not to be considered limiting of the scope of the embodiments described herein, as other embodiments are within the scope of the disclosure.DETAILED DESCRIPTION OF THE INVENTION

[0045] Before the present materials and methods are described, it is understood that this invention is not limited to the particular methodology, protocols, materials, and reagents described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[0046] As used in this specification and the claims, the singular forms “a,” “an,” and “the” include plural forms unless the context clearly dictates otherwise. For example, the term “a compound” should be interpreted to mean “one or more compounds” unless the context clearly dictates otherwise. As used herein, the term “plurality” means “two or more.”

[0047] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean up to plus or minus 10% of the particular term and “substantially” and “significantly” will mean more than plus or minus 10% of the particular term.

[0048] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing theAttorney Docket No. 650053.01255inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0049] Definitions

[0050] Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75thEd., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry', Thomas Sorrell, University' Science Books, Sausalito, 1999; Smith and March March ‘s Advanced Organic Chemistry', 5thEdition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modem Methods of Organic Synthesis, 3rdEdition, Cambridge University Press, Cambridge, 1987; the entire contents of each of which are incorporated herein by reference. Derivatives of the compounds as described herein include structural variants that can be obtained by modifications of the synthesis of such compounds, as generally understood in the art of organic synthesis. As an example, esters and amides of a compound having a reactive group (e.g., COOH, OH, or NH2) can be viewed as derivatives of the original compounds.

[0051] The term ‘“alkyl” as used herein refers to a monovalent saturated straight or branched hydrocarbon, such as a straight or branched group of 1-20, 1-12, 1-10, or 1-6 carbon atoms, referred to herein as C1-C20 alky l (or Ci-2oalkyl), C1-C12 alkyl (or Ci alky l), C1-C10 alkyl (or Ci-ioalkyl), or Ci-Ce alkyl (or Ci-ealkyl), respectively.

[0052] The term “alkylene” refers to a divalent saturated straight or branched hydrocarbon group, such as a straight or branched group having 1-20. 1-12, 1-10, or 1-6 carbon atoms, referred to herein as a C1-C20 alkylene (or Ci-2oalkylene), C1-C12 alkylene (or Ci- alkylene), C1-C10 alkylene (or Ci-ioalkylene), or Ci-Ce alkylene (or Ci-ealkylene), respectively. An exemplary alkylene group is -CH2CH2-.

[0053] The term “alkenyl” refers to a monovalent straight or branched hydrocarbon group having one or more double bonds. An alkenyl group having up to 20 carbon atoms is referred to as a C2-C20 alkenyl (or C2-2oalkenyl). Likewise, for example, an alkenyl having up to 6 carbon atoms is referred to as a C2-C6 alkenyl (or C2-6alkenyl).

[0054] The term “alkenylene” refers to a divalent straight or branched hydrocarbon group having one or more double bonds. An alkenylenyl group having up to 20 carbon atoms isAttorney Docket No. 650053.01255referred to as a C2-C20 alkenylene (or C2-2oalkenylene). Likewise, for example, an alkenylene having up to 6 carbon atoms is referred to as a C2-C6 alkenylene (or C2-6alkenylene).

[0055] The term "alkoxy" as used herein means an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, and hexyloxy.

[0056] The term ‘"carboxy” or "carboxyl" as used herein refers to the group -COOH or its corresponding salts, e.g. -COONa, etc.

[0057] The term "aryl" is art-recognized and refers to a carbocyclic aromatic group. Representative aryl groups include phenyl, naphthyl, and the like. The term "aryl" includes polycyclic ring systems having two or more carbocyclic rings in which two or more carbons are common to two adjoining rings (the rings are "fused rings") wherein at least one of the rings is aromatic and, e.g., the other ring(s) may be cycloalkyls, cycloalkenyls, cycloalkynyls, and / or aryls.

[0058] The term “arylene” refers to a divalent carbocyclic aromatic group. Representative arylene groups include -C6H4-, -C10H6-, and the like. The term "arylene" includes polycyclic ring systems having two or more carbocyclic rings in which two or more carbons are common to two adjoining rings (the rings are "fused rings") wherein at least one of the rings is aromatic and, e.g., the other ring(s) may be cycloalkyls, cycloalkenyls, cycloalkynyls, and / or aryls.

[0059] The term "phenyl" refers to a mono-substituted benzene ring and has a formula of -C6H5.

[0060] The term "heteroaryl" is art-recognized and refers to a heterocyclic aromatic group. Representative heteroaryl groups include pyridinyl, quinolinyl, furanyl, thionyl, and the like. The term "heteroaryl" includes polycyclic ring systems having two or more heterocyclic rings in which two or more carbon or heteroatom are common to two adjoining rings (the rings are "fused rings") wherein at least one of the rings is a heterocyclic aromatic group and, e.g., the other ring(s) may be cycloalkyls, cycloalkenyls, cycloalkynyls, and / or aryls. In certain embodiments, the heteroaryl group is a 6-10 membered ring structure. The term "pyridyl" refers to a group derived from pyridine by removal of a hydrogen atom from a ring carbon atom in pyridine. The pyridyl group has a formula -C5H4N.

[0061] The term "cycloalkyd" refers to a monovalent saturated cyclic, bicyclic, or bridged cyclic (e.g., adamantyl) hydrocarbon group of 3-12, 3-8, 4-8, or 4-6 carbons, referred to herein, e.g., as " C4-8-cycloalkyl," derived from a cycloalkane. Unless specified otherwise, cycloalkyl groups are optionally substituted at one or more ring positions with, for example, alkanoyl,Attorney Docket No. 650053.01255alkoxy, alkyl, haloalkyl, alkenyl, alkynyl, amido or carboxyamido, amidino, amino, aryl, arylalkyl, azido, carbamate, carbonate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halo, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, imino, ketone, nitro, phosphate, phosphonato, phosphinato, sulfate, sulfide, sulfonamide, sulfonyl or thiocarbonyl. In certain embodiments, the cycloalkyl group is not substituted, i.e., it is unsubstituted.

[0062] The term “cycloalkylene" refers to a divalent saturated cyclic, bicyclic, or bridged cyclic (e.g., adamantyl) hydrocarbon group having, for example, 3-12, 3-8, 4-8, or 4-6 carbons derived from a cycloalkane. An exemplary cycloalkylene group is -C3H4- Unless specified otherwise, cycloalkylene groups are optionally substituted at one or more ring positions with, for example, alkanoyl, alkoxy, alky l, haloalkyl, alkenyl, alkynyl, amido or carboxyamido, amidino, amino, aryl, arylalkyl, azido, carbamate, carbonate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halo, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, imino, ketone, nitro, phosphate, phosphonato, phosphinato, sulfate, sulfide, sulfonamide, sulfonyl or thiocarbonyl. In certain embodiments, the cycloalkylene group is not substituted, i.e., it is unsubstituted.

[0063] The terms "heterocycloalkyl" and "heterocyclic group" are art-recognized and refer to saturated, partially unsaturated, or aromatic 3- to 10-membered ring structures, alternatively 3-to 7-membered rings, whose ring structures include one to four heteroatoms, such as nitrogen, oxygen, and sulfur. The number of ring atoms in the heterocyclyl group can be specified using Cx-Cyor Cx-y nomenclature where x and y are integers specifying the number of ring atoms. For example, a C3-C7 (or C3-7) heterocyclyl group refers to a saturated or partially unsaturated 3- to 7-membered ring structure containing one to four heteroatoms, such as nitrogen, oxygen, and sulfur. The designation " C3-C7" or “C3-7’' indicates that the heterocyclic ring contains a total of from 3 to 7 ring atoms, inclusive of any heteroatoms that occupy a ring atom position. In one embodiment, the heterocyclyl is piperidinyl.

[0064] The term "halogen" refers to halogen atoms F, Cl, Br, and I, or halogen substituents fluoro (-F), chloro (-C1), bromo (-Br), and iodo- (-1).

[0065] The term “haloalky l” is art-recognized and refers to an alkyl group, as defined above, having halogen atoms, as defined above, replacing one or more hydrogen atoms. Representative haloalkyl groups include trifluoromethyl, dibromoethyl, monochloropropyl, and the like.

[0066] The term "hydroxy" refers to a group of the form -OH.

[0067] The term “hydroxy alkyl” refers to an alk l means an alkyl, as defined herein, in which a hydrogen atom is replaced by -OH. Representative examples of hydroxy alkyl include,Attorney Docket No. 650053.01255but are not limited to those derived from Ci-6 alky ls, such as -CH2OH, -CH2CH2OH, -CH2CH2CH2OH. and the like.

[0068] The term "nitro" refers to a group of the form -NO2.

[0069] The term "cyano" refers to a group of the form -CN.

[0070] Terms such as "alkyl," "cycloalkyl," "alkylene," "cycloalkylene," etc. may be preceded by a designation indicating the number of atoms present in the group in a particular instance (e.g., “Cwalkyl” ■’Ci-CTalky l ’. “Cs-ecycloalkyF; ‘" Cs-Cscycloalkyl’; " Cjualkylene". “Ci-C4alkylene”). These designations are used as generally understood by those skilled in the art. For example, the representation " C" followed by a subscripted number indicates the number of carbon atoms present in the group that follows. Thus, " Csalkyd" is an alkyl group with three carbon atoms (i.e., n-propyl. isopropyl). Where a range is given, as in " C1-C4" or " C1-4" the members of the group that follows may have any number of carbon atoms falling within the recited range. A " Ci-C4alkyl" or " Ci-4alkyl," for example, is an alkyl group having from 1 to 4 carbon atoms, however arranged (i.e., straight chain or branched).

[0071] If a group is described as being "substituted", a non-hydrogen substituent group is in the place of hydrogen on a carbon or nitrogen of that group. Thus, for example, a substituted alkyl is an alkyl in which at least one non-hydrogen group is in the place of a hydrogen on the alkyl. To illustrate, monofluoroalkyl is alkyl substituted with a fluoro group, and difluoroalkyl is alkyl substituted with two fluoro groups. It should be recognized that if there is more than one substitution on a substituent, each non-hydrogen group may be identical or different (unless otherwise stated). Substituent groups include, but are not limited to, halogen, =0, =S, cyano, nitro, fluoroalkyd, alkoxyfluoroalkyl, fluoroalkoxy, alkyd, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyd, cycloalkyd, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, acylamino, aminoalkyd, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, -COOH, ketone, amide, carbamate, and acyl.

[0072] When a group is referred to as "unsubstituted" or not referred to as "substituted" or "optionally substituted", it means that the group does not have any substituents. If a group is described as being "optionally substituted", the group may be either (1) not substituted or (2) substituted. If a group is described as being optionally substituted with up to a particular number of non-hydrogen substituents, that group may be either (1) not substituted; or (2) substituted by up to that particular number of substituent groups or by up to the maximum number of substitutable positions on that group, whichever is less.Attorney Docket No. 650053.01255

[0073] If substituents are described as being independently selected from a group, each substituent is selected independent of the other. Each substituent, therefore, may be identical to or different from the other substituent(s).

[0074] A person of ordinary skill in the art would be able to choose the substituents that fulfill the valency rules. For example, in a non-solvated or non-salt form of a compound, nitrogen typically has three bonds attached to it and oxygen typically has two bonds attached to it.

[0075] As used herein, "salt" refers to acid addition salts and basic addition salts. It may also refer to those salts that may be prepared in situ during the final isolation and purification of the present compounds.

[0076] The compounds of the disclosure may contain one or more chiral centers and / or double bonds and, therefore, exist as stereoisomers, such as geometric isomers, enantiomers or diastereomers. The term “stereoisomers” when used herein consist of all geometric isomers, enantiomers or diastereomers. These compounds may be designated by the symbols “R” or “S,” depending on the configuration of substituents around the stereogenic carbon atom. The present disclosure encompasses various stereo isomers of these compounds and mixtures thereof. Stereoisomers include enantiomers and diastereomers. Mixtures of enantiomers or diastereomers may be designated "(±)" in nomenclature, but the skilled artisan will recognize that a structure may denote a chiral center implicitly. It is understood that graphical depictions of chemical structures, e.g., generic chemical structures, encompass all stereoisomeric forms of the specified compounds, unless indicated otherwise.

[0077] The term “pharmaceutically acceptable salt thereof’ means a salt prepared by combining a compound of formula (I), (II), (III), (IV), (V), (VI), or (VII) with an acid whose anion, or a base whose cation, is generally considered suitable for human consumption. Pharmaceutically acceptable salts are particularly useful as products of the methods of the present invention because of their greater aqueous solubility relative to the parent compound. For use in medicine, the salts of the compounds of this invention are non-toxic “pharmaceutically acceptable salts”. Salts encompassed within the term “pharmaceutically acceptable salts” refer to non-toxic salts of the compounds of this invention which are generally prepared by reacting the free base with a suitable organic or inorganic acid.

[0078] Suitable pharmaceutically acceptable acid addition salts of the compounds of the present invention when possible include those derived from inorganic acids, such as hydrochloric, hydrobromic, hydrofluoric, boric, fluoroboric, phosphoric, metaphosphoric, nitric, carbonic, sulfonic, and sulfuric acids, and organic acids such as acetic, benzenesulfonic,Attorney Docket No. 650053.01255benzoic, citric, ethanesulfonic, fumaric, gluconic, glycolic, isothionic, lactic, lactobionic, maleic, malic, methanesulfonic, trifluoromethanesulfonic, succinic, toluenesulfonic, tartaric, and trifluoroacetic acids. Suitable organic acids generally include, for example, aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic, and sulfonic classes of organic acids. Specific examples of suitable organic acids include acetate, trifluoroacetate, formate, propionate, succinate, glycolate. gluconate, digluconate, lactate, malate, tartaric acid, citrate, ascorbate, glucuronate, maleate, fumarate, pyruvate, aspartate, glutamate, benzoate, anthranilic acid, stearate, salicylate, p-hydroxybenzoate, phenylacetate, mandelate, embonate (pamoate), methanesulfonate, ethanesulfonate, benzenesulfonate, pantothenate, toluenesulfonate, 2-hydroxyethanesulfonate, sufanilate, cyclohexylaminosulfonate, P-hydroxybutyrate. galactarate, galacturonate, adipate, alginate, butyrate, camphorate, camphorsulfonate, cyclopentanepropionate, dodecylsulfate, glycoheptanoate, glycerophosphate, heptanoate, hexanoate, nicotinate, 2-naphthalesulfonate, oxalate, palmoate, pectinate, 3-phenylpropionate, picrate, pivalate, thiocyanate, and undecanoate.

[0079] Furthermore, where the compounds of the invention cany’ an acidic moiety, suitable pharmaceutically acceptable salts thereof may include alkali metal salts, i.e., sodium or potassium salts; alkaline earth metal salts, e.g., calcium or magnesium salts; and salts formed with suitable organic ligands, e.g., quaternary' ammonium salts. In another embodiment, base salts are formed from bases which form non-toxic salts, including aluminum, arginine, benzathine, choline, di ethylamine, diolamine, glycine, lysine, meglumine, olamine, tromethamine and zinc salts.

[0080] Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like. Bases useful in preparing such salts include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide, calcium carbonate, and the like.

[0081] Organic salts may be made from secondary, tertiary' or quaternary' amine salts, such as tromethamine, diethylamine, N, N’-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and procaine. Basic nitrogen-containing groups may be quatemized with agents such as lower alkyl (Ci-Cs) halides (e.g. methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides), dialkyd sulfates (i.e., dimethyl, diethyl, dibutyl, and diamyl sulfates), long chain halides (e.g., decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides), arylalkyl halides (e.g.. benzyl and phenethyl bromides), and others.Attorney Docket No. 650053.01255

[0082] The particular counter-ion forming a part of any salt of a compound disclosed herein may not be critical to the activity of the compound, so long as the salt as a whole is pharmacologically acceptable and as long as the counter-ion does not contribute undesired qualities to the salt as a whole. Undesired qualities may include undesirably solubility or toxicity.

[0083] Pharmaceutically acceptable esters and amides of the compounds can also be employed in the compositions and methods disclosed herein. Examples of suitable esters include alkyl, aryl, and aralkyl esters, such as methyl esters, ethyl esters, propyl esters, dodecyl esters, benzyl esters, and the like. Examples of suitable amides include unsubstituted amides, monosubstituted amides, and disubstituted amides, such as methyl amide, dimethyl amide, methyl ethyl amide, and the like.

[0084] Site specific substitution of atoms having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number that predominates in nature can be regarded as a substituent of a compound of the present disclosure. A sample of a compound having such an isotope as a substituent has at least 50% isotope incorporation at the labelled position(s). The concentration of such isotopes, e.g., deuterium, may be defined by the isotopic enrichment factor. The term “isotopic enrichment factor” as used herein means the ratio between the isotopic abundance and the natural abundance of a specified isotope. For example, if a substituent in a compound of this invention is denoted deuterium, such compound has an isotopic enrichment factor for each designated deuterium atom of at least 3500 (52.5% deuterium incorporation at each designated deuterium atom), at least 4000 (60% deuterium incorporation), at least 4500 (67.5% deuterium incorporation), at least 5000 (75% deuterium incorporation), at least 5500 (82.5% deuterium incorporation), at least 6000 (90% deuterium incorporation), at least 6333.3 (95% deuterium incorporation), at least 6466.7 (97% deuterium incorporation), at least 6600 (99% deuterium incorporation), or at least 6633.3 (99.5% deuterium incorporation).

[0085] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications and patents specifically mentioned herein are incorporated by reference for all purposes including describing and disclosing the chemicals, cell lines, vectors, animals, instruments, statistical analysis and methodologies which are reported in the publications which might be used in connection with the invention. AllAttorney Docket No. 650053.01255references cited in this specification are to be taken as indicative of the level of skill in the art. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0086] Method of Use

[0087] Filarial nematodes, including Brugia malayi and Dirofilaria immitis, cause debilitating diseases in humans and animals and remain difficult to treat due to limited macrofilaricidal options, drug resistance, and host toxicity. Disclosed herein is the repurposing of mitochondria-targeted agents as a strategy to selectively disrupt parasite bioenergetics. For instance, mitochondria-targeted derivatives include, but are not limited to, atovaquone (Mito-ATO) and honokiol (Mito-HNK), both of which are conjugated to the triphenylphosphonium cation to enhance mitochondrial uptake. The mitochondria-targeted agents (e.g., Mito-ATO and Mito-HNK) disclosed herein may be used as potent inhibitors for treatment of parasitic infection particularly parasitic nematode infections.

[0088] The compounds as described herein may effectively inhibit the mobility', growth and proliferation of parasites in vivo, in particular the mobility, growth, and proliferation of parasitic nematodes within a subject.

[0089] In use, the compounds as described herein may be cytotoxic to the parasite but not to the subject being treated. Therefore, the compounds as described herein may show a favorable safety profile for therapeutic applications.

[0090] Suitable subjects include mammals and non-mammals. Mammals include any member of the class Mammalia. Examples of mammals include, but are not limited to, humans, non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, and swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice, guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, crustaceans, among others. A subj ect can be any suitable age or sex. In some embodiments, the subj ect is a human. In some embodiments, the subject is a non-human subject, such as a fish, a bird, a dog, a cat, or a livestock.

[0091] As used herein, "treating” or “treatment” describes the management and care of a subject for combating the disease, condition, or disorder. Treating includes the administration of the compound or composition described herein to reduce, prevent, ameliorate and / or improve the onset of the symptoms or complications, alleviating the symptoms or complications, or reducing or eliminating the disease, condition, or disorder associated with a parasitic infection, such as a parasitic nematode infection. The term treating as describedAttorney Docket No. 650053.01255herein includes the inhibiting or reducing growth and / or propagation / reproduction / egg-laying of parasites within a subject (i.e., in vivo).

[0092] Symptoms of parasitic infections include, but are not limited to, for example, stomach cramps and pain, nausea or vomiting, dehydration, weight loss, swollen lymph nodes, digestive problems including unexplained constipation, diarrhea or persistent gas, skin issues such as rashes, eczema, hives, and itching, continuous muscle and joint pain, fatigue, depression or feeling of apathetic, constant hunger, iron deficiency / anaemia, grinding teeth during sleep, unexplained feelings of anxiety, recurrent yeast infections, itching of the anus or vagina, itching, redness, irritation, and an unusual discharge from the genital area, trouble falling asleep or waking up multiple times during the night.

[0093] As used herein, the term “effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired result, including a desired therapeutic result, such as mitigation and / or treatment of a parasitic infection. An effective amount of the compounds as disclosed herein may vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the disclosed compounds to elicit a desired response in the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the compounds as disclosed herein are reduced as compared with known compounds and are outweighed by the therapeutically beneficial effects.

[0094] An effective amount can be readily determined by the attending diagnostician, as one skilled in the art, by the use of known techniques and by observing results obtained under analogous circumstances. In determining the effective amount or dose of compound administered, a number of factors can be considered by the attending diagnostician, such as: the species of the subject; its size, age, and general health; the degree of involvement or the severity of the disease or disorder involved; the response of the individual subject; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances.

[0095] The term “administering” or “administration” refers to introducing the present compounds or compositions into the body of the subject, such as by oral delivery. Suitable routes of administration include, but are not limited to, oral, topical, transdermal, buccal, sublingual, pulmonary, transdermal, transmucosal, rectal, as well as subcutaneous, intraperitoneal, intravenous, and intramuscular injection.Attorney Docket No. 650053.01255

[0096] The present methods may target mitochondrial respiration or glycolysis in a parasite. In some embodiments, the present methods target mitochondrial respiration of a parasite by disrupting / inhibiting oxidative phosphorylation (OXPHOS) across various life stages of the parasite. Advantageously, the present methods may inhibit both glycolysis and OXPHOS simultaneously across various life stages of the parasite, as parasites are capable of reprogramming their energy metabolism and may display stage-specific shifts between glycolysis and OXPHOS.

[0097] In one aspect, the present disclosure provides a method of inhibiting proliferation of a parasite in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound as described herein, such as a compound of formula (I), (II), (III), (IV), (V). (VI). VII), or (VIII). or a pharmaceutically acceptable salt thereof:Attorney Docket No. 650053.01255whereinR1is Mito;one of R2and R3is Mito, the other is H;each of R4, R5, R6, R7, and R8is Mito;Z is NH or OL is Ci-2oalkylene, C2-2oalkenylene, LI-RC-L2, or amino acid;Li and L2 are each independently absent or C1-C10 alkylene;Rcis -(CH2CH2O)q-, arylene, or cycloalkylene;q is 1-20;Y at each occurrence is independently -CF3, Me, Cl. OMe, C(O)CH3. NO2. N(Me)2, COOH, F, Br, I, or OH;m at each occurrence is independently 0, 1, 2, 3, 4, or 5; andX is a counterion.

[0098] In another aspect, the present disclosure provides a method of treating a disease caused by a parasite in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound as described herein, such as a compound of formula (I), (II), (III), (IV), (V), (VI), (VII), or (VIII), or a pharmaceutically acceptable salt thereof:Attorney Docket No. 650053.01255(VIII) whereinR1is Mito;one of R2and R3is Mito, the other is H;each of R4, R5, R6, R7, and R8is Mito;Z is NH or O;Attorney Docket No. 650053.01255L is Ci-2oalkylene, C2-2oalkenylene, LI-RC-L2, or amino acid;Li and L2 are each independently absent or C1-10 alkylene;Rcis - (CH2CH2O)q-, arylene, or cycloalkylene;q is 1-20;Y at each occurrence is independently -CF3, Me, Cl, OMe, C(O)CH3, NO2, N(Me)2, COOH, F, Br, I, or OH;m at each occurrence is independently 0, 1, 2, 3, 4, or 5; andX is a counterion.

[0099] In some embodiments, the compound is a compound of formula (I-a), or a pharmaceutically acceptable salt thereof:wherein u is 2-16.

[0100] In some embodiments, u is 4, 10, 12, or 16.

[0101] In some embodiments, u is 10.

[0102] In some embodiments, the compound is a compound of formula (II) or (III), or a pharmaceutically acceptable salt thereof.

[0103] In some embodiments, the compound is a compound of formula (IV-a), or a pharmaceutically acceptable salt thereof:Attorney Docket No. 650053.01255MeO©MeO' (CH2)V— p(Ph)3X©(IV-a)wherein v is 1-20.

[0104] In some embodiments of formula (IV-a), v is 2-16.

[0105] In some embodiments of formula (IV-a), v is 10.

[0106] In some embodiments, the compound is a compound of formula (V-a), or a pharmaceutically acceptable salt thereof:P(Ph)>N'6* (V-a) wherein n is 1-20.

[0107] In some embodiments of formula (V-a), n is 2-10.

[0108] In some embodiments of formula (V-a), n is 10.

[0109] In some embodiments, the compound is a compound of formula (II-a), or a pharmaceutically acceptable salt thereof:©,(CH2)W- P(Ph)3x°(II-a) wherein w is 1-20.

[0110] In some embodiments, w is 2-10.

[0111] In some embodiments, w is 10.

[0112] In some embodiments, X is halogen, trifluoroacetate, or acetate.

[0113] In some embodiments, X is bromide.

[0114] In some embodiments, the parasite comprises parasitic nematode, parasitic protozoa, parasitic trematode, parasitic cestode, or parasitic conoidasida.Attorney Docket No. 650053.01255

[0115] In some embodiments, the parasite includes but is not limited to Brugia malayi, Diroftlaria Immitis, Leishmania spp., Trypanosoma cruzi, Borrelia burgdorferi, Toxoplasma, Pneumocystis, Plasmodium sp, amoebae, Giardia, Paramecium Balantidium, Trypanosoma, Trichomonas, Cryptosporidium, Babesia, filariae, Brugia, Onchocerca, Wuchereria, Mansonella, Loaiasis, Necator, Ancyclostoma, Schistosoma, Fasciola, Clonorchis, Paragonimus, Dirofilariae, Dictocalus, hookworm, Strongyloides, Onchocerca, ascariasis, cestodes, Coccidia, or a combination thereof.

[0116] In some embodiments, the parasite comprises Brugia malayi, Dirof aria immitis, or a combination thereof.

[0117] The present compound may be used to effectively control mobility', growth, and / or reproduction of adult parasites and / or larvae. In some embodiments, the parasite comprises both male and female microfilaria, and the compound or a pharmaceutically acceptable salt thereof inhibits movement of both the male and female microfilaria. In some embodiments, the parasite comprises both adult male and adult female parasites, and the compound or a pharmaceutically acceptable salt thereof inhibits movement of both the adult male and adult female parasites.

[0118] In some embodiments, the parasite comprises both male and female parasites, and the compound or a pharmaceutically acceptable salt thereof reduces the number of microfilariae released by the female parasite.

[0119] compounds as disclosed herein may provide treatment for a disease caused by parasites in multiple species (such as humans).

[0120] In some embodiments, the disease is transmitted by mosquitoes infected by the parasite.

[0121] In some embodiments, the disease is elephantiasis, dirofilariasis, leishmaniasis, chagas disease, Lyme disease, gastroenteritis, colitis, parasitemia, lymphatic filariasis, ascariasis, hookworm, strongyloidiasis, malaria, babesiosis, or trypanosomiasis. In some embodiments, the disease is elephantiasis or dirofilariasis.

[0122] In some embodiments, the subject is a human, a dog. a cat, a cow, a horse, a sheep, a pig, a goat, a donkey, or a mule. In some embodiments, the subject is a human. In some embodiments, the subject is a dog. In some embodiments, the subject is a cat.

[0123] The disclosed compounds may be administered with additional therapeutic agents. The additional therapeutic agent may include, for example, one or more known agent for treating parasitic infection. In some embodiments of the disclosed methods, one or more additional therapeutic agents are administered with the disclosed compounds, where the additionalAttorney Docket No. 650053.01255therapeutic agent is administered prior to, concurrently with, or after administering the disclosed compounds.

[0124] In some embodiments, the therapeutic agent includes an adulticide, such as melarsomine. In some embodiments, the therapeutic agent includes, but is not limited to, melarsomine, 2-deoxyglucose, or a combination thereof.

[0125] In some embodiments, co-administration with the therapeutic agent achieves dual targeting of glycolysis and OXPHOS in the parasite and leads to synergistic antiparasitic effects.

[0126] For the present methods, the compounds as described herein, or a salt thereof, or the composition as described herein may be introduced to a parasite that is in an infected subject (e.g., to inhibit the growth of. or treat an infection caused by, the parasite in the subject). In some embodiments, the subject is a human. In some embodiments, the subject is a dog. In some embodiments, the subject is a cat. Alternatively, the compound or composition may be introduced to a parasite that is in an environment (outside the body of a subject), such as a river, a lake, a water reservoir, a grassland, or a forest. In these cases, the effective amount of the compound or composition refers to an amount that provides desired effect of inhibiting the growth, mobility, and / or reproduction of the parasite in such environment.

[0127] The compounds utilized in the methods disclosed herein may be administered in conventional dosage forms prepared by combining the active ingredient with standard pharmaceutical carriers or diluents according to known procedures, including for example mixing, granulating, compressing, and / or dissolving various ingredients as appropriate to the desired preparation.

[0128] In some embodiments, the subject may be administered a dose of the disclosed compound as low as 1.25 mg, 2.5 mg, 5 mg. 7.5 mg, 10 mg, 12.5 mg. 15 mg. 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35 mg, 37.5 mg, 40 mg, 42.5 mg, 45 mg, 47.5 mg, 50 mg, 52.5 mg, 55 mg, 57.5 mg, 60 mg, 62.5 mg, 65 mg, 67.5 mg, 70 mg, 72.5 mg, 75 mg, 77.5 mg, 80 mg, 82.5 mg, 85 mg, 87.5 mg, 90 mg, 100 mg, 200 mg, 500 mg, 1000 mg, or 2000 mg once daily, twice daily, three times daily, four times daily, once weekly, twice weekly, or three times per week in order to treat the disease or disorder in the subject. In some embodiments, the subject may be administered a dose of the disclosed compound as high as 1.25 mg, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35 mg, 37.5 mg, 40 mg, 42.5 mg, 45 mg, 47.5 mg, 50 mg, 52.5 mg, 55 mg, 57.5 mg. 60 mg. 62.5 mg, 65 mg, 67.5 mg, 70 mg, 72.5 mg, 75 mg, 77.5 mg, 80 mg, 82.5 mg, 85 mg, 87.5 mg, 90 mg, 100 mg, 200 mg, 500 mg, 1000 mg, or 2000 mg, once daily, twiceAttorney Docket No. 650053.01255daily, three times daily, four times daily, once weekly, twice weekly, or three times per week in order to treat the disease or disorder in the subject. Minimal and / or maximal doses of the compounds may include doses falling within dose ranges having as endpoints any of these disclosed doses (e.g., 2.5 mg - 200 mg).

[0129] Oral administration is an illustrative route of administering the compounds employed in the compositions and methods disclosed herein. Other illustrative routes of administration include transdermaL percutaneous, intravenous, intramuscular, intranasal, buccal, intrathecal, intracerebral, or intrarectal routes. The route of administration may be varied in any way, limited by the physical properties of the compounds being employed and the convenience of the subj ect and the caregiver.

[0130] Suitable formulations include those that are suitable for more than one route of administration. For example, the formulation can be one that is suitable for both oral and intravenous administration. Alternatively, suitable formulations include those that are suitable for only one route of administration as well as those that are suitable for one or more routes of administration, but not suitable for one or more other routes of administration. For example, the formulation can be one that is suitable for oral, topical, transdermaL percutaneous, intravenous, intramuscular, intranasal, buccal, and / or intrathecal administration but not suitable for intracerebral administration.EXAMPLES

[0131] The following examples are shown to further illustrate the disclosed invention and are not intended to limit the disclosed invention, as recited in the claims.

[0132] Example 1

[0133] It was hypothesized that Mito-ATO and other mitochondria-targeted drugs (Mito-honokiol [Mito-HNK] and pegylated analogs of Mito-ATO and Mito-HNK) could be used as antiparasitic drugs. To this end, we tested the anti-filarial activity of ATO and Mito-ATO in a model system using B. malayi.

[0134] Results show that Mito-ATO is considerably more potent than ATO with nanomolar activity against male, female and microfdarial parasites and decreases the number of microfilaria released from gravid females.

[0135] FIGS. 2A-2B show that Mito-ATO dose-dependently inhibits the movements of the adult B. Malayi. Even at nanomolar concentrations of Mito-ATO, the movements of the adult B. Malayi are inhibited by Mito-ATO treatments as compared with the control group (FIG.2B).Attomey Docket No. 650053.01255

[0136] FIGS. 3A-3C show that Mito-ATO inhibits the movements of both male and female B. Malayi at low micromolar to high nanomolar concentrations. FIGS. 3D-3F shows that ATO inhibits female B. Malayi movement at 10-fold higher concentrations as compared with Mito-ATO. Additionally, even at 10-fold higher levels, ATO did not affect the movements of adult male B. Malayi.

[0137] FIG. 4 shows that Mito-ATO can potently inhibit the number of microfilaria larvae released from the adult female B. Malayi as soon as 48 hours after treatment. Furthermore, the movement of the microfilaria larvae was abrogated in Mito-ATO-treated group.

[0138] FIG. 5 shows the effect of Mito-ATO on microfilaria larvae released from the female B. Malayi into the culture media after five-day treatment. Mito-ATO inhibits both the total number of microfilaria larvae released and the mobility of the microfilaria larvae compared with the control group.

[0139] FIGS. 6A-6B show that fluorescence staining images of mitochondria (using the cell-permeant Mito-tracker deep red dye) in live B. Malayi cells and the SYTOX Green nucleic acid stain in Mito-ATO-treated cells. The SYTOX Green labels the nuclei of dead cells and the intense staining of the SYTOX Green indicate the extent of cell death in Mito-ATO-treated B. Malayi.

[0140] All the control groups are un-treated.

[0141] Prior to this present study with Brugia malayi, no reports of ATO activity against helminth parasites can be found. Preliminary data shows mitochondria-targeted drugs inhibit the movement of both male and female microfilaria by depriving them of energy. These worms consume a lot of energy and Mito-ATO targets the mitochondria of microfilaria and inhibits their proliferation. Mito-ATO is highly effective in both male and female parasites. Further, preliminary data shows a dose and time-dependent killing of both the adult and baby worms, which can get into the blood stream of mosquito infected with B. Malayi. Therefore, mitochondria-targeted drugs (Mito-ATO, Mito-HNK, Mito-metformin, Mito-magnolol, Mito-lonidamine and related pegylated analogs) could be developed as alternative drugs.

[0142] Further studies can be conducted to evaluate the pharmacokinetics and efficacy’ of various new mitochondria-targeted drugs for use in treatment of diseases, such as cancer chemotherapy and parasitic infections. In particular for anti -parasitic therapy, the present disclosure includes additional approaches to (a) increase the potency of anti-protozoal drugs such as ATO, and (b) broaden the spectrum of selected anti-protozoal drugs to include helminths or other parasites that share specific mitochondrial characteristics.

[0143] ReferencesAttorney Docket No. 650053.01255A. Kwarteng, S. T. Ahuno, F. O. Akoto, Killing filarial nematode parasites: role of treatment options and host immune response, Infectious Diseases of Poverty 5(1) (2016) 86.X. Qing, K. Kulkeaw, S. Wongkamchai, S. K.-W. Tsui, Mitochondrial Genome of Brugia malayi Microfilariae Isolated From a Clinical Sample, Frontiers in Ecology and Evolution 9 (2021).U. Strubing, R. Lucius, A. Hoerauf, K. M. Pfarr, Mitochondrial genes for heme-dependent respiratory chain complexes are up-regulated after depletion of Wolbachia from filarial nematodes, Int. J. Parasitol. 40(10) (2010) 1193-202.R. Zhang, J. Chen, D. Wang, Z. Q. Fu, Hijacking of host mitochondria by Toxoplasma gondii and SARS-CoV-2, Trends Parasitol. 38(4) (2022) 269-271.L. Monzote, L. Gille, Mitochondria as a promising antiparasitic target, Curr Clin Pharmacol 5(1) (2010) 55-60.H. Kenji, K. Keisuke, S. Shigeo, K. Kiyoshi, Mitochondria of Malaria Parasites as a Drug Target, in: S. Amidou (Ed.), An Overview of Tropical Diseases, IntechOpen, Rijeka, 2015, p. Ch. 2.G. L. Nixon, D. M. Moss, A. E. Shone. D. G. Lalloo. N. Fisher, P. M. O'Neill, S. A. Ward. G. A. Biagini, Antimalarial pharmacology and therapeutics of atovaquone, J. Antimicrob. Chemother. 68(5) (2013) 977-85.G. Cheng, M. Hardy, P. Topchyan, R. Zander, P. Volberding, W. Cui, B. Kalyanaraman, Potent inhibition of tumour cell proliferation and immunoregulatory function by mitochondria-targeted atovaquone Sci. Rep. 10(1) (2020) 17872.D. Xiong, Z. Yin, M. Huang, Y. Wang, M. Hardy, B. Kalyanaraman, S. T. Wong, M. You, Mitochondria-targeted atovaquone promotes anti-lung cancer immunity by reshaping tumor microenvironment and enhancing energy metabolism of anti-tumor immune cells, Cancer Commun (Lond) 44(3) (2024) 448-452.Gang Cheng, Micael Hardy, Paystar Topchyan, Ryan Zander, Peter Volberding, Weiguo Cui and Balaraman Kalyanaraman. Potent inhibition of tumour cell proliferation and immunoregulatory function by mitochondria-targeted atovaquone. Scientific Reports (2020) 10:17872Donghai Xiong, Zheng Yin, Mofei Huang, Yian Wange, Micael Hardy, Balaraman Kalyanaraman, Stephen Wong, Ming You. Mitochondria tagged atovaquone promotes antilung cancer immunity by reshaping tumor microenvironment and enhancing energy metabolism of anti-tumor immune cells. Cancer Communications (2024):44;448-452.Attorney Docket No. 650053.01255M Fry and D Jenkins. Nematoda: Aerobic respiratory pathways of adult parasitic species. Experimental Parasitology (1984) 57:86-924Gemma Nixon, Darren Moss, Alison Shone, David Lalloo, Nicholas Fisher, Paul O Neil, Stephen Ward and Giancarlo Biagini. Antimalarial pharmacology and therapeutics of atovaquone. J Antimicrobial Chemo (2013);68:977-985

[0144] Example 2

[0145] Here, we evaluate two MTAs — mitochondria-targeted atovaquone (Mito-ATO) and mitochondria-targeted honokiol (Mito-HNK) — where TPP+conjugation enhances mitochondrial uptake of the parent compounds (FIG. 1 and FIG. 13). Atovaquone (ATO) is an FDA-approved antiparasitic for malaria, toxoplasmosis, and pneumocystis pneumonia, while honokiol (HNK), a magnolia-derived polyphenol, has demonstrated antimicrobial and antiparasitic properties.

[0146] Using the human filarial parasite B. malayi as a model, we show that Mito-ATO and Mito-HNK exhibit substantially enhanced activity7compared with their parent drugs against both adult and larval stages. These MTAs disrupt mitochondrial function, inhibit parasite motility, and deplete ATP in a dose- and time-dependent manner, supporting their potential as macrofilaricidal agents. Additionally, we provide evidence that melarsomine, used clinically for canine heartworm disease, exerts effects primarily on microfilarial mitochondria.

[0147] Results

[0148] Adult female B. malayi were scored for movement before and after treatment using a standardized system (normal activity to complete immobility; FIG. 2A). Exposure to 1 pM Mito-ATO progressively inhibited motility over 1-5 days, a concentration well below human therapeutic plasma levels (5-10 pM after a single oral dose).

[0149] Using this scoring system, we compared worm motility under control, Mito-ATO, and ATO treatments at varying concentrations over five days (FIG. 3A and FIG. 3D). Mito-ATO inhibited mobility of both male and female worms at high nanomolar to low micromolar concentrations (FIG. 3 A). By contrast, ATO alone had no detectable effect, even at 10-fold higher concentrations, over two days (FIG. 3D). Similarly, Mito-HNK inhibited mobility in both sexes at comparable concentrations (FIG. 9A).

[0150] Effects on microfilariae

[0151] Mito-ATO treatment for five days significantly reduced both the number of microfilariae released by female worms and the motility of the larvae compared with theAttorney Docket No. 650053.01255control group (FIG. 4 and FIG. 5). Mito-HNK also inhibited microfilarial mobility across the high nanomolar to low micromolar range (FIG. 9A).

[0152] Mitochondrial activity and cell death

[0153] Mitochondrial function and cell viability were assessed using MitoTracker Deep Red (active mitochondria) and SYTOX Green (cell death marker). MitoTracker Deep Red is a cellpermeant probe that selectively stains active mitochondria in live cells, allowing for their visualization in fluorescent imaging. SYTOX Green, a nucleic acid stain, serves as a marker for cell death. This dye cannot penetrate the membranes of live cells but can enter cells with compromised membranes, binding to nucleic acids and emitting strong green fluorescence (>500-fold fluorescence enhancement).

[0154] FIG. 9D displays fluorescence images of live B. malayi stained with MitoTracker Deep Red and SYTOX Green. Untreated B. malayi showed robust mitochondrial staining with minimal SYTOX uptake, whereas Mito-ATO-treated worms exhibited intense SYTOX Green fluorescence, indicating extensive cell death (FIG. 9D).

[0155] Isolation and testing of D. immitis

[0156] Microfilariae were isolated from the blood of a naturally infected dog, collected via cephalic venipuncture, and processed through filtration and centrifugation. Blood was collected from the cephalic vein of a naturally infected dog identified at the University of Georgia (UGA) College of Veterinary Medicine, using a heparinized syringe, then diluted 1:10 with RPMI 1640 medium containing penicillin and streptomycin. The sample was filtered through an Isopore 5.0 pm polycarbonate membrane (Merck & Co. Inc., Rahway, New Jersey) and washed with fresh RPMI medium. After centrifugation, the microfilariae were collected from the membrane and resuspended in an appropriate culture medium.

[0157] Mito-HNK markedly reduced D. inmitis microfilarial mobility at high nanomolar to low micromolar concentrations, whereas unconjugated HNK was nearly 10-fold less potent (FIG. 9B).

[0158] Longitudinal bioenergetics by Seahorse XF96 Analyzer

[0159] To monitor mitochondrial respiration (oxygen consumption rate [OCR]) and glycolysis (extracellular acidification rate [ECAR]), microfilariae (4×104per well) were cultured with daily Seahorse XF96 measurements over six days, minimizing disruption to viability (FIG. 14). Control OCR values (pmol / min) declined modestly from 107±9 (Day 1) to 89±16 (Day 6), consistent with stable viability.

[0160] Correlation of OCR with microfilariae mobilityAttorney Docket No. 650053.01255

[0161] Mito-HNK dose-dependently suppressed OCR, nearly abolishing it at 5 pM, while parent HNK caused only modest inhibition (FIG. 10A). Mitochondrial OCR strongly correlated with microfilarial mobility in B. malayi (FIG. 10B), supporting Seahorse XF96 as a high-throughput proxy for parasite viability.

[0162] Mito-HNK effects on OCR and glycolysis

[0163] Concomitantly, Mito-HNK induced a robust compensatory rise in ECAR within two-to-three days, reflecting glycolytic upregulation (FIG. 10C). HNK induced only slight ECAR changes at much higher doses.

[0164] To confirm glycolytic involvement, we co-treated B. malayi microfilariae with Mito-HNK and 2-deoxyglucose (2-DG), a hexokinase (HK) inhibitor in the glycolytic pathway. As shown in FIG. 10D. 2-DG potentiated OCR suppression and significantly attenuated Mito-HNK-induced ECAR elevation, supporting the interpretation that ECAR increases reflect enhanced glycolytic flux.

[0165] Dose-dependent effects of 2-DG and Mito-HNK on OCAR / ECAR on B. malayi microfilariae

[0166] B. malayi microfilariae were treated with different concentrations of 2-DG alone and in combination with Mito-HNK. Mitochondrial OCR and ECAR were monitored for seven days. FIG. 11 A shows the effects of 2-DG alone on OCR. At 0.1 mM concentration, 2-DG did not significantly affect OCR. However, at higher concentrations (5-20 mM). 2-DG decreased OCR in a dose- and time-dependent manner.

[0167] Mito-HNK (0.5 pM) alone decreased OCR (50% inhibition) after three days (FIG.1 IB). In the presence of the lowest concentration of 2-DG (0.1 mM), Mito-HNK synergistically inhibited OCR (FIG. 11C).

[0168] Dose-response experiments showed that while low-dose 2-DG alone had little effect, its combination with Mito-HNK synergistically suppressed OCR, ECAR, and microfilarial motility' (FIG. 11D).

[0169] Dual treatment with Mito-HNK and 2-DG suppressed the mobility in microfilariae

[0170] Dual treatment with Mito-HNK and 2-DG inhibited microfilariae mobility greater than treatment with either agent alone (FIG. 11). Synergistic effects were most pronounced within two days, coinciding with peak ECAR elevations.

[0171] In canine D. Inmitis microfilariae, dual treatment similarly suppressed mobility more effectively than single agents, with a notably higher sensitivity to 2-DG than observed in B. malayi (FIG. 1 ID).Attomey Docket No. 650053.01255

[0172] Despite its antiparasitic mechanism, one of the major concerns with its use is its toxicity (hypoglycemia and metabolic disruption) to host cells. One way to mitigate 2-DG toxicity to normal cells is by enhancing the synergistic toxicity in combination with Mito-HNK. At two days of treatment with either Mito-HNK (0.5 pM) or 2-DG (1 mM), OCR was not affected in B. malayi microfilaria. However, in combination, OCR was inhibited by >70%. Under these conditions, the mobility of B. malayi was similarly affected.

[0173] Effect of melarsomine alone and in combination with Mito-HNK and 2-DG in B. malayi microfilariae

[0174] As melarsomine was reported to inhibit glucose uptake and metabolism in adult parasites, we decided to investigate its effects alone and in combination with Mito-HNK in B. malayi microfilariae. Microfilariae were treated with different concentrations of melarsomine and OCR and ECAR were monitored for 7 days (FIGS. 12A and 12B). Melarsomine (0.01 mg / ml) slightly enhanced OCR at day 1, followed by a decrease with time. At 0.03 mg / ml concentration, there was a marked decrease in OCR (25%) on day 1 and at 0.1 mg / ml OCR was totally inhibited.

[0175] Under these conditions, ECAR was significantly elevated, up by 200% on day 2 after treatment with 0.03 mg / ml melarsomine. These results suggest that melarsomine primarily inhibits OXPHOS rather than glycolysis in microfilariae. This is in contrast with its proposed antiglycolytic mechanism of action in heartworm parasites.

[0176] Combining melarsomine (0.01 mg / ml and 0.03 mg / ml) with Mito-HNK (0.5 pM) produced additive suppression of OCR and motility (FIG. 12C), supporting overlapping mitochondrial targets.

[0177] DISCUSSION

[0178] Parasitic filarial nematodes, such as D. immitis and B. malayi, have evolved to survive within diverse human and non-human hosts through remarkable metabolic plasticity. Central to this adaptability is mitochondrial function, which supports stage-specific shifts between glycolysis and OXPHOS. Some parasitic nematodes even possess parasite-specific respiratory systems, complicating the design of broad-spectrum antiparasitic agents that effectively disrupt energy metabolism across different hosts and life stages.

[0179] Mitochondrial targeting of ATO and HNK

[0180] Conjugation of ATO or HNK to the lipophilic TPP+cation promotes mitochondrial accumulation driven by the negative membrane potential (FIG. 13). In this study, both Mito-ATO and Mito-HNK disrupted parasite respiration and mobility at submicromolar concentrations, considerably outperforming their parent compounds. Mechanistically, Mito-Attorney Docket No. 650053.01255HNK inhibits ETC complex I, while Mito-ATO inhibits complexes I and III in cancer cells. Cancer is increasingly conceptualized as a parasitic disease due to its metabolic demands and ability to hijack host bioenergetic resources. Parasitic cells often maintain a strongly negative plasma and mitochondrial membrane potential, depending on life cycle stage. This facilitates the accumulation of Mito-ATO and Mito-HNK within mitochondria, where they disrupt respiration, impair mobility, and induce parasite death at submicromolar concentrations. Notably, both compounds are far more potent than their parent molecules (HNK and ATO) in selectively suppressing tumor cell proliferation while sparing normal cells, suggesting a favorable therapeutic index. Other mitochondrial complex I inhibitors (e.g., IACS-010759) have been associated with peripheral neurotoxicity. In contrast, published data using Mito-HNK and Mito-ATO show a favorable safety profile.

[0181] Correlation between parasite mobility and OCR

[0182] A positive correlation between mitochondrial OCR and microfilariae mobility (FIG.10D) demonstrates that Mito-HNK-induced inhibition of mitochondrial function directly compromises mobility and viability of B. malayi microfilariae. However, the specific mechanism varies based on the type of drug and the parasite it targets. Parasitic worms (helminths) possess nervous and neuromuscular systems that control their mobility. A variety of antiparasitic drugs (pyrantel pamoate, levamisole) induce paralysis in parasites by targeting neuromuscular systems. Under these conditions, a positive correlation between parasite mobility and mitochondrial respiration may be unlikely.

[0183] Dual targeting of glycolysis and OXPHOS

[0184] Parasites, like cancer cells, often rely on aerobic glycolysis (the “Warburg effect1’) despite oxygen availability. When glycolysis is inhibited, parasites may compensate by shifting to mitochondrial pathways. Conversely, in parasites with compromised mitochondrial function, glycolysis or glutaminolysis becomes the dominant energy source. Such metabolic flexibility is often accompanied by increased glucose uptake to sustain glycolytic flux. Interestingly, in the protozoan parasite Plasmodium falciparum, glycolytic inhibition paradoxically enhances mitochondrial activity, marked by increased oxygen consumption and ATP production. These findings suggest that simultaneous inhibition of glycolysis and OXPHOS may achieve synergistic antiparasitic effects in certain parasites. Results from this study show- that combining Mito-HNK with 2-DG enhances suppression of parasite metabolism and motility, confirming the benefits of dual targeting of bioenergetics.

[0185] Parasites are capable of reprogramming their energy metabolism in response to different life cycle stages, host environments, and external conditions. The life cycle ofAttorney Docket No. 650053.01255heartworms ( / ). immitis) is closely related to their bioenergetic state, as they modify their energy needs across developmental stages.

[0186] Mechanism of melarsomine in microfilariae

[0187] Melarsomine, an organic arsenical compound and an FDA-approved adulticidal agent for canine heartworm disease inhibits glucose metabolism by targeting phosphofructokinase, a rate-limiting enzy me in glycolysis (FIG. 15). Compared with older arsenicals like thiacetarsamide, melasomine is more effective and safer for treating adult heartworm. However, as an arsenic-based drug, melarsomine poses significant toxicity concerns. Although classically described as an antiglycolytic agent through phosphofructokinase inhibition, our results indicate melarsomine primarily inhibits OXPHOS in microfilariae. This aligns with evidence that arsenic-based drugs can disrupt mitochondrial ETC complexes, suggesting a broader mechanism than previously recognized. Clearly, arsenic is proposed as the key active and cytotoxic component in killing adult and immature heartworms.

[0188] Melarsomine is used as an adulticide, targeting primarily adult and mature heartworms (L5 stage, over four months old), not the microfilariae (LI stage). Although the precise mechanism is not fully known, melarsoimine was shown to disrupt the glycolytic pathway in heartworms and the inhibit energy production needed for survival. Arsenic-based antineoplastic drugs are known to inhibit both glycolysis and OXPHOS mechanisms. Arsenicbased drugs also react with intracellular glutathione and redox-regulating thiol -containing enzymes such as thioredoxin, and are effective at cross-linking vicinal thiols at the active site of tyrosine phosphatases. The interference with cellular metabolism, glycolysis, and OXPHOS, can have a wide range of therapeutic implications in parasitology. The primary mechanism of 2-DG involves its uptake and phosphorylation by hexokinase, an enzyme that normally acts on glucose (FIG. 15). Upon phosphorylation, 2-deoxyglucose-6-phosphate (2-DG-6-P) is formed. However, unlike glucose-6-phosphate, 2-DG-6-P cannot be isomerized to fructose-6-phosphate and accumulates within the cell that results in the inhibition of glycolysis and ATP formation. This effect is well characterized in cancer cells that rely heavily on glycolysis for energy (z.e., the Warburg effect). However, the antiglycolytic effects of 2-DG are not limited to cancer cells. Pathogens that rely on host cell glucose metabolism for survival can also be affected by 2-DG. Combination strategies incorporating melarsomine with MT As and / or 2-DG may enhance efficacy while enabling dose reduction and mitigating toxicity.

[0189] Conclusions and additional resultsAttorney Docket No. 650053.01255

[0190] Mito-ATO and Mito-HNK offer several key advantages: mitochondrial specificity, potent activity at low concentrations, and low host toxicity. Their potential synergy with agents such as melarsomine or 2-DG supports development of rational combination therapies. Their distinct mechanisms of action also raise the possibility of synergistic use with standard antiparasitic agents, potentially leading to more effective and better-tolerated combination therapies.

[0191] The present work suggests that melarsomine inhibits mitochondrial respiration in microfilariae, not glycolytic mechanism. This differs from the previous findings in microfilariae where melarsomine was reported to target the glycolytic mechanism. The present findings are also consistent with previous results using arsenic-based antitumor drugs that target mitochondrial electron transport chain. Additional studies can illustrate the mechanism of action of melarsomine in heartworm parasites.

[0192] The present work also suggests that mapping parasite bioenergetics across developmental stages using OCR / ECAR “bioenergetic profiling” to identify vulnerabilities (high- or low-glycolytic and high- or low-OXPHOS) can provide guidance for treatment design. Such approaches could accelerate the development of safer, more effective therapies for filarial diseases.

[0193] Methods

[0194] Synthesis of Mito-HNK

[0195] Mito-ort / io-honokiol (z.e., Mito-HNK) was prepared by reacting 10-bromodecyl-triphenylphosphonium with EINK in the presence of potassium carbonate in dimethylformamide DMF (Scheme 1).Scheme 1. Synthesis of Mito-HNK and HNKio. Reagents and conditions: i, K2CO3, DMF, 40°C, 24h, 40%.; ii, K2CO3, DMF, 40°C, 24h. 10%.

[0196] 10-Bromodecyltriphenylphosphonium bromide. A mixture of triphenylphosphine (1 g, 3.8 mmol) and dibromodecane (5.7 g, 19 mmol) was heated at 90°C for 6 h. After cooling,Attorney Docket No. 650053.01255the crude product was purified by flash chromatography (pentane, ether [EtzO], and dichloromethane [CH2Ch] / ethanol [EtOH] 9:1) to afford the corresponding phosphonium salt as a white solid (1 g, 47% yield).

[0197] 31P (400.13 MHz, CDCl3) δ 24.32.1H NMR (400.13 MHz, CDCl3) δ 7.85-7.65 (15H, m), 3.73-3.66 (2H, m), 3.40-3.34 (2H, m), 1.80-1.75 (4H, m), 1.31-1.20 (12H, m).

[0198] To a mixture of HNK (1.3 g, 4.9 mmol), anhydrous potassium carbonate (0.69 g. 4.9 mmol) in DMF (40 mL) was added 10-bromodecyl-triphenylphosphonium bromide (2.8, 4.9 mmol). The mixture was stirred at 40°C for 24 h. The solvent was removed under vacuum, and the residue was taken up into water and extracted with CH2CI2. The organic layer was dried over sodium sulfate (Na2SO4), and the solvent was removed under reduced pressure. Purification by flash chromatography (Et20, CH2CI2, and CH2C12 / EtOH) delivered the corresponding Mito-HNK (400 mg, 10% yield).

[0199] The characterization of the ortho-isomer was performed by two-dimensional nuclear magnetic resonance (NMR) (1H, COSY, HSQCed, HMBC, NOESY,13C) and purity was determined by both NMR and high-performance liquid chromatography (HPLC) (>99%). HRMS calculated for Mito-HNK C46H52O2P [MH]+667.3699, found, 667.3699.

[0200] Mito-HNK.31P (400.13 MHz, CDCl3) δ 24.58.1H NMR (400.13 MHz, CDCl3) δ 7.86-7.65 (15H, m), 7.35-7.21 (2H, m). 7.04-6.89 (4H, m), 6.04-5.92 (2H, m), 5.57 (1H. s), 5.13-4.98 (4H. m). 3.99 (2H, t, J= 6.4). 3.87-3.75 (2H, m). 3.45-3.28 (4H. m). 1.84-1.74 (2H. m), 1.62-1.56 (3H, m), 1.51-1.12 (11H, m).13C NMR (125 MHz, CDCl3) δ 156.3, 151.1, 137.8, 136.7, 134.9, 133.7, 131.8, 130.4, 130.3, 118.8, 118.2, 115.7, 115.6, 115.4, 111.7, 68.0, 39.4, 34.5, 30.4, 30.3, 29.3, 29.2, 29.1, 29.08, 29.04, 25.9, 22.7 (J= 49.2), 22.6 (J= 4.2).

[0201] Synthesis of Mito-ATO

[0202] Mito-ATO was prepared by reacting the appropriate bromoalkyltriphenylphosphonium bromide with ATO in the presence of potassium carbonate in DMF. In contrast, the non-targeting ATO derivative (ATO-C10) was prepared for negative controls by using the same procedure but in the presence of the corresponding alkyl bromides (Scheme 2).Attorney Docket No. 650053.01255Scheme 2. Synthesis of Mito-ATO and ATO-Cio. Reagents and conditions: i, (10-bromodecyl)-triphenylphosphonium bromide, K2CO3. DMF, 70°C, 9h, 52%; ii. Bromodecane, K2CO3, DMF, 70°C, 7h, 81%.

[0203] To a mixture of ATO (0.4 g, 1.1 mmol) and potassium carbonate (0.17 g, 1.2 mmol) in DMF (5 mL) was added (10-bromodecyl)-triphenylphosphonium bromide (0.61 g, 1.1 mmol). The mixture was stirred at 60°C overnight. Then, CH2CI2 was added to the mixture as well as H2O (20 mL). The organic layer was washed twice with water and dried over Na2SO4. The solvent was removed under reduced pressure. Then, Et20 was added to the mixture to precipitate the compound. Purification by flash chromatography (CH2Cl2 / EtOH 9 / 1) delivered the corresponding Mito-ATO (0.48 g, 52% yield). Purity' was determined by both NMR and HPLC (>99%). HRMS calculated for Mito-ATO C50H53ClO3P+[M]+767.3415, found, 767.3420.

[0204] LC-MS experiments

[0205] Typically, 2 pL of a sample was injected into a Kinetex EVO C18 column (100 A, 100mm x 2.1 mm, 1.7 pm) maintained at 25°C. The column was equilibrated with 95% water and 5% acetonitrile containing 0.1% of formic acid. Compounds were separated using a linear gradient from 5% to 100% over 8 min at a flow rate of 0.21 mL / min, followed by an isocratic gradient at 100% acetonitrile from 8 min to 15 min. The absorption traces were collected at 254 nm and 230 nm.

[0206] Microfilariae

[0207] Adult male, adult female, and microfilarial larvae of B. malayi and microfilaria of D. immitis were obtained from National Institutes of Health-supported and authenticated facilities. (Note: Microfilariae and adult stages of both B. malayi and D. immitis are noninfectious to humans.)

[0208] The Filariasis Research Reagent Resource Center (FR3). formerly known as the Filariasis Repository, was established by the National Institutes of Health / National Institute ofAttorney Docket No. 650053.01255Allergy and Infectious Diseases in 1968. The overall objective of the FR3 is to facilitate and stimulate research in filarial diseases, and the FR3 has served as a unique supply source of filarial research materials, training, and technical information for investigators. A wide range of organisms including both human and canine / feline pathogens such as B. malayi and Brugia pahangi are available as live parasites. The UGA College of Veterinary7Medicine provides access to live D. immitis.

[0209] We have in place institutional approval and material transfer agreements between the Medical College of Wisconsin, FR3, UGA, and Biological and Emerging Infections (BEI) Research Resources (ATCC). Our studies only utilized parasites that can be handled safely in a BSL2 facility.

[0210] In vivo measurements of OCR / ECAR using the Seahorse XF96 analyzer

[0211] The 96-well Seahorse extracellular flux 96 (XF96) analyzer, housed in the Center for Immunology / Department of Microbiology7Core Facility at the Medical College of Wisconsin, was used to simultaneously measure the cellular OCR (pmol / min) — indicative of OXPHOS — and ECAR (mpH / min) — a proxy for glycolytic activity7. Each XF assay kit includes a disposable sensor cartridge embedded with 96 pairs of fluorescent biosensors for oxygen and pH, coupled to a fiber optic waveguide (532 nm for oxygen and 470 nm for pH detection). The system allows for the delivery7of reagents or inhibitors to each well during the assay.

[0212] To accommodate the longitudinal nature of this in vivo study, we modified the experimental design so that OCR and ECAR measurements were taken once daily from the same set of microfilariae. This approach minimizes disruption to the wells and maintains a consistent number of microfilariae across six days, ensuring that observed metabolic changes reflect actual physiological shifts rather than reductions in viable parasites, which were concurrently monitored microscopically (FIG. 15, panel B).

[0213] Longitudinal measurements of OCR and ECAR

[0214] Microfilariae (4×104per well) were suspended in an XF96 plate and incubated with MT As and other inhibitors (as indicated) using unbuffered assay media (RPMI 1640 supplemented with 5.5 mM glucose and 2 mM L-glutamine), devoid of pyruvate and sodium bicarbonate, at 37° C. Bioenergetic function was assessed once every724 h. Three baseline OCR and ECAR measurements were recorded each day. After each measurement, half of the medium in each well was replaced with fresh RPMI 1640, with or without treatment, to recondition the parasites.

[0215] Isolation of heartworms (D. immitis)Attorney Docket No. 650053.01255

[0216] Blood was collected from the cephalic vein of a naturally infected dog identified at the UGA College of Veterinary Medicine, using a heparinized syringe, then diluted 1:10 with RPMI 1640 medium containing penicillin and streptomycin. The sample was filtered through an Isopore 5.0 pm polycarbonate membrane (Merck) and washed with fresh RPMI medium. After centrifugation, the microfilariae were collected from the membrane and resuspended in an appropriate culture medium.

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[0218] For reasons of completeness, various aspects of the invention are set out in the following numbered clauses:

[0219] Clause 1. A method of inhibiting proliferation of a parasite in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound of formula (I), (II). (III). (IV), (V), (VI), VII). or (VIII), or a pharmaceutically acceptable salt thereof,Attorney Docket No. 650053.01255(VIII) whereinR1is Mito;one of R2and R3is Mito, the other is H;each of R4, R5, R6, R7, and R8is Mito;Z is NH or O;Attorney Docket No. 650053.01255L is Ci-2oalkylene, C2-2oalkenylene, LI-RC-L2, or amino acid;Li and L2 are each independently absent or C1-C10 alkylene;Rcis - (CH2CH2O)q-, arylene, or cycloalkylene;q is 1-20;Y at each occurrence is independently -CF3, Me, Cl, OMe, C(O)CH3, NO2, N(Me)2, COOH, F, Br, I, or OH;m at each occurrence is independently 0, 1, 2, 3, 4, or 5; andX is a counterion.

[0220] Clause 2. A method of treating a disease caused by a parasite in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound of formula (I), (II), (III), (IV), (V), (VI), (VII), or (VIII), or a pharmaceutically acceptable salt thereof,Attorney Docket No. 650053.01255(VIII) whereinR1is Mito;one of R2and R3is Mito, the other is H;each of R4, R5, R6, R7, and R8is Mito;Z is NH or O;L is C i-2oalkylene, C2-2oalkenylene, LI-RC-L2, or amino acid;Li and L2 are each independently absent or C1-10 alkylene:Rcis - (CH2CH2O)q-, arylene, or cycloalkylene;q is 1-20;Y at each occurrence is independently -CF3, Me, Cl, OMe, C(O)CH3, NO2, N(Me)2, COOH, F, Br, I, or OH;m at each occurrence is independently 0, 1, 2, 3. 4, or 5: andX is a counterion.Attorney Docket No. 650053.01255

[0221] Clause 3. The method of any one of clauses 1-2, wherein the compound is a compound of formula (I-a), or a pharmaceutically acceptable salt thereof,wherein u is 2-16.

[0222] Clause 4. The method of clause 3, wherein u is 4, 10, 12, or 16.

[0223] Clause 5. The method of clause 3, wherein u is 10.

[0224] Clause 6. The method of any one of clauses 1-2, wherein the compound is a compound of formula (II) or (III), or a pharmaceutically acceptable salt thereof.

[0225] Clause 7. The method of any one of clauses 1-2, wherein the compound is a compound of formula (IV-a), or a pharmaceutically acceptable salt thereof.X X ® MeO^xrf "(CH2)v-P(Ph)3(IV-a)wherein v is 1-20.

[0226] Clause 8. The method of clause 7, wherein v is 2-16.

[0227] Clause 9.. The method of clause 7, wherein v is 10.

[0228] Clause 10. The method of any one of clauses 1-2, wherein the compound is a compound of formula (V-a), or a pharmaceutically acceptable salt thereof,O,>-■0. P(Ph)3’ \ X X6* (V-a) wherein n is 1-20.

[0229] Clause 11. The method of clause 10, wherein n is 2-10.

[0230] Clause 12. The method of clause 10. wherein n is 10.Attorney Docket No. 650053.01255

[0231] Clause 13. The method of any one of clauses 1-2, wherein the compound is a compound of formula (Il-a), or a pharmaceutically acceptable salt thereof,©

[0232] Clause 14. The method of clause 13, wherein w is 2-10.

[0233] Clause 15. The method of clause 13, wherein w is 10.

[0234] Clause 16. The method of any one of clauses 1-15, wherein X is halogen, trifluoroacetate, or acetate.

[0235] Clause 17. The method of any one of clauses 1-16, wherein X is bromide.

[0236] Clause 18. The method of any one of clauses 1-17, wherein the parasite comprises parasitic nematode, parasitic protozoa, parasitic trematode, parasitic cestode, or parasitic conoidasida.

[0237] Clause 19. The method of any one of clauses 1-18, wherein the parasite comprises Brugici malayi, Dirofilaria immitis, Leishmania spp.. Trypanosoma cruzi, Borrelia burgdorferi. Toxoplasma, Pneumocystis, Plasmodium sp, amoebae, Giardia, Paramecium Balantidium, Trypanosoma, Trichomonas, Cryptosporidium, Babesia, filariae, Brugia, Onchocerca, Wuchereria, Mansonella, Loaiasis, Necator, Ancyclostoma, Schistosoma, Fasciola, Clonorchis, Paragonimus, Dirofllariae, Dictocalus, hookworm, Strongyloides, Onchocerca, ascariasis, cestodes, Coccidia. or a combination thereof.

[0238] Clause 20. The method of any one of clauses 1-19, wherein the parasite comprises Brugia malayi, Dirofilaria immitis, or a combination thereof.

[0239] Clause 21. The method of any one of clauses 1-20, wherein the parasite comprises both male and female microfilaria, and wherein the compound, or a pharmaceutically acceptable salt thereof, inhibits movement of both the male and female microfilaria.

[0240] Clause 22. The method of any one of clauses 1-20, wherein the parasite comprises both adult male and adult female parasites, and wherein the compound, or a pharmaceutically acceptable salt thereof, inhibits movement of both the adult male and adult female parasites.Attorney Docket No. 650053.01255

[0241] Clause 23. The method of any one of clauses 1-20, wherein the parasite comprises both male and female parasites, and wherein the compound, or a pharmaceutically acceptable salt thereof, reduces the number of microfilariae released by the female parasite.

[0242] Clause 24. The method of any one of clauses 2-23, wherein the disease is transmitted by mosquitoes infected by the parasite.

[0243] Clause 25. The method of any one of clauses 2-24, wherein the disease is elephantiasis, dirofilariasis, leishmaniasis, chagas disease, Lyme disease, gastroenteritis, colitis, parasitemia, lymphatic filariasis, ascariasis, hookworm, strongyloidiasis, malaria, babesiosis, or trypanosomiasis.

[0244] Clause 26. The method of any one of clauses 2-25, wherein the disease is elephantiasis or dirofilariasis.

[0245] Clause 27. The method of any one of clauses 1-26, wherein the subject is a human, a dog, a cat, a cow, a horse, a sheep, a pig, a goat, a donkey, or a mule.

[0246] Clause 28. The method of any one of clauses 1-27, further comprising administering a therapeutic agent to the subject.

[0247] Clause 29. The method of clause 28, wherein the therapeutic agent comprises melarsomine, 2-deoxyglucose, or a combination thereof.

Claims

Attorney Docket No. 650053.01255CLAIMSWhat is claimed is:

1. A method of inhibiting proliferation of a parasite in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound of formula (I), (II), (III), (IV), (V), (VI), VII), or (VIII), or a pharmaceutically acceptable salt thereof,whereinR1is Mito;one of R2and R3is Mito, the other is H;each of R4, R5, R6, R7, and R8is Mito;Attorney Docket No. 650053.01255Z is NH or O;L is C i-2oalkylene, C2-2oalkenylene, LI-RC-L2, or amino acid;Li and L2 are each independently absent or C1-C10 alkylene;Rcis - (CH2CH2O)q-, arylene, or cycloalkylene;q is 1-20;Y at each occurrence is independently -CF3, Me, Cl, OMe, C(O)CH3, NO2, N(Me)2, COOH, F, Br, I, or OH;m at each occurrence is independently 0, 1, 2, 3, 4, or 5; andX is a counterion.

2. A method of treating a disease caused by a parasite in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound of formula (I), (II), (III), (IV), (V), (VI), (VII), or (VIII). or a pharmaceutically acceptable salt thereof,Attorney Docket No. 650053.01255(VIII) whereinR1is Mito;one of R2and R3is Mito, the other is H;each of R4, R5, R6, R7, and R8is Mito;Z is NH or O;L is C i-2oalkylene, C2-2oalkenylene, LI-RC-L2, or amino acid;Li and L2 are each independently absent or C1-10 alkylene:Rcis - (CH2CH2O)q-, arylene, or cycloalkylene;q is 1-20;Y at each occurrence is independently -CF3, Me, Cl, OMe, C(O)CH3, NO2, N(Me)2, COOH, F, Br, I, or OH;m at each occurrence is independently 0, 1, 2, 3. 4, or 5: andX is a counterion.Attorney Docket No. 650053.012553. The method of claim 1, wherein the compound is a compound of formula (I-a), or a pharmaceutically acceptable salt thereof,wherein u is 2-16.

4. The method of claim 3, wherein u is 4.

10.

12. or 16.

5. The method of claim 3, wherein u is 10.

6. The method of claim 1, wherein the compound is a compound of formula (II) or (III), or a pharmaceutically acceptable salt thereof.

7. The method of claim 1, wherein the compound is a compound of formula (IV-a), or a pharmaceutically acceptable salt thereof,OMeO^ Jl zI X ® MeOx' YZICH2)v-P(Ph)3O yQx(IV-a)wherein v is 1-20.

8. The method of claim 7, wherein v is 2-16.9.. The method of claim 7, wherein v is 10.

10. The method of claim 1, w herein the compound is a compound of formula (V-a), or a pharmaceutically acceptable salt thereof,Attorney Docket No. 650053.01255(CH2)n. P(Ph)3X©o* (V-a) wherein n is 1-20.

11. The method of claim 10, wherein n is 2-10.

12. The method of claim 10, wherein n is 10.

13. The method of claim 1, wherein the compound is a compound of formula (II-a), or a pharmaceutically acceptable salt thereof,©14. The method of claim 13, wherein w is 2-10.

15. The method of claim 13, wherein w is 10.

16. The method of claim 1, wherein X is halogen, trifluoroacetate, or acetate.

17. The method of claim 1, wherein X is bromide.

18. The method of claim 1, wherein the parasite comprises parasitic nematode, parasitic protozoa, parasitic trematode, parasitic cestode, or parasitic conoidasida.

19. The method of claim 1, wherein the parasite comprises Brugia malayi, Dirofilaria immitis, Leishmania spp., Trypanosoma cruzi. Borrelia burgdorferi, Toxoplasma, Pneumocystis, Plasmodium sp, amoebae, Giardia, Paramecium Balantidium, Trypanosoma, Trichomonas, Cryptosporidium, Babesia, filariae, Brugia, Onchocerca, Wuchereria,Attorney Docket No. 650053.01255Mansonella, Loaiasis, Necator, Ancyclostoma, Schistosoma, Fasciola, Clonorchis, Paragonimus, Dirofllariae, Dictocalus, hookworm, Strongyloides, Onchocerca, ascariasis, cestodes, Coccidia, or a combination thereof.

20. The method of claim 1, wherein the parasite comprises Brugia malayi, Dirofilaria immitis, or a combination thereof.

21. The method of claim 1, wherein the parasite comprises both male and female microfilaria, and wherein the compound, or a pharmaceutically acceptable salt thereof, inhibits movement of both the male and female microfilaria.

22. The method of claim 1, wherein the parasite comprises both adult male and adult female parasites, and wherein the compound, or a pharmaceutically acceptable salt thereof, inhibits movement of both the adult male and adult female parasites.

23. The method of claim 1, wherein the parasite comprises both male and female parasites, and wherein the compound, or a pharmaceutically acceptable salt thereof, reduces the number of microfilariae released by the female parasite.

24. The method of claim 2, wherein the disease is transmitted by mosquitoes infected by the parasite.

25. The method of claim 2, wherein the disease is elephantiasis, dirofilariasis, leishmaniasis, chagas disease, Lyme disease, gastroenteritis, colitis, parasitemia, lymphatic filariasis. ascariasis, hookworm, strongyloidiasis, malaria, babesiosis, or trypanosomiasis.

26. The method of claim 2, wherein the disease is elephantiasis or dirofilariasis.

27. The method of claim 1, wherein the subject is a human, a dog, a cat, a cow, a horse, a sheep, a pig, a goat, a donkey, or a mule.

28. The method of claim 1, further comprising administering a therapeutic agent to the subject.

29. The method of claim 28, wherein the therapeutic agent comprises melarsomine, 2-deoxy glucose, or a combination thereof.

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