Antiparasitic Drugs Global Market Report 2024

28 Oct.,2024

 

Antiparasitic Drugs Global Market Report

Antiparasitic Drugs Market Driver: Rising Incidence Of Vector-Borne Diseases Sparks Growth In The Antiparasitic Drugs Market

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The increasing incidence of vector-borne diseases is expected to propel the growth of the antiparasitic drugs market going forward. Vector-borne diseases refer to infectious diseases transmitted to humans or other animals through the bite of arthropods, primarily insects like mosquitoes, ticks and flies. Some examples of vector-borne diseases include malaria, dengue and yellow fever. Antiparasitic drugs play an essential role in stopping the spread of vector-borne diseases by targeting and eliminating the parasites responsible. A few antiparasitic drugs, including chloroquine, doxycycline and atovaquone-proguanil, are commonly used to cure malaria. For instance, in August , according to the Pan American Health Organization, a US-based specialized agency of the United Nations, dengue incidences had significantly increased in the US so far in . More than 3 million new infections have been reported so far, exceeding the numbers for &#;the year with the most significant recorded incidence of the disease in the area, with 3.1 million cases, including 28,203 severe cases and 1,823 deaths. Furthermore, in December , according to the World Malaria Report published by the World Health Organization, a Switzerland-based intergovernmental organization, 247 million malaria cases were recorded worldwide in , up from 245 million cases in . Therefore, the increasing incidence of vector-borne diseases drives the growth of the antiparasitic drugs market.

Antiparasitic Drugs Market Driver: High Prevalence Of Parasitic Infections Raise Antiparasitic Drug Market

The high prevalence of parasitic infections is expected to drive the growth of the antiparasitic drug market. Parasitic infections are caused by microscopic parasites or larger organisms that live and thrive at the expense of their host organism, often causing harm or disease. Antiparasitic drugs are essential tools in the management and control of parasitic infections, contributing to improved health outcomes and a reduced burden of these diseases on affected individuals and communities. For instance, in August , according to the National Library of Medicine, a US-based medical library operated by the federal government, out of 136 participants, 46.3% of the participants in this study had intestinal parasites. The most prevalent parasite was Entamoeba histolytica (33.3%, or 21/63) and the least prevalent was Giardia lamblia (11.1%, or 7/63). Therefore, the high prevalence of parasitic infections drives the growth of the antiparasitic drug market.

Global Antiparasitic Drugs Market Major Players

Major players in the antiparasitic drugs market are Pfizer Inc., Johnson & Johnson, Cadila Pharmaceuticals, Merck & Co.Inc., Bayer AG, Novartis AG, Sanofi S.A., GlaxoSmithKline PLC, Takeda Pharmaceutical Company Limited, Boehringer Ingelheim group, Zoetis Inc., Eisai Co. Ltd., Covetrus Inc., IDEXX Laboratories Inc., Dr. Reddy's Laboratories, Intas Pharmaceuticals Ltd., Alkem Laboratories Ltd., Ceva Sante Animale S.A., Elanco Animal Health Incorporated, Phibro Animal Health Corporation, Norbrook Laboratories Ltd., Mankind Pharma, Neogen Corporation, Bimeda Inc., Indoco Remedies Ltd., ECO Animal Health, Lincoln Pharmaceuticals Ltd., Laboratorios Calier.

Global Antiparasitic Drugs Market Restraint: Challenges And Constraints In The Antiparasitic Drugs Market Due To Rising Resistance To Antimalarial Medications

The rising resistance to antimalarial drug regimens is higher than antiparasitic drugs which can act as a challenge and may restrain the market's growth during the forecast period. The development of combination medicines, which are more efficient but also more expensive, may be influenced by the increasing resistance to antimalarial medications. These factors may have a negative impact on market expansion by restricting access to antiparasitic drugs and boosting the cost of patient treatment. For instance, in January , according to Biomed Central Ltd., a for-profit scientific open access publisher, from 47,382 samples, 18,706 SNPs of anti-malarial medication resistance indicators were genotyped, resulting in a pooled prevalence of 35.4% (95% CI 29.1-42.3%). Therefore, the rising resistance to antimalarial drug regimens is hindering the growth of the antiparasitic drugs market.

Global Antiparasitic Drugs Market Trend: US FDA Approval Marks A Milestone For Parapro's Natroba In The Antiparasitic Drugs Market

Major companies operating in the antiparasitic drugs market are focused on developing innovative products and solutions to sustain their position in the market. For instance, in August , ParaPRO LLC, a US-based pharmaceutical company, launched Natroba Topical Suspension, the first antiparasitic product (new drug application) to be approved by the U.S. Food and Drug Administration (FDA) for the treatment of scabies. Its active ingredient, Spinosad, acts on the lice's nervous system, causing paralysis and their demise. It is applied topically to the scalp and hair and may only require one application. Spinosad, the active ingredient in Natroba, is delivered to the stratum corneum, the outermost layer of the epidermis and targets mites at the site of infestation without penetrating deeply into the dermis or entering systemic circulation, which is what makes this therapeutic option unique.

Antiparasitic Drugs Market Merger And Acquisition: Orion Corporation Expands Its Reach With Million Acquisition Of Inovet BV's Animal Health Business

In June , Orion Corporation, a Finland-based pharmaceutical company, acquired the animal health business of Inovet BV for $141 million (&#;130 million). The acquisition is expected to help Orion Corporation strengthen its geographical presence in Western Europe and export markets and gain a production unit specializing in veterinary medicine manufacturing. Inovet BV is a Belgium-based veterinary pharmaceutical company that provides a range of medicines and health products, including antiparasitic, antibiotics and vaccines.

Regional Outlook For The Global Antiparasitic Drugs Market

North America was the largest region in the antiparasitic drugs market in . Asia-Pacific is expected to be the fastest-growing region in the forecast period. The regions covered in antiparasitic drugs market report are Asia-Pacific, Western Europe, Eastern Europe, North America, South America, Middle East and Africa.

The countries covered in the antiparasitic drugs market report are Australia, Brazil, China, France, Germany, India, Indonesia, Japan, Russia, South Korea, UK, USA, Canada, Italy, Spain.

The antiparasitic drugs market consists of sales of antimalarial drugs, antiamoebic drugs, anti-trematode drugs and veterinary products. Values in this market are &#;factory gate&#; values, that is, the value of goods sold by the manufacturers or creators of the goods, whether to other entities (including downstream manufacturers, wholesalers, distributors and retailers) or directly to end customers. The value of goods in this market includes related services sold by the creators of the goods.

The market value is defined as the revenues that enterprises gain from the sale of goods and/or services within the specified market and geography through sales, grants, or donations in terms of the currency (in USD unless otherwise specified).

The revenues for a specified geography are consumption values that are revenues generated by organizations in the specified geography within the market, irrespective of where they are produced. It does not include revenues from resales along the supply chain, either further along the supply chain or as part of other products.

The antiparasitic drugs market research report is one of a series of new reports from The Business Research Company that provides antiparasitic drugs market statistics, including antiparasitic drugs industry global market size, regional shares, competitors with an antiparasitic drugs market share, detailed antiparasitic drugs market segments, market trends and opportunities and any further data you may need to thrive in the antiparasitic drugs industry. This antiparasitic drugs market research report delivers a complete perspective of everything you need, with an in-depth analysis of the current and future scenario of the industry.

An Antiparasitic Compound from the Medicines for Malaria ...

Abstract

The few frontline antileishmanial drugs are poorly effective and toxic. To search for new drugs for this neglected tropical disease, we tested the activity of compounds in the Medicines for Malaria Venture (MMV) &#;Pathogen Box&#; against Leishmania amazonensis axenic amastigotes. Screening yielded six discovery antileishmanial compounds with EC50 values from 50 to 480 nM. Concentration&#;response assays demonstrated that the best hit, MMV, had mid-nanomolar cytocidal potency against intracellular Leishmania amastigotes, Trypanosoma brucei, and Plasmodium falciparum, suggesting broad antiparasitic activity. We explored structure&#;activity relationships (SAR) within a small group of MMV analogs and observed a wide potency range (20&#; nM) against axenic Leishmania amastigotes. Compared to MMV, our most potent analog, SW41, had ~5-fold improved antileishmanial potency. Multiple lines of evidence suggest that MMV selectively disrupts Leishmania tubulin dynamics. Morphological studies indicated that MMV and analogs affected L. amazonensis during cell division. Differential centrifugation showed that MMV promoted partitioning of cellular tubulin toward the polymeric form in parasites. Turbidity assays with purified Leishmania and porcine tubulin demonstrated that MMV promoted leishmanial tubulin polymerization in a concentration-dependent manner. Analogs&#; antiparasitic activity correlated with their ability to facilitate purified Leishmania tubulin polymerization. Chemical cross-linking demonstrated binding of the MMV scaffold to purified Leishmania tubulin, and competition studies established a correlation between binding and antileishmanial activity. Our studies demonstrate that MMV is a potent antiparasitic compound that preferentially promotes Leishmania microtubule polymerization. Due to its selectivity for and broad-spectrum activity against multiple parasites, this scaffold shows promise for antiparasitic drug development.

Keywords: Leishmania, tubulin, MMV, drug discovery, parasite, pathogen

Graphical Abstract

Human leishmaniasis is endemic in nearly 100 countries, and 350 million people worldwide are at risk for this disfiguring (cutaneous or mucocutaneous) or lethal (visceral) disease.1 Leishmaniasis is caused by obligate intracellular single-celled parasites of the Leishmania genus, which have two life cycle stages. The fast-growing promastigote, which lives in sandflies, transforms into the slow-growing amastigote inside human phagocytic cells, causing clinical disease.2 The primary species of Leishmania used in this manuscript, Leishmania amazonensis, which belongs to the L. mexicana complex, can be grown in the tissue culture setting in vitro as promastigotes, axenic amastigotes (amastigotes in tissue culture media alone, without host cells present), or intracellular amastigotes (amastigotes grown inside macrophages (Mϕ)).

Current treatments are few and have significant side effects. Since resistance has emerged to these antileishmanial drugs3-5 and effective Leishmania vaccines are decades away, there is an urgent need for novel chemotypes to find replacements for the drugs that are currently available. To accelerate drug discovery, MMV has coordinated screens of over 5 million compounds against Plasmodium, generating approximately 20 000 starting points for drug discovery and development.6-9 Previously, a representative set of 400 compounds, called the Malaria Box, made a significant impact beyond the malaria field and stimulated medicinal chemistry efforts against many diseases, including leishmaniasis.6,10,11 Due to the success of the Malaria Box, the MMV distributed another collection of 400 drug-like compounds, the Pathogen Box (www.PathogenBox.org), which are likely to show acceptable oral absorption and target an expanded set of pathogens.12,13 Its name derives from the fact that each compound in the box has known activity against one or more pathogenic bacterial, fungal, or parasitic organisms. Known antiparasitic drugs and current antiparasitic lead compounds are also included. Similar to the Malaria Box, these compounds also reflect a cross-section of the chemical diversity available in MMV&#;s 20 000 hits, providing 374 starting points for oral drug discovery.

Here, we assessed the Pathogen Box for activity against L. amazonensis axenic amastigotes, which yielded six discovery antileishmanial compounds with EC50 values ranging from 50 to 480 nM. The top hit, MMV, also killed intracellular Leishmania spp., T. brucei, and P. falciparum at nanomolar concentrations. We describe this screen and our initial SAR studies that provide proof-of-concept for future optimization of the scaffold.

We next engaged in target identification studies for MMV and derivatives. Using assays in intact parasites, we demonstrated that our scaffold affects Leishmania cell division, as well as selectively promotes microtubule polymerization within Leishmania parasites, resulting in several additional morphological changes in the parasite. Since Leishmania requires microtubule dynamics for multiple critical functions to survive throughout its life cycle, including chromosome segregation, flagellar motility, cell division, and structure maintenance,14 parasite microtubule polymerization is a plausible antiparasitic drug target. Furthermore, our assays on purified tubulin demonstrate that MMV and derivatives directly bind to tubulin and facilitate its polymerization, with selectivity for leishmanial over porcine tubulin, providing one plausible mechanism for this scaffold&#;s antiparasitic activity.

In summary, our studies demonstrate that MMV and its analogs are potent antiparasitic compounds that promote Leishmania microtubule polymerization. Although depolymerizing antileishmanial agents have been studied in the past, to our knowledge, no drugs currently on the market or in development selectively facilitate tubulin polymerization in parasites. Since MMV has activity against multiple parasites, this scaffold shows promise for future antiparasitic drug development.

DISCUSSION

To search for tractable chemotypes for the neglected tropical disease leishmaniasis, we used a phenotypic screen to test the activity of compounds in the MMV &#;Pathogen Box&#; against L. amazonensis axenic amastigotes. We identified and validated a hit compound, MMV, which also kills other protozoan parasites: Leishmania donovani, Plasmodium falciparum, and Trypanosoma brucei. We showed that MMV and active analogs affect Leishmania cell division and morphology and increase the percentage of polymerized microtubules in parasites. Finally, we determined that this scaffold directly binds to tubulin and selectively promotes polymerization of purified Leishmania tubulin. To our knowledge, we are the first group reporting that a particular scaffold preferentially promotes microtubule polymerization in parasites. Our initial SAR studies on this scaffold highlight potential for improvements in potency and selectivity for future lead optimization studies.

We optimized several assays for screening the MMV Pathogen Box compounds against L. amazonensis axenic amastigotes and identified several compounds with comparable in vitro potency to that of the gold-standard antileishmanial drug, amphotericin B. Most antileishmanial drug screening campaigns have utilized microscopy-based techniques;70 however, one difficulty with these methods is that they rely on detection of parasite burden as a proxy for parasite viability. We therefore employed multiple more direct ways to report parasite viability. First, we used an optimized alamarBlue assay to measure metabolic activity of extracellular L. amazonensis axenic amastigotes. Second, as previously reported for other parasites,37-40 we generated transgenic L. amazonensis parasites stably expressing a luciferase reporter gene, which provided a tractable parasite bioluminescent marker. This assay is rapid and sensitive, and allowed us to validate the activity of our hit compound against intracellular parasites. Finally, we used a washout assay to ensure that our compound&#;s activity was cytocidal rather than cytostatic. Using these improved techniques, we narrowed our focus to one verified hit.

Because it was identified via a phenotypic screen, the target of MMV was initially unknown. Through a series of experiments using intact parasites and purified protein, we were able to narrow one likely target of our compound from one that affected Leishmania cell division and morphology to one that affected Leishmania microtubules, and then to one that facilitated polymerization of purified Leishmania tubulin. We then confirmed that our scaffold directly binds to tubulin. There are several plausible mechanisms that could underlie why a microtubule stabilizer would result in parasite death. One possibility is that these stabilizers may directly act as antimitotics in Leishmania, which would be similar to the mechanism by which paclitaxel exerts its effects on cancer cells. Another possibility is that parasite death is instead caused by affecting the subpellicular leishmanial microtubular array. If Leishmania is unable to remodel the subpellicular microtubule array during parasite cell division due to treatment with these compounds, the number of parasites that were arrested at some point during cell division would also increase, even though the compounds are not acting directly as an antimitotic. The methodologies employed in this manuscript do not distinguish between these possibilities. Furthermore, we cannot be certain whether tubulin polymerization is the primary or only mechanism that leads to parasite death, as it is impossible to completely rule out additional targets for MMV based on existing data. However, several lines of evidence detailed in this manuscript indicate that MMV enhances tubulin polymerization in Leishmania.

First, MMV promoted the partitioning of cellular tubulin toward the polymeric form in intact parasites, confirming that this scaffold enhances tubulin polymerization in parasites (Figure 4). One caveat is that we used 0.1% Triton X-100 in buffer to separate the polymeric form from the dimeric form in Leishmania amastigote compound-treated and untreated samples, as has previously been done in the field.41 However, based on our data (Figure 4) and the results in Jayanarayan et al.,41 Leishmania tubulin polymers seem to tolerate 0.1% Triton X-100 better than mammalian tubulin polymers do. Additionally, all comparisons were made relative to the untreated DMSO control, which was prepared in the same buffer. It is likely that the concentration of Triton X-100 in our buffers led us to underestimate rather than overestimate the degree to which tubulin was polymerized in parasites.42

Second, the antiparasitic activity of MMV and analogs (L. amazonensis axenic amastigote EC50) strongly correlated with their effects on purified Leishmania tubulin as estimated by turbidity assays (Figure 5), further supporting the findings shown in intact parasites (Figure 4) that tubulin is the target of MMV. Turbidity measurements at A340 are affected by the tubulin polymer type in addition to the amount of polymer. Because we did not compare dimer to polymer concentrations after our turbidity measurements, the possibility that compounds may induce polymer shape changes (sheet polymers), which would also increase the A340 plateau, could not be completely ruled out. Regardless, our data suggest that MMV and its analogs exert an effect on tubulin polymerization behavior. Furthermore, it should be noted that Leishmania tarentolae and porcine tubulin EC50 values were estimated at 30 °C for leishmanial versus 37 °C for mammalian tubulin, respectively, as is typical in the literature.32,33 Because increased temperature makes tubulin polymerization more likely and may affect the lag time, polymerization rate, and steady-state level of the A340 curve in turbidity assays, it is difficult to directly compare MMV&#;s selectivity between Leishmania and mammalian tubulin based on turbidity data only. However, the >20-fold selectivity of MMV for purified Leishmania tubulin over porcine tubulin (0.5 vs 11 μM EC50) is consistent with the in vitro selectivity of MMV for L. amazonensis over mammalian cells (79 vs nM).

Finally, MMV showed direct binding to tubulin in cross-linking experiments (Figure 6). We noticed that the 100-fold excess of the competitor MMV (Figure 6B) resulted in only a 2-fold decrease in the degree of fluorescence of the probe band, as is typical in these studies.43 The reason is that the probe compound SW22 is expected to initially bind to proteins reversibly. However, irradiation of the samples generates a covalent adduct between the probe and its binding partners, rendering the binding irreversible. By contrast, the competitors only bind reversibly. Accordingly, the interaction with the cross-linking probe is not done under equilibrium conditions, and large excesses of competitor are required to block specific interactions with biologically relevant binding partners, such as tubulin in this case. Therefore, the direct tubulin binding seen in our cross-linking studies that can be competed with active but not inactive analogs strongly suggests that tubulin is a target of MMV.

Our SAR studies suggest that the benzamide moiety on the MMV scaffold is required for its ability to bind tubulin and promote polymerization. Replacing the substituted N-aryl ring with a simple acetate (SW23) or an unsubstituted benzamide (SW100) resulted in a loss of activity. Replacing the benzamide with a pyridyl amide (SW74) slightly elevated the EC50 value relative to the initial hit, while the urea in compounds SW102 and SW101 decreased activity. The position of the benzoyl group appears important because the O-acylated isomer SW10 was largely devoid of activity, although this analog also lacks the ethyl group. Interestingly, by contrast, disubstituted benzamide (SW41) improved potency and therapeutic index relative to the initial hit. At the other extreme, SW10, which is an isomer of MMV, and the truncated analog SW23 are nearly inactive against the Leishmania parasite.

Tubulin is an excellent antiparasitic drug target, as evidenced by the first-line antihelminthic benzamidizoles (e.g., albendazole). Despite a high degree of amino acid sequence conservation for both the α- and β-subunits among eukaryotes,44-47 certain tubulin-targeting compounds selectively bind to phylogenetically restricted tubulin subsets. Comparison of tubulin sequences from Tetrahymena, Plasmodium, Toxoplasma, Euglena, Trypanosoma, and Leishmania has demonstrated that the protozoan tubulins form a distinct group, and these tubulin proteins are more similar to tubulins from plants than vertebrate or fungal tubulins.48 As such, plant antitubulin compounds, such as dinitroanilines, selectively disrupt microtubules from diverse protozoa and plants but not vertebrates and fungi.49-53 Other studies have identified tubulin as an antiprotozoal target in the past.51,53-56 However, all compounds that we found reported in the literature targeted trypanosomatid microtubules to prevent tubulin polymerization. By contrast, MMV promotes tubulin polymerization, which provides a unique mechanism for this scaffold. Furthermore, our scaffold has potent antiparasitic activity against both kinetoplastids (Leishmania spp., T. brucei) and apicomplexans (Plasmodium), providing significant potential for eventual use as a broad-spectrum antiparasitic agent. In addition, MMV was initially reported as an anti-mycobacterial agent,57 and a recent study also identified MMV as a hit against Staphylococcus aureus,28 although its mechanism of action was not described. Thus, it is plausible that the MMV scaffold could be employed as an antibiotic as well.

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CONCLUSION

In conclusion, we have determined that a novel antileishmanial compound from the Pathogen Box binds to Leishmania tubulin, induces parasite microtubule polymerization, and affects Leishmania morphology and cell division. Such a mechanism of action would be unique among antiparasitic agents. Further genetic and biochemical studies using MMV will help us understand differences in its mechanism of action compared to other tubulin-active drugs, as well as differences in tubulin structure, regulation, or dynamics between protozoa and other organisms that may be suggested by the selectivity differences for parasitic and mammalian tubulin seen here. We will also continue our ongoing iterative medicinal chemistry studies in an effort to identify a lead compound that is effective in a murine model of cutaneous leishmaniasis. Due to tubulin&#;s conservation across the protozoa and the MMV scaffold&#;s activity against multiple parasites, there is significant potential for this scaffold to allow the development of a broad-spectrum antiparasitic agent that will treat a multitude of devastating protozoal infections.

METHODS

Compounds.

The Pathogen Box was generously provided by the MMV58 as 10 mM stocks in DMSO (10 μL each) and stored at &#;20 °C. The antileishmanial reference drugs amphotericin B and miltefosine (Sigma) were prepared in deionized water and stored at &#;20 °C. The maximum final DMSO concentration was 0.2% v/v in all experiments.

Parasite Cultures.

Leishmania amazonensis promastigotes (strain IFLA/BR/67/PH8, provided by Norma W. Andrews, University of Maryland, College Park, MD) and L. tarentolae (Parrot strain, ATCC) were maintained at 26 °C in Schneider&#;s Drosophila medium supplemented with 15% heat-inactivated, endotoxin-free FBS and 10 μg/mL gentamicin.20,21 L. amazonensis amastigotes were grown axenically at 32 °C in M199 (Invitrogen) at pH 4.5, supplemented with 20% FBS, 1% penicillin&#;streptomycin, 0.1% hemin (25 mg/mL in 50% triethanolamine), 10 mM adenine, 5 mM l-glutamine, 0.25% glucose, 0.5% trypticase, and 40 mM sodium succinate.20,21

A transgenic luciferase-expressing line of L. amazonensis parasites (L. amazonensisluc) was generated similar to the method in refs 39 and 40. Briefly, the 1.66 kb luciferase-coding region of pGL3-Basic (Promega) was cloned in the expression vector pLEXSY.hyg2 (Jenabioscience). The final construct containing the luciferase gene and hygromycin resistance marker was integrated into the 18S rRNA locus of the nuclear DNA of L. amazonensis using the Human T-Cell Nucleofector kit and the Amaxa Nucleofector electroporator (program U-033). Following transfections, after 24 h at 26 °C, transfectants were selected with 100 μg/mL hygromycin in Schneider&#;s Drosophila medium. Clones were isolated by limiting dilution. L. amazonensisluc parasites were maintained as above, but the media was supplemented with 100 μg/mL hygromycin. L. amazonensis virulence was maintained by passage in C57B/6 mice.20

For other parasites, L. donovani, strain MHOM/SD/62/1S-C12D, was kindly provided by Robert Duncan (Food & Drug Administration) and was grown as previously described.59 Plasmodium falciparum parasites of the 3D7 strain (kindly provided by Margaret A. Phillips, UT Southwestern) were cultured in RPMI medium supplemented with 37.5 mM HEPES, 10 mM d-glucose, 2 mM l-glutamine, 100 μM hypoxanthine, 25 μg/mL gentamicin, 4% (v/v) human serum, and 0.25% (v/v) Albumax II, at a 2% hematocrit in an atmosphere of 1% O2, 3% CO2, and 96% N2 as described previously.10 Staging and parasitemia of the in vitro culture were assessed by light microscopy of Giemsa-stained thin blood smears. The parasites were synchronized using sequential sorbitol lysis treatment,22,60 with experiments carried out at least one intra-erythrocytic cycle later. T. brucei single marker (SM) cells were maintained at log phase growth (<1.5 × 106 cells/mL) in HMI-19 media61 supplemented with 10% FBS and 2.5 μg/mL G418 (Life Technologies) at 37 °C and 5% CO2.

Cell Cultures.

RAW 264.7 cells (ATCC TIB-71) were grown in Dulbecco&#;s modified Eagle&#;s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Invitrogen) as described previously.20,21

Concentration&#;Response Assays in L. amazonensis, T. brucei, and P. falciparum.

Leishmania amazonensis axenic amastigotes (100 μL, 2 × 106 cells/mL) were added to 96-multiwell plates containing 100 μL of amastigote culture medium with an appropriate compound dilution series. DMSO at 0.1% (no drug) served as a positive control (100%), and 5 μM amphotericin B served as a negative growth control (0%). Only internal wells were used to minimize edge effects from evaporation. Axenic amastigotes were incubated at 32 °C with a compound or drug for 72 h prior to measurement. Similarly, the cytotoxicity of each compound was determined against RAW 264.7 cells, following 72 h incubation at 37 °C (promastigotes at 26 °C). AlamarBlue (10%, Thermo Fisher Scientific) was used to measure the growth of both parasite and mammalian cells. Conversion from a blue oxidized state to a pink reduced state was assessed visually at 6 h.17,62,63 The fluorescence signal was measured with a BioTek Synergy H1 plate reader (530 nm excitation, 570 nm emission; BioTek Instruments). For bioluminescence assays using L. amazonensisluc, relative luminescence units (RLU) were measured using Britelite Plus (PerkinElmer, USA).38 Luminescence produced by this luciferase reaction is proportional to the amount of luciferase-expressing, viable L. amazonensisluc.38 Following 5 min incubation at room temperature, the luminescence was measured with a BioTek Synergy H1 plate reader. Compounds were tested against the blood-stage Trypanosoma brucei (48 h end point) and intra-erythrocytic asexual stages of Plasmodium falciparum (3D7) using CellTiter-Glo luminescent and Malaria SYBR green I fluorescence (MSF) assays, respectively, as described previously.21,22,64

All experiments were conducted as three technical replicates on the same plate, with at least three independent biological repeats of each plate performed. For all assays, percent growth was expressed as a proportion of the untreated (positive) control (i.e., 100%) as described previously10 and plotted against drug or compound concentration. Concentrations were then log10 transformed, and EC50 values for each biological repeat were determined using nonlinear regression (sigmoidal dose&#;response/variable slope equation) in GraphPad Prism v5.0 (GraphPad Software, Inc.).10 Values from the three biological replicates were used to calculate the mean EC50 values ± SE shown.

Intramacrophage L. amazonensis Assays.

Intracellular EC50 values were estimated with L. amazonensisluc parasites. Briefly, RAW 264.7 cells were starved overnight and then infected with metacyclic promastigotes at a multiplicity of infection (MOI) of 15 and incubated for another 24 h at 37 °C. The plates containing infected RAW 264.7 cells were washed five times with serum-free DMEM. Serially diluted compounds were added, and plates were incubated at 37 °C for 72 h. The bioluminescence signal was measured as described above.

We measured intracellular LD50 values using adaptations of protocols described by Paape et al.17 and Jain and colleagues.65 Briefly, RAW 264.7 cells were seeded to 96-multiwell plates at a density of 2 × 105 cells/mL (200 μL) and starved overnight at 37 °C. The cells were infected with metacyclic promastigotes at an MOI of 15 and incubated for another 24 h at 37 °C. The plates containing infected RAW 264.7 cells were washed five times with serum-free DMEM. Serially diluted compounds were added, and plates were incubated at 37 °C for 72 h. Wells were washed five times with DMEM, and the RAW 264.7 cells were lysed with 100 μL of 2 mg/mL saponin in DMEM for 5 min at room temperature,17 and further lysis was stopped with 100% FBS. After centrifugation, 200 μL of acidic promastigote media was replaced, and plates were incubated at 26 °C for 96 h. Fluorescence intensity (alamarBlue) was measured as described above.

Microscopy.

Promastigotes or amastigotes were treated with the indicated compound concentrations and allowed to adhere to poly(l-lysine) coated plates. To estimate effects on cell division or morphology, we used the estimated ECh concentrations of compounds but treated parasites only for 48 h. For studies of flagellar length, we used the estimated ECh concentrations of compound but treated parasites for only 24 h. Thus, for each experiment, each active compound was used at its respective ECh concentration; for inactive compounds, the values shown in Table 4 were used. All cells were fixed with 4% paraformaldehyde and permeabilized and blocked with 0.01% Triton X-100 and 2% BSA in PBS. Promastigotes and amastigotes were incubated with mouse anti-GP46 or anti-P8, respectively (both kind gifts from Diane McMahon-Pratt, Yale University) at 1:50 or 1: and rat anti-α-tubulin monoclonal YL1/2 antibody (cat. no. MA1-, Invitrogen) at 1:. Samples were then probed with A568 anti-mouse and A488 anti-rat secondary antibodies (Molecular Probes). DNA was labeled with Hoechst . For representative images, samples were visualized on a Zeiss LSM 880 inverted confocal Airyscan microscope at 63×; full Z-thicknesses through parasites were obtained. Maximal intensity projections were formulated and quantified with ImageJ (1.52a, http://imagej.nih.gov/ij). Parasites in representative images were selected from the maximal intensity projections and linearly processed in Adobe Photoshop CS6 (version 13.0.6).

For quantification, images were obtained with a BioTek Cytation 5 confocal imaging reader and analysis was performed by an observer blinded to experimental condition. For the promastigotes shown in Figure 3C, parasites in 2F1K1N, 2F2K1N, and 2F2K2N were all considered to be undergoing cell division (mid-cell division). For the amastigotes shown in Figure 3C, parasites that appeared to be in 2K1N or 2K2N were considered to be in mid-cell division. For both categories, at least 200 parasites were analyzed per condition per experiment after 48 h incubation at compounds&#; ECh, and the mean percentage in mid-cell division plus standard error for three experiments was calculated. To calculate the average flagellar length shown in Figure S1, the flagellar length was measured in the gp46 channel in ImageJ for at least 50 parasites per experimental condition for each of 3 experiments after 24 h incubation at compounds&#; ECh; mean lengths for each experimental category (normalized to DMSO control flagellar lengths) plus SE are shown. To calculate the percentages of pear-shaped or rounded parasites shown in Figure S1, parasites with cell bodies that had a width of &#;70% their length were counted. At least 200 parasites were analyzed per condition per experiment after 48 h incubation at compounds&#; ECh, and the mean percentage of pear-shaped parasites per experimental category plus SE for three experiments was calculated.

Polymerized versus Unpolymerized Tubulin in Parasites.

Soluble (unpolymerized) and insoluble (polymerized) tubulin fractions were separated by high-speed centrifugation, as described previously,41,66,67 with minor modifications. Amastigotes or promastigotes of L. amazonensis were seeded at a density of 2 × 107 cells in 2 mL and treated with the specified compounds for 24 h at their EC50 concentration. Parasites were lysed in buffer containing 100 mM 2-(N-morpholino) ethanesulfonic acid (MES, pH 6.9), 1 mM EGTA, 1 mM MgSO4, 0.1% Triton X-100, and protease inhibitors (1 mM benzamidine, 0.5 mM phenylmethylsulfonyl fluoride, 25 μg/mL leupeptin) and centrifuged at 100 000g to separate the polymerized and unpolymerized tubulin fractions. The pellets were dissolved in 0.5% SDS in 25 mM Tris, pH 6.8, in a volume equal to that of the supernatant.

Electrophoresis, Western Blotting, and Protein Assays.

SDS-PAGE was performed using 12% polyacrylamide gels as described previously.32,54 Protein purity and concentration were assessed using Coomassie Blue staining and the bicinchoninic acid (BCA) (Pierce Biotechnology) assay, respectively, following the manufacturers&#; protocols. Bovine serum albumin (BSA) was included to generate a standard curve. Western blotting was performed as described.41,61 Total proteins were resolved by polyacrylamide gel electrophoresis and transferred to PVDF membranes using a Mini Trans-Blot Cell (BioRad). The membranes were blocked in 5% nonfat dry milk in Tris-buffered saline (TBS) (20 mM Tris (pH 7.6), 150 mM NaCl). The membranes were incubated with mouse anti-α-tubulin antibody (monoclonal antibody B-5-1-2, cat. no. 32-, Invitrogen) at 1: in 5% BSA and TBS-T overnight at 4 °C. The membranes were then incubated with a 1: dilution of goat anti-mouse HRP-conjugated secondary antibody in TBS-T (5% nonfat dry milk) for 1 h. Finally, membranes were incubated in SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) for 5 min and visualized by the ImageQuant LAS (GE Healthcare). GAPDH antibodies (Santa Cruz) were used at a 1: dilution as a loading control.

Tubulin Purification from Leishmania tarentolae.

L. tarentolae (Parrot strain from ATCC) tubulin was purified as originally described by Yakovich et al.32 and others.54 Promastigotes of L. tarentolae were grown to a high density (~1 × 108 cells/mL), harvested, and resuspended in PME + P buffer containing 100 mM piperazine N, N&#;-bis (2-ethanesulfonic acid) (PIPES) buffer (pH 6.9), 1 mM glycol ether diamine tetraacetic acid (EGTA), 1 mM MgCl2, 1 mM benzamidine, 0.5 mM phenylmethylsulfonyl fluoride, and 25 μg/mL leupeptin. The resulting suspension was lysed using an Emulsiflex C5 homogenizer (Avestin) or extensively sonicated on ice with a probe sonicator (Misonix), cooled on ice for 30 min, and centrifuged at 40 000g for 1 h at 4 °C using an ultracentrifuge (Beckman). The resulting supernatant was filtered through a glass wool or 0.45 μm filter. Using the peristaltic pump, the sample was loaded onto an equilibrated DEAE-Sepharose Fast Flow matrix (Amersham Biosciences). The column was washed with two column volumes of PME + P and subsequently four column volumes PME + P containing 0.1 M KCl and 0.25 M glutamate (pH 6.9). Tubulin that still contained some additional proteins was then eluted with two column volumes of PME + P containing 0.3 M KCl and 0.75 M glutamate (pH 6.9). Samples for studies shown in Figure S7 were removed at this point. For full tubulin purification for the reminder of studies shown, the column was connected to the AKTA fast performance liquid chromatography system (GE), and tubulin was eluted with two column volumes of PME + P containing 0.3 M KCl and 0.75 M glutamate (pH 6.9). The purified fractions were confirmed using SDS-page and Coomassie blue staining. The tubulin-rich fractions were confirmed by tubulin polymerization assays as described previously. The assembly competent tubulin fractions were pooled together and subjected to dialysis overnight in 1× PME buffer. Tubulin was concentrated by using Amicon ultracentrifugal filters (Millipore Sigma), flash frozen in liquid nitrogen, and stored at &#;80 °C.

Tubulin Polymerization Assays.

Tubulin polymerization assays were adapted from protocols from Cytoskeleton, Inc., and others14,32,54,68 using 96-well half-area microplates (Costar) in a final volume of 100 μL. Leishmanial or porcine tubulin (>99% pure, cat. no. T240, Cytoskeleton, Inc.) in 50 μL volume was pretreated with drugs on ice for 5 min before adding 50 μL of ice cold buffer to provide a final concentration of 3 mg/mL tubulin in 80 mM PIPES (pH 6.9), 2 mM MgCl2, 0.5 mM EGTA, 1 mM GTP, and 1% DMSO unless specified otherwise. The absorbance at 340 nm was recorded in a Synergy H1 microplate reader (BioTek) for up to 45 min at 37 °C (porcine tubulin) or 30 °C (L. tarentolae tubulin). To estimate the EC50, tubulin was exposed to 8 concentrations of drug as above. A control sample with 10% DMSO was included to create the Vmax 100% standard (positive control, maximal tubulin polymerization). Untreated sample was used as a negative control (0%) as described in refs 14, 32, 54, and 68. The Vmax data at each drug concentration used were converted into percent of the control Vmax. Percent tubulin polymerization was then expressed as a proportion of the untreated control and plotted against log10 transformed drug or compound concentration. EC50 values were estimated using nonlinear regression (sigmoidal dose&#;response/variable slope equation) in GraphPad Prism v5.0.10

Fluorescent Microtubule Assembly Assays.

Fluorescence images of microtubule assembly were acquired as described.32,33 Briefly, 1.5 mg/mL purified porcine tubulin (>99% pure, cat. no. T240, Cytoskeleton, Inc.) was treated for 40 min in assembly buffer containing 80 mM PIPES (pH 6.9), 1 mM EGTA, 1 mM MgCl2, 1 mM GTP, and 1% DMSO at 37 °C. The mixture was cross-linked by diluting it 10-fold using assembly buffer containing 1% glutaraldehyde. After 3 min, the reaction was quenched by diluting 5-fold with assembly buffer containing 20 mM Tris, pH 6.8. Pedestals were inserted into centrifuge tubes, and a poly(l-lysine) coated coverslip was placed on top. Glycerol, 20% in assembly buffer with no GTP, was made, and the poly(l-lysine) coated coverslips were covered with 3 mL of cushion. The quenched, cross-linked reactions (50 μL) were gently layered on top and spun through the cushion onto the poly(l-lysine) coated coverslips using an ultracentrifuge (22 500g at 20 °C for 45 min or g for 12 h at 20 °C). Coverslips were washed three times with assembly buffer (no GTP), fixed with ice-cold methanol, and stained for 20 min with FITC-DM1α (anti-α tubulin, cat. no. F Sigma-Aldrich) diluted 250× in PBS + BSA. The coverslips were washed three times with assembly buffer and imaged by epifluorescence as described by Ayaz et al.36

Chemical Cross-Linking Experiments.

A probe compound (SW22) with benzophenone and alkyne modifications was synthesized (for details, see the synthesis of N-(1-(5-ethyl-4-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)-3-methyl-1H-pyra-zol-5-yl)-4-(4-(prop-2-yn-1-yloxy) benzoyl) benzamide). A click-chemistry protocol was adapted from Theodoropoulos et al.69 Purified parasite tubulin at 10 μM (starting concentration) was plated in 96 well plates, treated with the probe in the presence or absence of highly active and less active competitors, and polymerized for 1 h at 30 °C. The samples were UV cross-linked by placing the 96 well plates on ice approximately 3&#;4 in. below the bulbs in a Stratalinker (Stratagene) and then exposing them to 15 min of UVB radiation. The samples were immediately solubilized in 1% SDS with benzonase (Sigma) diluted 1: in buffer containing 50 mM HEPES, pH 7.4, 10 mM KCl, and 2 mM MgCl2. The samples were normalized for protein concentrations using the BCA assay (Life Technologies). Equal amounts of sample were subjected to a click reaction with 100 μM TBTA (dissolved in 4:1 DMSO/t-butanol), 1 mM TCEP, 2 mM CuSO4 and 25 μM Alexafluor-532 azide (see Theodoropoulos et al.69 for synthesis of Alexafluor-532 azide) for 1 h at 25 °C with agitation. SDS sample buffer was then added to the samples to quench the reaction, and proteins were resolved by SDS-PAGE. Samples in Figure 6 and Figure S6A were run using a 4&#;12% gel (BioRad precast), while those in Figure S6B,C were run using a 12% gel (custom-made). A Typhoon scanner with a 532 nm excitation laser and a 555 nm emission filter was used to scan the gels for fluorescently labeled proteins.

Synthesis of MMV Analogs.

A total of 11 analogs were synthesized, and all tested compounds have a purity of >95% as judged by HPLC analysis (UV detection at 210 nM) (see Supplemental Methods for full details).

Supplementary Material

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ACKNOWLEDGMENTS

We thank Margaret A. Phillips (M.A.P.) and Luke M. Rice (L.M.R.) for use of reagents and constructive comments on our experiments and the manuscript. We appreciate the technical assistance provided by Emma L. Rhodes, Emily T. Mamula, and Rebecca A. Kernen, and Leah Imlay&#;s assistance with manuscript revision. We thank the MMV for providing the Pathogen Box used in this study and Diane McMahon-Pratt for providing antileishmanial antibodies. We thank Norma W. Andrews, Robert Duncan, and Margaret A. Phillips for providing additional parasite strains used in this manuscript. In addition, we appreciate the efforts of our three anonymous peer reviewers, whose constructive feedback was incorporated into several areas of this revised manuscript. I.U. was supported by a travel grant from the UT Southwestern Postdoctoral Society. H.N. was supported by the NCI Simmons Center Cancer Support Grant. S.M. was supported by the National Institutes of Health (NIH; R01GM) and the Robert A. Welch Foundation (I-) (to L.M.R). C.L. was supported by NIH R01 AI and AI (to M.A.P) and GM (to C.L). S.G., B.H., and J.M.R. were supported by the Robert A. Welch Foundation (I-) and NIH R01 CA (to J.M.R). I.U., L.M.B, J.M.B, and D.M.W. were supported by NIH K08 AI, a Children&#;s Clinical Research Advisory Committee (CCRAC) Junior Investigator Award, a CCRAC Early Investigator Award, a Harrington Scholar-Innovator Award, NIH R01 AI, and funds from the UT Southwestern Department of Pediatrics (to D.M.W.). The authors have filed a provisional patent application on parts of this work.

ABBREVIATIONS USED

CuAAC

copper-catalyzed alkyne&#;azide cycloaddition

MMV

Medicines for Malaria Venture

macrophage

SI

selectivity index

Footnotes

The authors declare no competing financial interest.

Supporting Information

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10./acsinfecdis.0c.

Supplemental Methods (Synthesis Methods) (PDF)

Morphological changes in Leishmania promastigotes treated with MMV and analogs, effect of paclitaxel on porcine microtubule assembly in vitro, relative polymerization activity of MMV in presence or absence of detergent, effect of miltefosine on purified tubulin assembly in vitro, relative polymerization activity of MMV in the presence or absence of detergent, fluorescent images of purified tubulin treated with paclitaxel and MMV, full fluorescence and Coomassie blue gels from Figure 6, competition-sensitive fluorescent binding of MMV analogs to tubulin and other proteins, screening of the MMV Pathogen Box, and cytotoxicity data (CC50 values) for selected &#;hit&#; compounds. (PDF)

Contributor Information

Imran Ullah, Department of Pediatrics and Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas , United States.

Suraksha Gahalawat, Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas , United States.

Laela M. Booshehri, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas , United States

Hanspeter Niederstrasser, Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas , United States.

Shreoshi Majumdar, Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, Texas , United States.

Christopher Leija, Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas , United States.

James M. Bradford, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas , United States

Bin Hu, Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas , United States.

Joseph M. Ready, Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas , United States.

Dawn M. Wetzel, Department of Pediatrics and Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas , United States.

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

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S1

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