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Background: Ascorbate peroxidase (APX) has emerged as a promising target for chemotherapy because of its absence in humans and crucial role in the antioxidant defence of trypanosomatids. APXs, which are class I haeme-containing enzymes, reduces hydrogen peroxide using ascorbate to produce water and monodehydroascorbate, thereby preventing cell damage caused by H 2 O 2 ., Methods: We aimed to create an APX-knockout L. infantum line using CRISPR/Cas9. Despite unsuccessful attempts at full knockouts, we achieved deletion of chromosomal copies post-APX episomal insertion, yielding LiΔchrAPX::LbAPX parasites. We performed phenotypic characterization to assess the impact of these genetic modifications, which included the determination of APX transcript expression levels using quantitative PCR, drug sensitivity, infectivity, and parasite survival in macrophages., Results: Quantitative polymerase chain reaction (PCR) analysis revealed a 10- to 13-fold reduction in APX transcript expression in LiΔchrAPX::LbAPX compared with wild-type (LiWT) and APX-overexpressing (Li::Cas9::LbAPX) parasites, respectively. The episomes in those knockdown parasites remained stable even after 20 drug-free passages in vitro. Li::Cas9::LbAPX parasites showed increased resistance to trivalent antimony (Sb III ) and isoniazid, reduced tolerance to H 2 O 2 , and unchanged macrophage infectivity compared with LiWT. In contrast, LiΔchrAPX::LbAPX parasites were more sensitive to Sb III and isoniazid, exhibited greater susceptibility to H 2 O 2 -induced oxidative stress, and 72 h post-infection, showed fewer infected macrophages and intracellular amastigotes compared with LiWT parasites., Conclusions: Our findings hint at the indispensability of APX in L. infantum and raise the possibility of its potential as a therapeutic target for leishmaniasis., Competing Interests: Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Background: Acanthamoeba spp. is a waterborne, opportunistic protozoan that can cause amebic keratitis and granulomatous amebic encephalitis. Knema retusa is a native tree in Malaysia, and its extracts possess a broad range of biological activities. Niosomes are non-ionic surfactant-based vesicle formations and suggest a future targeted drug delivery system. Copolymer micelle (poly(ethylene glycol)-block-poly( ɛ -caprolactone); PEG- b -PCL) is also a key constituent of niosome and supports high stability and drug efficacy. To establish Knema retusa extract (KRe) loading in diverse nanocarriers via niosome, PEG- b -PCL micelle, and their combination and to study the effect of all types of nanoparticles (NPs) on Acanthamoeba viability, adherent ability, elimination of adherence, and cytotoxicity., Methods: In this study, we characterized niosomes, PEG- b -PCL, and their combination loaded with KRe and tested the effect of these NPs on Acanthamoeba triangularis stages. KRe-loaded PEG- b -PCL, KRe-loaded niosome, and KRe-loaded PEG- b -PCL plus niosome were synthesized and characterized regarding particle size and charge, yield, encapsulation efficiency (EE), and drug loading content (DLC). The effect of these KRe-loaded NPs on trophozoite and cystic forms of A. triangularis was assessed through assays of minimal inhibitory concentration (MIC), using trypan blue exclusion to determine the viability. The effect of KRe-loaded NPs was also determined on A. triangularis trophozoite for 24-72 h. Additionally, the anti-adhesion activity of the KRe-loaded niosome on trophozoites was also performed on a 96-well plate. Cytotoxicity activity of KRe-loaded NPs was assessed on VERO and HaCaT cells using MTT assay., Results: KRe-loaded niosome demonstrated a higher yielded (87.93 ± 6.03%) at 286 nm UV-Vis detection and exhibited a larger size (199.3 ± 29.98 nm) and DLC (19.63 ± 1.84%) compared to KRe-loaded PEG- b -PCL (45.2 ± 10.07 nm and 2.15 ± 0.25%). The EE (%) of KRe-loaded niosome was 63.67 ± 4.04, which was significantly lower than that of the combination of PEG- b -PCL and niosome (79.67 ± 2.08). However, the particle charge of these NPs was similar (-28.2 ± 3.68 mV and -28.5 ± 4.88, respectively). Additionally, KRe-loaded niosome and KRe-loaded PEG- b -PCL plus niosome exhibited a lower MIC at 24 h (0.25 mg/mL), inhibiting 90-100% of Acanthamoeba trophozoites which lasted 72 h. KRe-loaded niosome affected adherence by around 40-60% at 0.125-0.25 mg/mL and removed Acanthamoeba adhesion on the surface by about 90% at 0.5 mg/mL. Cell viability of VERO and HaCaT cells treated with 0.125 mg/mL of KRe-loaded niosome and KRe-loaded PEG- b -PCL plus niosome exceeded 80%., Conclusion: Indeed, niosome and niosome plus PEG- b -PCL were suitable nanocarrier-loaded KRe, and they had a greater nanoparticle property to test with high activities against A. triangularis on the reduction of adherence ability and demonstration of its low toxicity to VERO and HaCaT cells., Competing Interests: Sónia MR Oliveira is an Academic Editor for PeerJ., (©2024 Chimplee et al.)

The current treatments for toxoplasmosis are only active against fast-growing tachyzoites, present in acute infections, with little effect on slow-growing bradyzoites within tissue cysts, present in latent chronic infections. The mitochondrion of Toxoplasma gondii is essential for its survival, and one of the major anti-parasitic drugs, atovaquone, inhibits the mitochondrial electron transport chain at the coenzyme Q:cytochrome c oxidoreductase site. Coenzyme Q (also known as ubiquinone [UQ]) consists of a quinone head and a lipophilic, isoprenoid tail that anchors UQ to membranes. The synthesis of the isoprenoid unit is essential for cell growth and is inhibited by lipophilic bisphosphonates, which inhibit the parasite growth. In this work, we investigated the effect of lipophilic bisphosphonates on the chronic stages of T. gondii . We discovered that three lipophilic bisphosphonates (BPH-1218, BPH-1236, and BPH-1238), effective for the acute infection, were also effective in controlling the development of chronic stages. We showed effectiveness by testing them against in vitro cysts and in vivo derived tissue cysts and, most importantly, these compounds reduced the cyst burden in the brains of chronically infected mice. We monitored the activity of infected mice non-invasively and continuously with a novel device termed the CageDot. A decrease in activity accompanied the acute phase, but mice recovered to normal activity and showed signs of hyperactivity when the chronic infection was established. Moreover, treatment with atovaquone or BPH-1218 ameliorated the hyperactivity observed during the chronic infection.IMPORTANCETreatment for toxoplasmosis is challenged by a lack of effective drugs to eradicate the chronic stages. Most of the drugs currently used are poorly distributed to the central nervous system, and they trigger allergic reactions in a large number of patients. There is a compelling need for safe and effective treatments for toxoplasmosis. Bisphosphonates (BPs) are analogs of inorganic pyrophosphate and are used for the treatment of bone disorders. BPs target the isoprenoid pathway and are effective against several experimental parasitic infections. Some lipophilic BPs can specifically inhibit the mitochondrial activity of Toxoplasma gondii by interfering with the mechanism by which ubiquinone is inserted into the inner mitochondrial membrane. In this work, we present the effect of three lipophilic BPs against T. gondii chronic stages. We also present a new strategy for the monitoring of animal activity during disease and treatment that is non-invasive and continuous., Competing Interests: The authors declare no conflict of interest.

Vector-borne protozoal diseases (VBPD) represent an enormous health and economic burden, particularly in low- and middle-income countries. Their control requires integrated approaches that consider not only therapeutic interventions for affected human and animal populations but also preventive tools. Environmental contamination can lead to therapeutic ineffectiveness. Effective intervention must consider in-depth knowledge of the environmental factors that regulate the exposure, transmission and pathogenicity of VBPD within a One Health approach. In recent decades, the incidence and prevalence of VBPD have been substantially reduced in many regions of the world, although there are still hot spots and emerging epidemiological cycles. Except for a partially protective vaccine against malaria, vaccination is not available for any other human VBPD, and therefore epidemiological control and chemotherapy are the main control tools. Current therapeutics have several drawbacks, including reduced efficacy, toxicity and high price of safer formulations. In addition, the industrial pipeline is limited, and no therapeutic breakthroughs are expected. Integrated control of VBPD requires multitarget control systems adapted to the disease and region. In this scenario, harmonized surveillance systems, accurate reporting and increased public and private investment will ensure more rational use of the few available and new drugs.

Visceral leishmaniasis, caused by Leishmania infantum in New World countries, is the most serious and potentially fatal form of leishmaniasis, if left untreated. There are currently no effective prophylactic measures, and therapeutic options are limited. Therefore, we investigated whether the aromatase inhibitor letrozole (LET), which is already used to treat breast cancer, has an antileishmanial activity and/or immunomodulatory potential and therefore may be used to treat L. infantum infection. LET was active against L. infantum promastigote and amastigote life cycle stages in an i n vitro infection model using human THP-1 cell-derived macrophages. In human peripheral blood leukocytes ex vivo , LET reduced the internalized forms of L. infantum by classical monocytes and activated neutrophils. Concomitantly, LET stimulated the production of IL-12/TNF-α and decreased the production of IL-10/TGF-β by peripheral blood phagocytes, while in T and B cells, it promoted the production of TNF-α/IFN-γ and decreased that of IL-10. In a murine infection model, LET significantly reduced the parasite load in the liver after just 5 days and in the spleen after 15 days. During in vivo treatment with LET, the production of TNF-α/IFN-γ also increased. In addition, the proportion of developing granulomas decreased and that of mature granulomas increased in the liver, while there was no significant change in organ architecture in the spleen. Based on these data, repositioning of LET may be promising for the treatment of visceral leishmaniasis in humans., Competing Interests: The authors declare no conflict of interest.

Leishmaniasis is a parasitic neglected tropical disease, affecting 12 million people. Available treatments present several limitations, with an increasing number of resistance cases. In the search for new chemotherapies, the natural product dehydrodieugenol B was used as a scaffold for the synthesis of a series of derivatives, resulting in the discovery of the promising analog [4-(4-(5-allyl-3-methoxy-2-((4-methoxybenzyl)oxy)phenoxy)-3-methoxybenzyl)morpholine, 1 ]. In this work, we investigated the effect of compound 1 on cell signaling in Leishmania (L.) infantum , culminating in cell death, as well as its immunomodulatory effect in the host cell. Additionally, we performed a pharmacokinetic profile study in an animal model. After treatment, compound 1 induced the alkalinization of acidocalcisomes and concomitant Ca 2+ release in the parasite. These events may induce depolarization of the mitochondrial potential, with successive collapse of the bioenergetic system, leading to a reduction of ATP and reactive oxygen species (ROS) levels. The analysis of total proteins and protein profile by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) demonstrated that compound 1 also altered the parasite proteins after treatment. Transmission electron microscopy studies revealed ultrastructural damage to mitochondria; together, these data suggest that compound 1 may promote autophagic cell death. Additionally, compound 1 also induced an immunomodulatory effect in host cells, with a reduction of Th1 and Th2 cytokine response, characterizing an anti-inflammatory compound. The obtained pharmacokinetic profile in rats enhances the potential of the compound, with a mean plasma half-life (T 1/2 ) of 21 h. These data reinforce the potential of compound 1 as a new lead for future efficacy studies., Competing Interests: The authors declare no conflict of interest.

Diarrhoeal disease caused by Cryptosporidium is a major cause of morbidity and mortality in young and malnourished children from low- and middle-income countries, with no vaccine or effective treatment. Here we describe the discovery of EDI048, a Cryptosporidium PI(4)K inhibitor, designed to be active at the infection site in the gastrointestinal tract and undergo rapid metabolism in the liver. By using mutational analysis and crystal structure, we show that EDI048 binds to highly conserved amino acid residues in the ATP-binding site. EDI048 is orally efficacious in an immunocompromised mouse model despite negligible circulating concentrations, thus demonstrating that gastrointestinal exposure is necessary and sufficient for efficacy. In neonatal calves, a clinical model of cryptosporidiosis, EDI048 treatment resulted in rapid resolution of diarrhoea and significant reduction in faecal oocyst shedding. Safety and pharmacological studies demonstrated predictable metabolism and low systemic exposure of EDI048, providing a substantial safety margin required for a paediatric indication. EDI048 is a promising clinical candidate for the treatment of life-threatening paediatric cryptosporidiosis., (© 2024. The Author(s).)

Ten compounds, six extracts and five fractions obtained from three Nigerian plants were assayed for their in vitro antitrypanosomal and antileishmanial activities. Each plant was extacted with hexane, ethyl acetate, and methanol. Isolated compounds were characterized and identified based on their NMR chemical shifts and comparison to literature reports. The crude extracts, fractions and isolated compounds were tested against the kinetoplastid parasites: bloodstream forms of Trypanosoma brucei Lister 427WT and the derived multi-drug resistant clone B48, and promastigote forms of Leishmania mexicana cas9/T7 and the derived clone cas9ΔNT1. Column chromatography of the extracts using silica gel yielded ten compounds identified as curzerenone, epi-curzerenone, chloranthene F, isofuranodienone, 8(17)-12E-labdadiene-15, 16-dial and 15-hydroxy-8(17),12E-labdadiene-16-al from Siphonochilus aetiopicus, lupeol, linalolic acid and spinasterone from Calliandra portoricensis, and abruquinone B from Abrus precatorius. The assay results showed that the Siphonochilus aetiopicus and Calliandra portoricensis crude extracts, fractions and compounds displayed moderate activity against the Trypanosoma brucei but showed less activity against Leishmania mexicana. Abrus precatorius crude extract, fraction, and isolated compound exhibited only weak trypanocidal and leishmanicidal activities against both kinetoplastid parasites tested. These findings have provided evidence for the use of Siphonochilus aetiopicus and Calliandra portoricensis in traditional medicine relating to parasitic diseases., Competing Interests: Declaration of competing interest The authors declare no conflict of interest associated with this publication., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In this work, chitosan/collagen-based membranes loaded with 2,3-dihydrobenzofuran (2,3-DHB) were developed through a simple solvent-casting procedure for use in the treatment of cutaneous Leishmaniasis. The obtained membranes were characterized by elemental analysis, FTIR, TG, DSC, and XRD. Porosity, swelling, mechanical properties, hydrophilicity, and antioxidant activity were analyzed. In addition, assessment to the biocompatibility, through fibroblasts/keratinocytes and in vitro wound healing essays were performed. The obtained results show that the new 2,3-DHB loaded chitosan/collagen membrane presented high porosity and swelling capacity as well as maximum strength, hydrophilicity, and antioxidant activity higher in relation to the control. The tests of antileishmanial activity and the AFM images demonstrate great efficacy of inhibition growth of the parasite, superior to those from the standard therapeutic agent that is currently used: Amphotericin B. The new membranes are biocompatible and stimulated the proliferation of keratinocytes. SEM images clearly demonstrate that fibroblasts were able to adhere, maintained their characteristic morphology. The healing test evidenced that the membranes have adequate environment for promoting cell proliferation and growth. As the conventional treatments often use drugs with high toxicity, the as-developed new membranes proved to be excellent candidate to treat cutaneous Leishmaniasis and can be clearly indicated for further advanced studies in vivo., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Edvani Curti Muniz reports financial support was provided by National Council for Scientific and Technological Development. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: The protist Blastocystis species (sp.) inhabits the gastrointestinal tracts of humans and animals. In recent decades, alternative natural products derived from plants have demonstrated potential as effective treatments for Blastocystis infection. The anti-Blastocystis activity of three herbal ethanolic extracts- Odontites linkii subsp. cyprius, Ptilostemon chamaepeuce subsp. cyprius and Quercus alnifolia-were investigated in this study., Methods: Three distinct isolates of Blastocystis sp. maintained in vitro were molecularly subtyped. Cytotoxicity analysis was performed on individual Blastocystis sp. isolates using 250, 500, 1000, and 2000 μg/mL herbal ethanolic extracts for 24 and 48 hours. Quantitative, morphological, and size alterations of Blastocystis cells assessed the cytotoxicity of herbal anti-Blastocystis effect., Results: Following subtyping analysis, one strain of Blastocystis had ST3 and ST1 mixed subtypes, and two strains had ST1 subtypes. Starting after 24 h of incubation, P. cham. subsp. cyprius (1000 μg/mL) exhibited the most pronounced and consistent anti-Blastocystis cytotoxicity against all three strains, comparable to metronidazole. The Ptilostemon chamaepeuce subsp. cyprius anti-Blastocystis cytotoxicity was evident in parasite quantitative distress, morphological alterations, and significant reductions in cell size. Odontites linkii subsp. cyprius cytotoxicity varied among the three Blastocystis strains. The three Blastocystis strains were resistant to Quercus alnifolia., Conclusion: P. cham. subsp. cyprius was a potent and promising new herbal extract against Blastocystis sp. in vitro assays., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

CYP5122A1, an enzyme involved in sterol biosynthesis in Leishmania, was recently characterized as a sterol C4-methyl oxidase. Screening of a library of compounds against CYP5122A1 and CYP51 from Leishmania resulted in the identification of two structurally related classes of inhibitors of these enzymes. Analogs of screening hit N-(3,5-dimethylphenyl)-4-(pyridin-4-ylmethyl)piperazine-1-carboxamide (4a) were generally strong inhibitors of CYP51 but were less potent against CYP5122A1 and typically displayed weak inhibition of L. donovani promastigote growth. Analogs of screening hit N-(4-(benzyloxy)phenyl)-4-(2-(pyridin-4-yl)ethyl)piperazine-1-carboxamide (18a) were stronger inhibitors of both CYP5122A1 and L. donovani promastigote proliferation but also remained selective for inhibition of CYP51. Two compounds in this series, N-(4-((3,5-bis(trifluoromethyl)benzyl)oxy)phenyl)-4-(2-(pyridin-4-yl)ethyl)piperazine-1-carboxamide (18e) and N-(4-((3,5-di-tert-butylbenzyl)oxy)phenyl)-4-(2-(pyridin-4-yl)ethyl)piperazine-1-carboxamide (18i) showed modest selectivity for inhibiting L. donovani promastigote proliferation compared to J774 macrophages and were effective against intracellular L. donovani with EC 50 values in the low micromolar range. Replacement of the 4-pyridyl ring present in 18e with imidazole resulted in a compound (4-(2-(1H-imidazol-1-yl)ethyl)-N-(4-((3,5-bis(trifluoromethyl)benzyl)oxy)phenyl)piperazine-1-carboxamide, 18p) with approximately fourfold selectivity for CYP5122A1 over CYP51 that inhibited both enzymes with IC 50 values ≤ 1 µM, although selective potency against L. donovani promastigotes was lost. Compound 18p also inhibited the proliferation of L. major promastigotes and caused the accumulation of 4-methylated sterols in L. major membranes, indicating that this compound blocks sterol demethylation at the 4-position in Leishmania parasites. The molecules described here may therefore be useful for the future identification of dual inhibitors of CYP51 and CYP5122A1 as potential antileishmanial drug candidates and as probes to shed further light on sterol biosynthesis in Leishmania and related parasites., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Karl Werbovetz has patent #WO2024059333A1 pending to Ohio State Innovation Foundation, University of Kansas. Pankaj Sharma has patent #WO2024059333A1 pending to Ohio State Innovation Foundation, University of Kansas. Chris La Rosa has patent #WO2024059333A1 pending to Ohio State Innovation Foundation, University of Kansas. Michael Zhuo Wang has patent #WO2024059333A1 pending to Ohio State Innovation Foundation, University of Kansas. Yiru Jin has patent #WO2024059333A1 pending to Ohio State Innovation Foundation, University of Kansas. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Trichomonas vaginalis is a protist parasite of the urogenital tract, responsible for human trichomoniasis, an infection sexually transmitted that affects approximately 156 million people worldwide. This pathology is more evident in females and can cause miscarriages, premature births, and infertility. The disease can also lead to a greater predisposition to HIV infection and cervical and prostate cancer. Metronidazole (MTZ) is a drug that treats human trichomoniasis. The data from studies involving human subjects are limited regarding MTZ use during pregnancy. In addition to the toxicity of the treatment, some isolates have become resistant to MTZ. Therefore, searching for new compounds active for treating trichomoniasis becomes necessary. In the present study, we report results obtained using new phospholipid analogs. Two cardanol-based compounds designated LDT117 and LDT134 were active against T. vaginalis with an IC 50 of 4.58 and 10.24 μM, respectively. These compounds were not toxic to epithelial cells in culture. Scanning electron microscopy observations revealed a rounding of the cells, a shortening of the flagella, and protrusions on the surface of drug-treated cells. Transmission electron microscopy of treated cells revealed alterations in the plasma membrane with formations of blebs, protrusions, depressions, and vacuoles with myelin figures and vacuolization in the cytoplasm after incubation. Furthermore, after treatments with the compounds LDT117 and LDT134, the parasites presented a positive reaction for TUNEL, indicating death by a mechanism like apoptosis. Given the results obtained, further in vivo studies using animal experimental models are necessary to validate that these compounds are effective for treating human trichomoniasis., Competing Interests: Declaration of competing interest We do not have any interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Leishmaniasis is a neglected tropical disease caused by parasites of the genus Leishmania and is responsible for more than 1 million new cases and 70,000 deaths annually worldwide. Treatment has high costs, toxicity, complex and long administration time, several adverse effects, and drug-resistant strains, therefore new therapies are urgently needed. Synthetic compounds have been highlighted in the medicinal chemistry field as a strong option for drug development against different diseases. Organic salts (OS) have multiple biological activities, including activity against protozoa such as Leishmania spp. This study aimed to investigate the in vitro leishmanicidal activity and death mechanisms of a thiohydantoin salt derived from l-arginine (ThS) against Leishmania amazonensis. We observed that ThS treatment inhibited promastigote proliferation, increased ROS production, phosphatidylserine exposure and plasma membrane permeabilization, loss of mitochondrial membrane potential, lipid body accumulation, autophagic vacuole formation, cell cycle alteration, and morphological and ultrastructural changes, showing parasites death. Additionally, ThS presents low cytotoxicity in murine macrophages (J774A.1), human monocytes (THP-1), and sheep erythrocytes. ThS in vitro cell treatment reduced the percentage of infected macrophages and the number of amastigotes per macrophage by increasing ROS production and reducing TNF-α levels. These results highlight the potential of ThS among thiohydantoins, mainly related to the arginine portion, as a leishmanicidal drug for future drug strategies for leishmaniasis treatment. Notably, in silico investigation of key targets from L. amazonensis, revealed that a ThS compound from the l-arginine amino acid strongly interacts with arginase (ARG) and TNF-α converting enzyme (TACE), suggesting its potential as a Leishmania inhibitor., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Six novel tri-aryl antimony(V) hydroximato complexes (3-8) with composition [SbAr 3 (O 2 NCR)] (3: Ar = Ph, R = o-(OH)Ph, 4: Ar = Ph, R = Me, 5: Ar = Ph, R = Ph; 6: Ar = Mes, R = Me, 7: Ar = Mes, R = Ph, 8: Ar = Mes, R = o-(OH)Ph (where Ph = phenyl, Me = methyl, Mes = mesityl)), were synthesised and evaluated for anti-parasitic activity towards Leishmania major (L. major) promastigotes and amastigotes. Complexes of the form [SbAr 3 (O 2 NCR)], with the dianionic hydroximato ligand binding O,O'-bidentate to the Sb(V) centre, exist in the solid-state for the mesityl-derived complexes. In contrast, the phenyl-ligated Sb(V) complexes crystallise as the hexacoordinate, hydroxamato species [SbPh 3 (O 2 NHC(OH))], with the OH ligand derived from entrained H 2 O in the crystallisation solvent. It is found that both the aryl and hydroximato ligands are found to influence the bioactivity of the Sb(V) complexes. Complexes 3-8 exhibited varied anti-promastigote activity with IC 50 values ranging from 1.53 μM for 6 to 36.0 μM for 3, also reflected in varied anti-amastigote activity with a percentage infection range of 5.50% for 6 to 29.00% for 3 at a concentration of 10 μM. The complexes were relatively non-toxic to human fibroblasts with an IC 50 value range of 59.3 μM (7) to ≥100 μM (3-6, 8), and exhibited varied toxicity towards J774.1 A macrophages (IC 50 : 3.97 (6) to ≥100 (8) μM). All complexes showed enhanced activity compared to the parent hydroxamic acids., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Abstract: We determined the in vitro minimum lethal concentration of secnidazole and assessed the correlation with clinical susceptibility among Trichomonas vaginalis isolates obtained from 71 women, of whom 66 were successfully treated with this medication. A minimum lethal concentration ≤12.5 μg/mL correlated with clinical susceptibility in this study., Competing Interests: Conflict of Interest and Sources of Funding: This study was supported by an investigator-initiated award granted to C.A.M. by Lupin Pharmaceuticals. Lupin Pharmaceuticals had no influence on the conduct of the study or the results reported. K.J.G. is currently funded by an F31 award from the National Institute of Allergy and Infectious Diseases (1F31AI161785 01A1)., (Copyright © 2024 American Sexually Transmitted Diseases Association. All rights reserved.)

Ampelozizyphus amazonicus Ducke, known as "saracura-mirá" in the Amazon rainforest, is valued for its traditional use in malaria prevention and treatment. The plant's roots and bark are employed for these purposes, while the wood is often overlooked. Given the global importance of leishmaniasis, this research focused on A. amazonicus anti-Leishmania amazonensis potential. The ethyl acetate extract from the bark (EAEB) exhibited the most effective inhibition of intracellular amastigote growth with IC 50 7.0 μg mL -1 but showed high toxicity (CC 50 9.0 μg mL -1 ). The wood ethanol (EW) and ethyl acetate (EAEW) extracts demonstrated promising results against L. amazonensis with IC 50 15.8 μg mL -1 and 10.0 μg mL -1 , CC 50 50.5 μg mL -1 and 22.7 μg mL -1 , respectively, with EW showing the greatest promise. High-speed countercurrent chromatography (HSCCC) combined with mass spectrometry (MS) off-line detection was employed for the bioassay-guided fractionation of EAEB and EAEW using HEMWat as a solvent system. These extracts showed to be rich in saponins and triterpenes, besides the presence of glycosylated flavonoids recently reported as minor compounds. The fractions obtained from HSCCC showed high toxicity. However, this technique proved effective in obtaining fractions with a high purity level, leading to the identification of a di-glycosylated triterpene saponin from EAEB, and melaleucic acid from EAEW. These findings highlight A. amazonicus as a potential source for developing new therapeutic alternatives for leishmaniasis and HSCCC as a technique enabling better separation efficiency of complex saponin matrices., (© 2024 Wiley-VHCA AG, Zurich, Switzerland.)

Chalcone (E)-1,3-diphenyl-prop-2-en-1-one and a series of 14 methoxylated derivatives have been synthesized via Claisen-Schmidt aldol condensation and characterized by FTIR, CG/MS/DIC, 1D ( 1 H and 13 C), 2D (COSY, HSQC, and HMBC) NMR, and EMAR techniques. All molecules were tested at 1 mM concentration for antifungal (Sclerotium sp., Macrophomina phaesolina and Colletotrichum gloeosporioides), antibacterial (Acidovorax citrulli two strains), and antiprotozoal (Phytomonas serpens) activities. Unmodified chalcone (CH0) and derivatives CH1, CH2, CH8 stood out in terms of antifungal activity. CH0 presented IC 50 values of 47.3 μM (9.8 μg/mL) for the fungus C. gloeosporioides. In addition, fluorescence microscopy indicated that CH0 promoted loss of hyphal cell membrane integrity. The CH1 and CH2 derivatives promoted the inhibition of Sclerotium sp. with IC 50 of 127.5 μM (32.9 μg/mL) and 110.4 μM (29.6 μg/mL), respectively. All molecules showed high activity against the phytoparasite P. serpens with IC 50 values of 0.98, 2.40, 10.25, and 3.11 μM for the derivatives CH2, CH3, CH5 and CH14 respectively. The results demonstrated that derivatives methoxylated in both rings (CH2) as well as derivatives with a furan ring associated with the methoxy group in ring A, as well as unmodified chalcone can be promising agricultural fungicides for controlling the fungi studied., (© 2024 Wiley-VHCA AG, Zurich, Switzerland.)

Leishmaniasis is recognized as a serious public health problem in Brazil and around the world. The limited availability of drugs for treatment, added to the diversity of side effects and the emergence of resistant strains, shows the importance of research focused on the development of new molecules, thus contributing to treatments. Therefore, this work aimed to identify leishmanicidal compounds using a peptide dimerization strategy, as well as to understand their mechanisms of action. Herein, it was demonstrated that the dimerization of the peptide TSHa, (TSHa) 2 K, presented higher potency and selectivity than its monomeric form when evaluated against Leishmania mexicana and Leishmania amazonensis . Furthermore, these compounds are capable of inhibiting the parasite cysteine protease, an important target explored for the development of antileishmanial compounds, as well as to selectively interact with the parasite membranes, as demonstrated by flow cytometry, permeabilization, and fluorescence microscopy experiments. Based on this, the identified molecules are candidates for use in in vivo studies with animal models to combat leishmaniasis.

Visceral leishmaniasis is a life-threatening parasitic disease, but current antileishmanial drugs have severe drawbacks. Antifungal azoles inhibit the activity of cytochrome P450 (CYP) 51 enzymes which are responsible for removing the C14α-methyl group of lanosterol, a key step in ergosterol biosynthesis in Leishmania. However, they exhibit varying degrees of antileishmanial activities in culture, suggesting the existence of unrecognized molecular targets. Our previous study reveals that, in Leishmania, lanosterol undergoes parallel C4- and C14-demethylation to form 4α,14α-dimethylzymosterol and T-MAS, respectively. In the current study, CYP5122A1 is identified as a sterol C4-methyl oxidase that catalyzes the sequential oxidation of lanosterol to form C4-oxidation metabolites. CYP5122A1 is essential for both L. donovani promastigotes in culture and intracellular amastigotes in infected mice. CYP5122A1 overexpression results in growth delay, increased tolerance to stress, and altered expression of lipophosphoglycan and proteophosphoglycan. CYP5122A1 also helps to determine the antileishmanial effect of antifungal azoles in vitro. Dual inhibitors of CYP51 and CYP5122A1 possess superior antileishmanial activity against L. donovani promastigotes whereas CYP51-selective inhibitors have little effect on promastigote growth. Our findings uncover the critical biochemical and biological role of CYP5122A1 in L. donovani and provide an important foundation for developing new antileishmanial drugs by targeting both CYP enzymes., (© 2024. The Author(s).)

On the basis of a streamlined route to the pyrroloiminoquinone (PIQ) core, we made 16 natural products spread across four classes of biosynthetically related alkaloid natural products, and multiple structural analogs, all in ≤8 steps longest linear sequence (LLS). The strategy features a Larock indole synthesis as the key operation in a five-step synthesis of a key methoxy-PIQ intermediate. Critically, this compound was readily diverged via selective methylation of either (or both) of the imine-like or pyrrole nitrogens, which then permitted further divergence by either O- demethylation to o- quinone natural products or displacement of the methoxy group with a range of amine nucleophiles. Based on a single, early report of their potential utility against the malaria parasite, we assayed these compounds against several strains of Plasmodium falciparum , as well as two species of the related protozoan parasite Babesia . In combination with evaluations of their human cytotoxicity, we identified several compounds with potent (low-nM IC 50 ) antimalarial and antibabesial activities that are much less toxic toward mammalian cells and are therefore promising lead compounds for antiprotozoal drug discovery.

Leishmania infantum belongs to the L. donovani complex, which includes species associated with visceral leishmaniasis. Traditionally, antimonial compounds have served as the primary antiparasitic treatment for all clinical forms of leishmaniasis. However, the global spread of resistance to these compounds has posed a significant challenge in the treatment in some regions. In this study, we aimed to investigate resistance to trivalent sodium stibogluconate in vitro using promastigotes from a wild strain of L. infantum. We compared the growth rates and proteomic profiles of wild-type and resistant line conducting label-free quantitative mass spectrometry-based proteomic analyses. Statistical and bioinformatics analyses were employed to evaluate the significance of protein concentration changes, protein identity annotation, GO term analysis, biosynthetic pathways, and protein-protein interactions. Our findings revealed that the resistant line displayed a notable reduction in growth rate. Proteomic data unveiled similar protein concentrations per cell in both groups but with differing molecule copy numbers. We identified 165 proteins with increased concentration, these were associated with transcription and translation activities, lipid metabolism, energy metabolism, and peroxisome biogenesis. In the decreased protein groups were 56 proteins linked to metal acquisition and metabolism, particularly iron. These results suggest a novel perspective on antimonial resistance, highlighting the importance of post-transcriptional and post-translational regulation, alongside energy expenditure compensation and alterations in organelle membrane lipid composition in antimonial-resistant parasites. Overall, our study provides insights into the proteomic profile of stibogluconate-resistant strain, contributing to our general understanding of the complex landscape of antiparasitic resistance in L. infantum. SIGNIFICANCE: Species within the Leishmania donovani complex are implicated in cases of visceral leishmaniasis in the world. Leishmania infantum is a species that predominates in regions spanning the Mediterranean Basin, the Middle East, Central Asia, South and Central America. Antimonials were the first treatment for leishmaniasis, however in the last decades, the resistance has emerged in subregions like India, where it is not a therapeutic option. In contrast, sodium stibogluconate (SbIII) remains the first-line treatment in the Americas. Unfortunately, the emergence of resistance has outpaced the development of new therapeutic options, thereby becoming a critical point in the struggle against the disease. In this study we performed an in-depth proteomic analysis with liquid chromatography mass-mass spectrometry (LC-MS/MS) on L. infantum with Sb-induced resistance in vitro. Results showed a complex proteomic adaptation in the resistant line, involving transcriptional and translational proteins, energy compensation, and homeostasis maintenance. These insights contribute to understanding the molecular adaptation in the parasite and provide information to new investigations related to therapeutics development., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

The present review article recapitulates for the first time the antipathogenic biological data of a series of lipidic natural products and synthetic analogues thereof characterized by the presence in their structure of an alkynylcarbinol unit. The cytotoxic properties of such natural and bioinspired compounds have been covered by several literature overviews, but to date, no review article detailing their activity against pathogens has been proposed. This article thus aims at providing a comprehensive overview of the field including early studies from the 1970s and 1980s with a specific focus on results published from the late 1990s until nowadays. Publications presenting the data of almost 50 different natural products are reported. Detailed activities encompass the fields of leishmanicidal, antiplasmodial, trypanocidal, fungicidal, and mainly antibacterial and antimycobacterial compounds. The few published studies aimed at exploring the structure-activity relationship in these series are also described. Around 15 different synthetic analogues of natural products, selected among the most active reported, are also presented. The rare data available regarding the antipathogenic mode of action of these products are recalled, and finally, a comparative analysis of the available biological data is proposed with the aim of identifying the key structural determinants for the bioactivity against pathogens of these unusual compounds.

Background: The fabrication of anti-Toxoplasma drugs with less side effects and desirable efficacy is one of the important research goals facing with toxoplasmosis. This study aimed to determine the anti-Toxoplasma effects of Cinnamon zeylanicum (CZ), Moringa oleifera (MO) oil encapsulated into solid lipid nanoparticles (SLNs)., Methods: Vero cells were cultured with serial concentrations (1 mg/mL to 100 µg/mL) of CZ-SLNs and MO-SLNs in DMEM culture medium. The morphological, physical, and chemical features of nanoparticles were calculated. The cell viability assays and anti-T. gondii effects of CZ-SLNs and MO-SLNs were evaluated. The CC 50 and IC 50 indices of SLNs-enveloped extracts were calculated., Results: The particle sizes of MO-SLNs and CZ-SLNs were 411.5 and 365 nm, while PDI indices were 0.53 and 0.7, respectively. Transmission electron microscopy (TEM) showed that both MO-SLNs and CZ-SLNs were smoothed spherical nanoparticles with rounded edges. The cytotoxicity assay showed the CC 50 value of MO-SLNs at concentrations of ˃10 mg/mL. In addition, 60% of T. gondii-infected Vero cells remained alive at the concentrations ≤ 1 mg/ml, while the MO-SLNs killed at least 90% of T. gondii tachyzoites with an IC 50  > 1 µg/ml. The cytotoxicity of CZ-SLNs extract showed the CC 50 at the concentration 0.1 mg/mL. More than 50% of Vero cells, infected with T. gondii tachyzoites, survived at a concentration less than 0.1 mg/mL (IC 50 ˂ 0.1 mg/mL), while the CZ-SLNs killed at least 85% of T. gondii tachyzoites in all concentrations., Conclusion: The current results represented that the use of SLNs as a nano-carrier for M. oleifera and C. zeylanicum could kill T. gondii tachyzoites with low cytotoxicity, suggesting the effectiveness of these nano-emulsions along with the chemical agents in the treatment of Toxoplasma., (© 2024. The Author(s).)

Primary amoebic meningoencephalitis (PAM) is a human brain infection caused by Naegleria fowleri with a 97% mortality rate. Quinazolinones resulting from a Mannich-coupled domino rearrangement were recently identified as inhibitors of the amoeba. Herein, we resolved the effective concentrations for 25 pilot compounds and then, using the Mannich protocol and a key late-stage, N -demethylation/functionalization, we synthesized 53 additional analogs to improve potency, solubility and microsomal stability. We established an antiamoebic quinazolinone pharmacophore, culminating in (±)- trans - 57b which featured the best combination of potency, selectivity index, solubility, and microsomal stability. Enantiomeric separation afforded (4a S ,13b R )- 57b ( BDGR-20237 ) with a 41-fold potency advantage over its enantiomer. ADME and mouse pharmacokinetic profiling for BDGR-20237 revealed high brain penetrance but a limited half-life which did not statistically enhance the mouse survival in a pilot efficacy study. The pharmacophoric model, supported by 88 quinazolinones, several of which exhibit subnanomolar potency, will guide further scaffold optimization.

Macrocycles are prominent among drugs for treatment of infectious disease, with many originating from natural products. Herein we report on the discovery of a series of macrocycles structurally related to the natural product hymenocardine. Members of this series were found to inhibit the growth of Plasmodium falciparum , the parasite responsible for most malaria cases, and of four kinetoplastid parasites. Notably, macrocycles more potent than miltefosine, the only oral drug used for the treatment of the neglected tropical disease visceral leishmaniasis, were identified in a phenotypic screen of Leishmania infantum . In vitro profiling highlighted that potent inhibitors had satisfactory cell permeability with a low efflux ratio, indicating their potential for oral administration, but low solubility and metabolic stability. Analysis of predicted crystal structures suggests that optimization should focus on the reduction of π-π crystal packing interactions to reduce the strong crystalline interactions and improve the solubility of the most potent lead.

Objective: Antimicrobial resistance is a real threat to humanity. Pentavalent antimonials are reported non-effective in leishmaniasis treatment today, in countries like India. New treatment options have been assessed worldwide lately. Antimicrobial peptides (AMP) are the leading antibiotic candidates due to their large spectrum, fast efficacy, and low resistance risks. Cathelicidins are the AMP with well-documented antimicrobial activities against bacteria, fungi, and protozoa, over their positively charged membranes. Here, we aim to design cathelicidine-like helical peptides (CLHP), and compare their anti- Leishmanial efficacies in vitro , with meglumine antimoniate (MA) on Leishmania tropica ., Methods: A total of five study [TN-1-5] and two control (MA and non-drug) groups were formed. Cryopreserved L. tropica isolate was thawed and cultivated in Novy-MacNeal-Nicolle medium and then in RPMI. Five different CLHPs (TN1-5) were diluted in dimethyl sulphoxide. A total of 150 uL of CLHPs and MA were added into the first wells of the test plaques, followed by serial dilutions that revealed doses within 4 and 512 ug/mL. Then, 100 uL of cultures including 1x10 8 /mL of L. tropica promastigotes were added into each well. Viability of promastigotes was checked with XTT, while the parasite count was assessed at 24 th and 48 th hours., Results: TN3 was effective at 32 ug/mL. All tested CLHPs exhibited varying degrees of anti- Leishmanial activities, except TN5, even at its highest dose., Conclusion: TN3 showed a particular efficacy against L. tropica in vitro . Further studies including in vivo testing of the candidate's both efficacy and toxicity are essential., Competing Interests: Conflict of Interest: No conflict of interest was declared by the authors.

Background: Trichomonas gallinae is a protozoan parasite responsible for canker in pigeons, a debilitating disease that causes significant economic losses. While metronidazole (MTZ) remains the primary treatment, the emergence of resistance is a growing concern. This study investigated the efficacy of eugenol and its nanoemulsion formulation against T. gallinae in both in vitro and in vivo settings., Aim: To evaluate the anti-trichomonal activity of eugenol, eugenol nanoemulsion, and MTZ against T. gallinae using in vitro and in vivo models., Methods: In vitro , T. gallinae trophozoites were exposed to varying concentrations of eugenol and eugenol nanoemulsion (0.625-10 μg/ml), as well as MTZ (25 μg/ml). Cytotoxicity was assessed using Vero cells. In vivo , 120 pigeons were experimentally infected and treated with either eugenol (10 mg/kg), eugenol nanoemulsion (10 mg/kg), MTZ (25 mg/kg), or left untreated. Treatments were administered daily for 5 days., Results: In vitro , both eugenol and its nanoemulsion at 10 μg/ml achieved 100% lethality of T. gallinae after 48 hours, while MTZ reached the same effect within 24 hours. In vivo , MTZ and eugenol (at 25 mg/kg and 10 mg/kg, respectively) resulted in 100% recovery of infected pigeons 5 days post-treatment. Notably, eugenol nanoemulsion (10 mg/kg) achieved 100% recovery within just 4 days post-treatment., Conclusion: This study highlights the potential of eugenol and its nanoemulsion as alternative treatments for T. gallinae infections in pigeons. The eugenol nanoemulsion, in particular, demonstrated promising results with faster recovery rates compared to both MTZ and eugenol, suggesting it may be especially effective against MTZ-resistant strains. Further research is warranted to explore the efficacy and safety of these agents for treating T. gallinae infections in pigeons., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Indazoles have previously been identified as molecules with antiprotozoal activity. In this study, we evaluate the in vitro activity of thirteen 3-alkoxy-1-benzyl-5-nitroindazole derivatives (series D) against L. amazonensis , L. infantum, and L. mexicana . In vitro, cytotoxicity against mouse peritoneal macrophages and growth inhibitory activity in promastigotes were evaluated for all compounds, and those showing adequate activity and selectivity were tested against intracellular amastigotes. Transmission and scanning electron microscopy were employed to study the effects of 3-alkoxy-1-benzyl-5-nitroindazole and 2-benzyl-5-nitroindazolin-3-one derivatives on promastigotes of L. amazonensis . Compounds NV6 and NV8 were active in the two life stages of the three species, with the latter showing the best indicators of activity and selectivity. 3-alkoxy-1-benzyl-5-nitroindazole derivatives (series D) showed in vitro activity comparable to that of amphotericin B against the promastigote stage of Leishmania spp. Two compounds were also found to be active the amastigote stage. Electron microscopy studies confirmed the antileishmanial activity of the indazole derivatives studied and support future research on this family of compounds as antileishmanial agents.

Leishmaniasis is considered one of the most critical health concerns in the world. Unfortunately, no protective vaccines exist and conventional treatments are relatively ineffective. Therefore, new strategies are necessary against leishmaniasis. In recent years, exosomes have shown promising therapeutic outcomes in various diseases, including infectious diseases. In this regard, we aimed to explore the effect of the exosome, pyrimethamine and their combination on the anti-parasitic function of RAW264.7 cells against Leishmania major. Exosomes were isolated from the C57BL/6 peritoneal macrophages. L. major infected and non-infected RAW264.7 cells treated with exosomes, pyrimethamine (PM), and exosomes along with PM. The effect of the treatments was analysed on phagocytosis, efferocytosis, the intracellular parasite count, arginase activity, nitric oxide (NO) and reactive oxygen species (ROS) production. Exosomes could significantly elevate the phagocytosis, efferocytosis, NO and ROS in both infected and non-infected groups (Pv < 0.05). The exosomes reduced the arginase activity in both groups (Pv < 0.05). The intracellular parasite count was significantly lower after treatment with exosomes (Pv < 0.05). These results demonstrate that MQ-derived exosomes can enhance in vitro anti-parasitic responses against L. major. This provides a potential pathway for more effective treatments and underscores the importance of further research in this area., (© 2024 John Wiley & Sons Ltd.)

Leishmaniasis is a parasitic disease spread by the bite of an infected sandfly and caused by protozoan parasites of the genus Leishmania. Currently, there is no vaccine available for leishmaniasis in humans, and the existing chemotherapy methods face various clinical challenges. The majority of drugs are limited to a few toxic compounds, with some parasite strains developing resistance. Therefore, the discovery and development of a new anti-leishmanial compound is crucial. One promising strategy involves the use of nanoparticle delivery systems to accelerate the effectiveness of existing treatments. In this study, Amphotericin B (AmB) was incorporated into functionalized carbon nanotube (f-CNT) and evaluated for its efficacy against Leishmania major in vitro and in a BALB/c mice model. The increase in footpad thickness was measured, and real-time PCR was used to quantify the parasite load post-infection. Levels of nitric oxide and cytokines IL-4 and IFN-γ were also determined. We found that f-CNT-AmB significantly reduced the levels of promastigotes and amastigotes of the Leishmania parasite. The nanoparticle showed strong anti-leishmanial activity with an IC 50 of 0.00494 ± 0.00095 mg/mL for promastigotes and EC 50 of 0.00294 ± 0.00065 mg/mL for amastigotes at 72 h post-infection, without causing harm to mice macrophages. Treatment of infected BALB/c mice with f-CNT-AmB resulted in a significant decrease in cutaneous leishmania (CL) lesion size in the foot pad, as well as reduced Leishmania burden in both lymph nodes and spleen. The levels of nitric oxide and IFN-γ significantly increased in the f-CNT-AmB treated groups. Also, our results showed that the level of IL-4 significantly decreased after f-CNT-AmB treatment in comparison to other groups. In conclusion, our results demonstrate that AmB loaded into f-CNT is significantly more effective than AmB alone in inhibiting parasite propagation and promoting a shift towards a Th1 response., (© 2024 John Wiley & Sons Ltd.)

Miltefosine is the first and only drug approved for the treatment of leishmaniasis. It is also known as a PI3K/AKT signaling pathway inhibitor utilized in anti-cancer or anti-viral therapies. However, the impact of miltefosine on male fertility has not been fully understood. Therefore, this study was performed to investigate the effects of miltefosine on sperm function during capacitation. Duroc spermatozoa were exposed to 0, 2.5, 5, 10, 20, 40, and 80 μM miltefosine and induced for capacitation. Our results showed that miltefosine dramatically increased the expression of PI3K/AKT signaling pathway-associated proteins. Sperm motility, motion kinetics, capacitation, and tyrosine phosphorylation were significantly suppressed by miltefosine. However, intracellular ATP levels and cell viability were not significantly affected. Our findings suggest that miltefosine may disrupt sperm function by abnormally increasing the levels of PI3K/AKT signaling pathway-associated proteins. Therefore, the harmful effects of miltefosine on male reproduction should be considered when using this drug., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Protozoal diarrhea caused by Tritrichomonas foetus (blagburni) is a prevalent, lifelong, and globally distributed burden in domestic cats. Treatment is limited to the use of 5-nitroimidazoles and treatment failure is common. The repurposed gold salt compound auranofin has killing activity against diverse protozoa in vitro but evidence of efficacy in naturally occurring protozoal infections is lacking. This exploratory study investigated the efficacy and safety of auranofin for treatment of cats with naturally occurring, 5-nitroimidazole-resistant, T. foetus infection. The minimum lethal concentration (MLC) of auranofin against 5 isolates of feline T. foetus was determined under aerobic conditions in vitro. Healthy cats and cats with T. foetus infection were treated with immediate release auranofin (range, 0.5-3 mg/cat for 7 days) or guar gum-coated auranofin capsules (0.5 or 3 mg/cat for 7 days). Adverse effects were monitored by clinical signs and clinicopathologic testing. Efficacy was determined by fecal consistency score, bowel movement frequency, and single-tube nested PCR of feces for T. foetus rDNA. Fecal samples were assayed for concentrations of auranofin, known and predicted metabolites of auranofin, gold containing molecules, and total gold content using HPLC, LC-MS, ion mobility-MS, and ICP-MS, respectively. Auranofin was effective at killing isolates of feline T. foetus at MLC ≥ 1 μg/ml. Treatment of cats with T. foetus infection with either immediate release auranofin or a colon-targeted guar gum-coated tablet of auranofin did not eradicate infection. Treatment failure occurred despite fecal concentrations of gold that met or exceeded the equivalent MLC of auranofin. Neither auranofin, known or predicted metabolites of auranofin, nor any gold-containing molecules >100 Da could be detected in fecal samples of treated cats. Adverse effects associated with auranofin treatment were common but minor. These studies identify that in vitro susceptibility test results of auranofin may not translate to treatment effectiveness in vivo even when achieving gold concentrations equivalent to the MLC of auranofin in the target environment. These studies further establish the absence of any predicted or unpredicted gold containing metabolites in feces after oral administration of auranofin., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Introduction: Over the past 20 years, significant progress has been made in anti-leishmanial therapy. Three new drugs/formulations are available for the treatment of various forms of leishmaniasis, namely oral miltefosine, paromomycin and liposomal amphotericin B. However, these advances in drug development have added considerable complexity for clinicians including toxicity, emergence of resistance and decreased sensitivity of available drugs. The development of newer drugs with less toxicity and more efficacy is urgently needed., Areas Covered: This review comprehensively examines the latest developments and current status of antileishmanial drugs for the treatment of leishmaniasis across the world. Several new investigational drugs that showed anti-leishmanial activity under in vitro or in vivo conditions and either underwent the phase-I/II clinical trials or are on the verge of entering the trials were reviewed. We also delve into the challenges of drug resistance and discuss the emergence of new and effective antileishmanial compounds., Expert Opinion: The available treatments for leishmaniasis are limited in number, toxic, expensive, and demand extensive healthcare resources. Every available antileishmanial drug is associated with several disadvantages, such as drug resistance and toxicity or high cost. Miltefosine is potentially teratogenic. New antileishmanial drugs/treatment modalities are sorely needed for expanding future treatment options.

Toxoplasma gondii (T.gondii) is an obligate intracellular protozoan that infects warm-blooded animals and has a global distribution. Acute toxoplasmosis is commonly reported in patients with acquired/congenital toxoplasmosis and immune deficiency. New methods are needed to prevent the sideffects of classical treatment. In this study, Rosuvastatin loaded chitosan nanoparticle (CH-NP-ROS) were synthesized and zeta potential and size were determined, and an MTT assay was performed to evaluate the cell toxicity on Macrophage cells (MQ) and anti-Toxoplasma activity using Trypan-blue staining by different concentrations of Rosuvastatin (ROS), and Rosuvastatin loaded chitosan nanoparticle (CH-NP-ROS). The cell viability assay demonstrated that CH-NP-ROS had lower cell toxicity (<15 %) compared to ROS (<30 %). Statistical analysis showed that CH-NP-ROS significantly killed 98.950 ± 1.344; P < 0.05) of Toxoplasma gondii tachyzoites. In vivo results of perituneal fluid showed that CH-NP significantly reduced the parasite load in the CH-NP-ROS group, compared to that in negative control group (P < 0.001). Growth inhibition rates of tachyzoites in mice receiving free ROS and CH-NP-ROS (injection and oral form) were found to be 166.125 + 4.066, 118.750 + 4.596 and 124.875 + 2.652, respectively, compared to mice in Sulfadiazine/Pyrimethamine treated group (positive control). In the infected untreated mice (control +), the mean tachyzoite counts per oil immersion field in the spleen was 8.25 respectively. The mean survival time in all the groups treated with ROS and CH-NP-ROS was longer than that in the negative control group Therefore, nanoformulation is a promising approach for the delivery and is safe for using therapeutic effects in acute toxoplasmosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Acanthamoeba spp. emerged as a clinically important pathogen related to amoebic keratitis. It is among the main causes of corneal transplantation and vision loss in ophthalmology. The treatment protocols have a low cure rate, high toxicity, and need for drug combination. Transition metal compounds have shown promising antiprotozoal effects. This study evaluates the amoebicidal activity of copper(II) coordination compounds in combination with chlorhexidine and the cytotoxicity to topical ocular application. These copper(II) coordination compounds were screened against Acanthamoeba castellanii trophozoites (ATCC 50492). The cytotoxicity on rabbit corneal cell line (ATCC-CCL 60) was performed. The compounds showed high amoebicidal potential, with inhibition of trophozoite viability above 80%. The Cp12 and Cp13 compounds showed Minimal Inhibitory Amoebicidal Concentration (MIAC) at 200 µM and mean inhibitory concentration (IC 50 ) values lower than 10 µM. Against the cysts, Cp12 showed a reduction in viability (48%) in the longest incubation period. A synergistic effect for Cp12 with chlorhexidine was observed. The compounds have a dose-dependent effect against rabbit corneal cells. Compound Cp12 has potential for future application in developing ophthalmic formulations against Acanthamoeba keratitis and its use in multipurpose solutions is highlighted., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Cryptosporidium is among the top causes of life-threatening diarrheal infection in public health and livestock sectors. Despite its high prevalence and economic importance, currently, there is no vaccine. Control of this protozoan is difficult due to the excretion of many resistant oocysts in the feces of the infected host, which contaminate the environment. Paromomycin shows inconsistent results and isn't considered a reliable therapy for cryptosporidiosis. Nitazoxanide (NTZ), the only FDA-approved drug against this parasite, is less productive in impoverished children and PLWHA (people living with HIV/AIDS). The absence of mitochondria and apicoplast, its unique location inside enterocytes separated by parasitophorous vacuole, and, most importantly, challenges in its genetic manipulations are some hurdles to the drug-discovery process. A library of compounds has been tested against Cryptosporidium during in vitro and in vivo trials. However, there has still not been sufficient success in finding the drug of choice against this parasite. Recent genome editing technologies based on CRISPR/Cas-9 have explored the functions of the vital genes by producing transgenic parasites that help to screen a collection of compounds to find target-specific drugs, provided the sufficient availability of in vitro culturing platforms, efficient transfection methods, and analytic techniques. The use of herbal remedies against Cryptosporidium is also an emerging area of interest with sufficient clinical success due to enhanced concern regarding anthelmintic resistance. Here, we highlighted present treatment options with their associated limitations, the use of genetic tools and natural products against it to find safe, effective, and inexpensive drugs to control the ever-increasing global burden of this disease., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Mexico's Yucatan Peninsula is an endemic area of cutaneous leishmaniasis, locally known as the chiclero's ulcer, and Mayan traditional medicine which refers to the use of Thouinia paucidentata Radlk, known as k'an chuunup. Aqueous and organic leaves extracts were evaluated against promastigotes and amastigotes of Leishmania mexicana. Toxicity tests of extracts were performed using Vero and J774A.1 macrophage cell lines. The composition of the most active extracts was analysed by GC-MS. The n -hexane and ethyl acetate extracts showed potent anti- Leishmania activity against the promastigote form, and remarkably, n -hexane extract exhibited potent activity against the amastigote form. Both extracts showed low toxicity on Vero both not on J774A.1 cells. Analysis of both bioactive extracts identified as more abundant compounds, germacrene D-4-ol and thunbergen in n -hexane, and thunbergol in ethyl acetate extracts. Our study presents T. paucidentata as anti- Leishmania phytomedicine supporting its medicinal use and contributes to the understanding of its phytochemical composition.

Extracts from leaves and stems of Thymelaea tartonraira (L.) All. growing in Tunisia were characterised for the presence of flavonoids and phenolic acids by LC-ESI-MS analysis. Twelve flavonoids and ten phenolic acids were detected for the first time in the aerial parts of this plant species, the most abundant compounds being gallic acid, kaempferol, catechin, chlorogenic acid, naringenin and acacetin. The extracts were subjected to in vitro antileishmanial, antifungal and cytotoxic assays, showing promising antileishmanial activity for the E6 dichloromethane extract from the stems (IC 50 values of 1.12 ± 0.50 and 5.41 ± 1.84 µg/mL on L. donovani axenic and intramacrophagic amastigotes, respectively) at the level of the reference drug miltefosine for axenic model. No antifungal activity was observed against Candida albicans (CAAL) and Aspergillus fumigatus (ASFU) strains, with the exception of the E6 dichloromethane extract (IC 50 value of 25.28 ± 4.89 µg/mL on CAAL93 strain). Low toxicity was also highlighted against macrophages Raw 264.7 cells. These promising results point out Thymelaea tartonraira (L.) All. extracts as a valuable source of new natural products to combat leishmaniasis.

Cryptosporidiosis, a prevalent gastrointestinal illness worldwide, is caused by the protozoan parasite Cryptosporidium parvum. Calcium-dependent protein kinase 1 (CpCDPK1), crucial for the parasite's life cycle, serves as a promising drug target due to its role in regulating invasion and egress from host cells. While potent Pyrazolopyrimidine analogs have been identified as candidate hit molecules, they exhibit limitations in inhibiting Cryptosporidium growth in cell culture, prompting exploration of alternative scaffolds. Leveraging the most potent compound, RM-1-95, co-crystallized with CpCDPK1, an E-pharmacophore model was generated and validated alongside a deep learning model trained on known CpCDPK1 compounds. These models facilitated screening Enamine's 2 million HTS compound library for novel CpCDPK1 inhibitors. Subsequent hierarchical docking prioritized hits, with final selections subjected to Quantum polarized docking for accurate ranking. Results from docking studies and MD simulations highlighted similarities in interactions between the cocrystallized ligand RM-1-95 and identified hit molecules, indicating comparable inhibitory potential against CpCDPK1. Furthermore, assessing metabolic stability through Cytochrome 450 site of metabolism prediction offered crucial insights for drug design, optimization, and regulatory approval processes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The present study is focused on the isolation and identification of new therapeutic candidates from Chrysanthellum americanum Vatke., and their efficacy against pteridine reductase-1 (PTR1), a valid chemotherapeutic target in the Leishmania parasite. Henceforth, a new compound, chrysanamerine (1), along with 7 known compounds, polyacetylene 2, and flavonoids 3-8, were isolated from C. americanum. Their structures were determined by chemical and spectroscopic analyses and compared with the reported spectroscopic data. All compounds were evaluated for their anti-leishmanial activity against PTR1 via biochemical mechanism-based assay. The in vitro results showed five potential hits including a new compound, chrysanamerine (1), and four known compounds against the PTR1 enzyme. Among them, compound 1 showed a potent enzyme inhibition with an IC 50 of 31.02 ± 2.36 μM, whereas a moderate inhibition was observed in cases of compounds 5 and 6 (IC 50  = 59.86 ± 3.32, and 45.32 ± 3.5 μM, respectively). Whereas, compounds 3 and 8 showed mild inhibition (IC 50  = 72.12 ± 1.12, and 97.18 ± 1.23 μM, respectively) against PTR1, compared with trimethoprim (positive control) (IC 50  = 21.07 ± 1.6 μM). Moreover, the results were further validated via molecular docking and molecular dynamics (MD) simulations. Compound 1 showed a strong affinity to the binding site with a docking score of -11.83, along with the formation of a stable protein-ligand complex over the trajectory of 100 ns. Besides, compounds 1-8 were found to be non-cytotoxic on BJ (human fibroblast) cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Geraniol, a primary component of several essential oils, has been associated with broad-spectrum antiprotozoal activities, although moderate to weak. This study primarily concentrated on the synthesis of hydrazinated geraniol derivatives as potential antiprotozoal agents. The synthesised compounds were tested in vitro against different parasitic protozoans of clinical relevance, including Trypanosoma brucei brucei, Trypanosoma brucei rhodesiense, Trypanosoma cruzi and Leishmania infantum. Compounds 6, 8, 13, 14 and 15 demonstrated low micromolar activity against the different parasites. Compounds 8, 13, 14 and 15 had the highest efficacy against Trypanosoma brucei rhodesiense, as indicated by their respective IC 50 values of 0.74, 0.56, 1.26 and 1.00 µM. Compounds 6, 14 and 15 displayed the best activity against Trypanosoma brucei brucei, with IC 50 values of 1.49, 1.48 and 1.85 µM, respectively. The activity of compounds 6, 14 and 15 also extended to intracellular Trypanosoma cruzi, with IC 50 values of 5.14, 6.30 and 4.90 µM, respectively. Compound 6, with an IC 50 value of 11.73 µM, and compound 14, with an IC 50 value of 8.14 µM, demonstrated some modest antileishmanial activity., (© 2024 The Author(s). Archiv der Pharmazie published by Wiley‐VCH GmbH on behalf of Deutsche Pharmazeutische Gesellschaft.)

DNA topoisomerase I (Topo I) is a ubiquitous enzyme that plays a crucial role in resolving the topological constraints of supercoiled DNA during various cellular activities, including repair, replication, recombination, transcription, and chromatin remodeling. Multiple studies have confirmed the essential role of Topo I in nucleic acid metabolism of Leishmania donovani , the kinetoplastid parasite responsible for visceral leishmaniasis or kala-azar. Inhibition of this enzyme has shown promise as a strategy for therapy against visceral leishmaniasis. However, current treatment options suffer from limitations related to effectiveness, cost, and side effects. To address these challenges, computational methods have been employed in this study to investigate the inhibition of Leishmania donovani DNA topoisomerase I (LdTopo I) by phytochemicals derived from Indian medicinal plants known for their anti-leishmanial activity. A library of phytochemicals and known inhibitors was assembled, and virtual screening based on docking binding affinities was conducted to identify potent phytochemical inhibitors. To assess the drug-likeness of the docked phytochemicals, their physicochemical properties were predicted. Additionally, molecular dynamics (MD) simulations were performed on the docked complexes for a duration of 100 ns to evaluate their stability, intermolecular interactions, and dynamic behavior. Among all the docked phytochemicals, three compounds, namely CID23266147 (withanolide N), CID5488537 (fagopyrine), and CID100947536 (isozeylanone), exhibited the highest inhibitory potential against LdTopo I. These findings hold promise for the development of novel inhibitors targeting LdTopo I, which could potentially lead to improved therapies for visceral leishmaniasis.Communicated by Ramaswamy H. Sarma.

Toxoplasmosis is a pervasive parasitic infection possessing a chief impact on both public health and veterinary medicine. Unfortunately, the commercially-available anti-Toxoplasma agents have either serious side effects or diminished efficiency, specifically on the Toxoplasma tissue cysts. In the present study, metformin (The first-line treatment for type 2 diabetes mellitus) was investigated for the first time against chronic cerebral toxoplasmosis in mice model experimentally-infected with ME49 strain versus spiramycin. Two metformin regimens were applied; starting one week before the infection and four weeks PI. Parasitological, ultrastructural, histopathological, immunohistochemical, immunological, and biochemical assessments were performed. The anti-parasitic effect of metformin was granted by the statistically-significant reduction in tissue-cyst burden in both treatment regimens. This was accompanied by markedly-mutilated ultrastructure and profound amelioration of the cerebral histopathology with remarkable decline in the brain CD4 + and CD8 + T cell count. Besides, diminution of anti-Toxoplasma IgG and brain GSH levels was evident. Ultimately, the present findings highlighted the powerful promising therapeutic role of metformin in the management of chronic toxoplasmosis on a basis of anti-parasitic, anti-inflammatory, and anti-oxidant possessions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The methanolic extract of the marine sponge Hemimycale sp . yielded two new compounds; 1-(2'-methyl heptadecyl) phenol ( 1 ) and a new pyrazole derivative; 4-(hydroxymethyl)-1 H -pyrazol-3-ol ( 2 ), together with previously isolated (2' R )-2'-hydroxy-N-((2 S ,3 S ,4 R )-1,3,4-trihydroxy-16-methylpentadecan-2-yl)docosanamide ( 3 ), cholesterol ( 4 ), 5, 8- epi- dioxycholest-6-en-3-ol ( 5 ) and 3-acetylsesterstatin 3 ( 6 ), which were firstly reported from family Hymedesmiidae. Their structure elucidation was based on extensive nuclear magnetic resonance spectroscopy and high resolution-electrospray ionization-mass spectrometry. The isolated compounds were evaluated for their anti-leishmanial and cytotoxic activities. Compound 5 showed remarkable anti-leishmanial activity with IC 50 value of 15.8 ± 0.92 µg/mL comparable with the standard miltefosine (IC 50 = 3.2 ± 0.07 µg/mL), while compound 3 exhibited noteworthy cytotoxicity against A594 cell line with IC 50 value of 29.6 ± 1.68 µg/mL compared to etoposide (IC 50 = 10.9 ± 1.30 µg/mL).

Cryptosporidium parvum (C. parvum), a protozoan parasite, is known to induce significant gastrointestinal disease in humans. Lactate dehydrogenase (LDH), a protein of C. parvum, has been identified as a potential therapeutic target for developing effective drugs against infection. This study utilized a computational drug discovery approach to identify potential drug molecules against the LDH protein of C. parvum. In the present investigation, we conducted a structure-based virtual screening of 55 phytochemicals from the Syzygium aromaticum (S. aromaticum). This process identified four phytochemicals, including Gallotannin 23, Eugeniin, Strictinin, and Ellagitannin, that demonstrated significant binding affinity and dynamic stability with LDH protein. Interestingly, these four compounds have been documented to possess antibacterial, antiviral, anti-inflammatory, and antioxidant properties. The docked complexes were simulated for 100 ns using Desmond to check the dynamic stability. Finally, the free binding energy was computed from the last 10ns MD trajectories. Gallotannin 23 and Ellagitannin exhibited considerable binding affinity and stability with the target protein among all four phytochemicals. These findings suggest that these predicted phytochemicals from S. aromaticum could be further explored as potential hit candidates for developing effective drugs against C. parvum infection. The in vitro and in vivo experimental validation is still required to confirm their efficacy and safety as LDH inhibitors., Competing Interests: Declaration of competing interest The author(s) declare no competing interests., (Copyright © 2024. Published by Elsevier Inc.)

Leishmaniasis is a disease caused by the parasite Leishmania donovani affecting populations belonging to developing countries. The present study explores drug repurposing as an innovative strategy to identify new uses for approved clinical drugs, reducing the time and cost required for drug discovery. The three-dimensional structure of Leishmania donovani Sterol C-24 methyltransferase (LdSMT) was modeled and 1615 FDA-approved drugs from the ZINC database were computationally screened to identify the potent leads. Fulvestrant, docetaxel, indocyanine green, and iohexol were shortlisted as potential leads with the highest binding affinity and fitness scores for the concerned pathogenic receptor. Molecular dynamic simulation studies showed that the macromolecular complexes of indocyanine green and iohexol with LdSMT remained stable throughout the simulation and can be further evaluated experimentally for developing an effective drug. The proposed leads have further demonstrated promising safety profiles during cytotoxicity analysis on the J774.A1 macrophage cell line. Mechanistic analysis with these two drugs also revealed significant morphological alterations in the parasite, along with reduced intracellular parasitic load. Overall, this study demonstrates the potential of drug repurposing in identifying new treatments for leishmaniasis and other diseases affecting developing countries, highlighting the importance of considering approved clinical drugs for new applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This study aimed to develop microemulsions (MEs) containing α-bisabolol for the topical treatment of cutaneous leishmaniasis (CL). Initially, pseudoternary phase diagrams were developed using α-bisabolol as the oil phase, Eumulgin® CO 40 as the surfactant, Polymol® HE as the co-surfactant, and distilled water as the aqueous phase. Two transparent liquid systems (TLS) containing 5% of α-bisabolol were selected and characterized (F5E25 and F5EP25). Next, skin permeation and retention assays were performed using Franz cells. The interaction of the formulation with the stratum corneum (SC) was evaluated using the FTIR technique. The cytotoxicity was evaluated in murine peritoneal macrophages. Finally, the antileishmanial activity of microemulsions was determined in promastigotes and amastigotes of L. amazonensis (strain MHOM/BR/77/LTB 0016). As a result, the selected formulations showed isotropy, nanometric size (below 25 nm), Newtonian behavior and pH ranging from 6.5 to 6.9. The MEs achieved a 2.5-fold increase in the flux and skin-permeated amount of α-bisabolol. ATR-FTIR results showed that microemulsions promoted fluidization and extraction of lipids and proteins of the stratum corneum, increasing the diffusion coefficient and partition coefficient of the drug in the skin. Additionally, F5E25 and F5EP25 showed higher activity against promastigotes (IC 50 13.27 and 18.29, respectively) compared to unencapsulated α-bisabolol (IC 50 53.8). Furthermore, F5E25 and F5EP25 also showed antileishmanial activity against intracellular amastigotes of L. amazonensis, with IC 50 50 times lower than free α-bisabolol and high selectivity index (up to 15). Therefore, the systems obtained are favorable to topical administration, with significant antileishmanial activity against L. amazonensis promastigotes and amastigotes, being a promising system for future in vivo trials., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Leishmaniasis is a widespread parasitic disease and is one of the major public health concerns in developing countries. Many drugs have been identified for leishmania as targets, but the potential toxicity and long-term treatment remain the most significant problems in terms of further development. The present study employed physicochemical investigations, structure-based virtual screening, ADMET analysis, molecular dynamics simulation, and MM-PBSA, to identify potential compounds for Leishmania. We evaluated 30,926 natural products from the NPASS database, and four potentials passed the pharmacokinetic ADMET studies and were verified using the molecular docking approach. Molecular docking results showed good binding interaction of the compounds with the active site of leishmania pteridine reductase enzyme PTR1, with compound TTC1 showing FRED and Autodock binding energies of -10.33 and -10.94, respectively, which were comparable with the antileishmania drugs of Allopurinol, Miltefosine and the original ligand, methotrexate. TTC1 compound was found to be favorable for hydrophobic interaction with PTR1. In addition, the physicochemical properties of the compounds were studied using the SwissADME web server. All compounds followed Lipinski's rule of five and can be considered as good oral candidates. The analysis of the 100 ns molecular dynamics simulation results based on the best-docked TTC1 with PTR1 receptor demonstrates stable interactions, and the complex undergoes low conformational fluctuations. The average of the calculated binding free energy of the TTC1-1e7w complex is (-68.67 kJ/mol), and the result demonstrated that the TTC1 promoted stability to the Leishmania-PTR1 complex. The potential compounds can be further explored for their antileishmanial activity.Communicated by Ramaswamy H. Sarma.

Visceral leishmaniasis (VL), caused by protozoa of the genus Leishmania , remains a significant public health concern due to its potentially lethal nature if untreated. Current chemotherapy options are limited by severe toxicity and drug resistance. Derivatives of 1,2,4-oxadiazole have emerged as promising drug candidates due to their broad biological activity. This study investigated the effects of novel 1,2,4-oxadiazole derivatives ( Ox1 - Ox7 ) on Leishmania infantum , the etiological agent of VL. In silico predictions using SwissADME suggest that these compounds have high oral absorption and good bioavailability. Among them, Ox1 showed the most promise, with higher selectivity against promastigotes and lower cytotoxicity towards L929 fibroblasts and J774.G8 macrophages. Ox1 exhibited selectivity indices of 18.7 and 61.7 against L. infantum promastigotes and amastigotes, respectively, compared to peritoneal macrophages. Ultrastructural analyses revealed severe morphological damage in both parasite forms, leading to cell death. Additionally, Ox1 decreased the mitochondrial membrane potential in promastigotes, as shown by flow cytometry. Molecular docking and dynamic simulations indicated a strong affinity of Ox1 for the L. infantum CYP51 enzyme. Overall, Ox1 is a promising and effective compound against L. infantum .

Drug repurposing is a promising approach towards the discovery of novel treatments against Neglected Tropical Diseases, such as Leishmaniases, presenting the advantage of reducing both costs and duration of the drug discovery process. In previous work, our group developed a Machine Learning pipeline for the repurposing of FDA-approved drugs against Leishmania parasites. The present study is focused on an in vitro validation of this approach by assessing the antileishmanial effects of 10 predicted drug candidates. First, we evaluated the drugs' activity against promastigotes from two strains of L. infantum and one of L. major , which caused distinct clinical manifestations, using an MTT assay. The standard anti- Leishmania drug Amphotericin B was used as a positive control. Five molecules demonstrated anti- Leishmania effects, out of which Acebutolol, Prilocaine and Phenylephrine are described herein for the first time. When tested on promastigote growth, Acebutolol displayed IC 50 values ranging from 69.28 to 145.53 µg/mL. Prilocaine exhibited IC 50 values between 33.10 and 45.81 µg/mL. Phenylephrine, on the other hand, presented IC 50 values >200 µg/mL. The two remaining drugs, Dibucaine and Domperidone, exhibited significantly low IC 50 values varying between 0.58 and 1.05 µg/mL, and 6.30 and 8.17 µg/mL, respectively. Both compounds were previously described as anti- Leishmania agents in vivo . All five compounds demonstrated no notable cytotoxic effects on THP-1-derived macrophages at the IC 50 concentrations, allowing for their testing on the intracellular form of L. major and L. infantum parasites. Interestingly, all compounds exhibited antileishmanial activity on amastigotes with enhanced IC 50 values compared to the corresponding promastigotes. Noticeably, Dibucaine and Domperidone displayed IC 50 values of at most 1.99 µg/mL. Acebutolol, Prilocaine and Phenylephrine showed IC 50 values ranging from 13.84 to 66.81 µg/mL. Our previously published Computer-Aided repositioning pipelines of FDA-approved drugs as antileishmanial agents identified Dibucaine and Domperidone as candidates in support of previous in vivo studies. This study consolidates such findings through the in vitro validation against 2 Leishmania species, highly prevalent in Africa and Middle East, and reveals Acebutolol, Prilocaine, and Phenylephrine as novel anti- Leishmania effectors, confirming the relevance of our approach and calling for further investigations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Oualha, Abdelkrim, Guizani and Harigua-Souiai.)